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M!

INVESTIGATION OF Ca“ SIGNAL TRANSDUCTION PATHWAY(S) IN
HUMAN FIBROBLASTS

By

Shixia Huang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Genetics Program

1997

ABSTRACT

INVESTIGATION OF Ca“ SIGNAL TRANSDUCTION PATHWAY(S) IN
HUMAN FIBROBLASTS

By

Shixia Huang

In serum-free medium containing serum replacements but totally lacking in protein
growth factors, diploid human fibroblasts remained quiescent if the extracellular Ca2+
concentration was only 0.1 mM. However, when the Ca2+ concentration in this medium was
increased to 1 mM, the cells replicated as rapidly as they do in medium supplemented with
protein growth factors. When quiescent cells in medium with only 0.1 mM Ca2+ were
exposed to 1 mM or 10 mM Ca2+ or 100 ng/ml EGF, the 42 kDa and 44 kDa forms of MAPK
were rapidly activated, as demonstrated by a characteristic electrophoretic mobility shift of
these proteins and by their enhanced ability to phosphorylate myelin basic protein (MBP).

Analysis of fractions from Mono Q anion-exchange chromatography of lysates of cells
exposed to 10 mM Ca2+ or 100 ng/ml EGF revealed a peak of MBP phosphorylation activity
that coeluted with p42 and p44 MAPK as shown by immunoblot analysis. Activation of
MAPK by extracellular Ca2+ was dose dependent and biphasic, with a peak of activation at
5-10 min afier exposure, followed by a period of sustained activation of MAPK at a lower
level. This pattern has been shown (Vouret-Craviari et al. [1993], Biochem J. 289, 209) to
correlate with the re-entry of mammalian cells into the cell cycle.

We then investigated the pathway of Cap-induced MAPK activation. We found the

exposure of human fibroblasts to extracellular Ca2+ increased cytosolic inositol (l, 4, 5)-

trisphosphate level and caused a transient rise in the concentration of intracellular Ca2+. Ca2+-
induced MAPK activation was partially abolished by treatment of the cells with pertussis
toxin which inhibits certain species of G proteins. It was also decreased by treatment with
thapsigargin which depletes intracellular Ca2+ stores, by exposure to phorbol 12—myristyl 13-
acetate that downregulates protein kinase C (PKC), by treatment with calmodulin
antagonists, N-(6-aminohexyl)-5-chloro—l~naphthalenesulfonamide HCL and calmidazolium,
as well as by exposure of the cells to lanthanum, a Ca“ channel inhibitor. We found that the
c-raf-l protein was not phosphorylated after Ca” stimulation, and the Ca2+ sensing receptor
(Brown et al. [1993] Nature 366, 575-580) was not involved in mediating MAPK activation
or cell growth. These results suggest that extracellular Ca“ stimulates MAPK activation
through a pathway(s) involving a pertussis toxin sensitive G protein, phospholipase C,

intracellular Ca2+, calmodulin, and PKC.

This work is dedicated to

my husband: Yi Li

my daughter: Grace (DanDan)
my unborn baby

my father: J ia Gu Huang

my mother: Dong Xiu Li

ACKNOWLEDGMENTS

I am grateful to my graduate advisor, Dr. J. Justin McCormick, for his guidance, his
encouragement, and his support throughout my graduate program. I also want to express my
deep appreciation to Dr. Veronica Maher for her advice, her support, and her selfless efforts
in revising my papers.

I would like to give special thanks to my other committee members: Dr. Susan
Conrad, Dr. David DeWitt, and Dr. Walter Esselman, for their advice, their encouragement,
and their precious time. I would like to thank Dr. Joseph Przybyszewski for his help in
editing Chapter I. I would also like to thank members of the Carcinogenesis Laboratory for
their help and friendship during my graduate years.

Finally, I am thankful for the support and help of my husband Yi. I want to thank my
daughter Grace (DanDan) who provides me with so much love and fun and makes my life
so enjoyable, and my unborn baby whose cooperation has made the writing of my
dissertation possible in a relatively short time. Special thanks also go to my parents whose

love and encouragement are very important to me.

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................................. ix
ABBREVIATIONS ............................................................................................................. x
INTRODUCTIONS ............................................................................................................. 1
List of references ..................................................................................................... 4
CHAPTER 1. LITERATURE REVIEW ............................................................................... 7
A. Extracellular Ca2+ as the first messenger ............................................................ 7

l. Extracellular Ca2+ and cell growth .......................................................... 7

2. Other functions of extracellular Ca2+ ..................................................... 10

B. Growth factor pathways

1. Growth factors and their receptors ......................................................... 11

2. Intracellular pathways
2.1 Overview of the pathways upon growth factor stimulation ............ 13
2.2 G proteins ...................................................................................... .18
2.3 Phospholipase C ............................................................................. .19
2.4 Protein kinase C ............................................................................. 22
2.5 Raf ................................................................................................. .24
2.6 Intracellular Ca2+ ............................................................................. 27
2.7 Calmodulin .................................................................................... .29
2.8 MAP kinase ................................................................................... .33

C. Ca2+ signaling in mammalian cells
1. Intracellular components involved in Ca2+ signaling and cell

differentiation/hormonal secretion ................................................... 35

2. Intracellular components involved in Ca2+ stimulated growth pathway..37

3. Ca2+-sensing receptor and its signaling .................................................. 38

4. Other possible Cay-sensing receptors ..................................................... 39

List of references .................................................................................................... 42

CHAPTER II. Extracellular Ca2+ stimulates the activation of mitogen-activated

protein kinase and cell growth in human fibroblasts ................................. 65
Abstract .................................................................................................................. 66
Abbreviations ......................................................................................................... 67
Introduction ................................................................................... 68

vi

Materials and methods

Materials ..................................................................................................... .70
Cells and cell culture .................................................................................. 70
Media for testing for growth stimulation ................................................... 70
Preparation of cell lysates ........................................................................... 71
Immunoblot analysis of MAPK ................................................................. 72
MBP phosphorylation as an assay for MAPK activity .............................. 72
Purification of MAPK by Mono Q column chromatography ..................... 73
Results and discussion
Evidence that extracellular Ca2+ stimulates cell growth ............................ .74
MAPK activation as a result of Ca” or EGF stimulation .......................... 74
Time course for Cay-induced MAPK activation ...................................... .81
Acknowledgments ................................................................................................... 85
References ............................................................................................................. .86

CHAPTER III. Characterization of the second messenger pathway of extracellular Ca“-
induced mitogen-activated protein kinase activation in human fibroblasts

................................................................................................................ 88
Abstract .................................................................................................................. 89
Abbreviations ................................................................................. 90
Introduction ........................................................................................................... .91
Material and methods
Materials ..................................................................................................... .94
Cells and cell culture ................................................................................. .94
Isolation and culture of mouse skin fibroblasts ......................................... 95
Media for testing for growth stimulation of mouse fibroblasts .................. 96
Preparation of cell lysates .......................................................................... 96
Immunoblot analysis of MAPK (Western Blot) ......................................... 96
MAP-2 phosphorylation assay (MAPK activity assay) .............................. 97
Intracellular Ca2+ measurement ................................................................. .97
Cytosolic 1P3 measurement ....................................................................... 98
Results and discussion
Evidence that activation of MAPK by extracellular Ca2+ is
partially inhibited by PMA ............................................................ 100
Evidence that extracellular Ca” c-raf-l phosphorylation .......................... 102
Evidence that extracellular Ca2+ induces intracellular Ca2+
mobilization .................................................................................. 104
Evidence that the mobilization of intracellular Ca2+ transient by extracellular
Ca” is partially abolished by thapsigargin ................................... 106
Evidence that activation of MAPK by extracellular Ca2+ is partially
inhibited by thapsigargin ............................................................. 107

Evidence that activation of MAPK by extracellular Ca” is abolished

by the channel blocker lanthanum, but not by verapamil

or nifedipine ................................................................................. 109
Evidence that activation of MAPK by extracellular Ca2+ is partially

vii

abolished by calmodulin antagonists ............................................ 112

The role of PLC ........................................................................................ 114
Evidence that MAPK activation by extracellular Ca2+ is partially

abolished by pertussis toxin .......................................................... 118

Evidence that the Cay-sensing receptor is not involved in extracellular Ca”-

induced MAPK activation in mouse skin fibroblasts ................... .120

References ............................................................................................................ . 125

viii

LIST OF FIGURES

CHAPTER 11

Figure 1. Growth stimulating activity of Ca2+ ................................................................... .75
Figure 2. MAPK activation as a result of Ca2+ or EGF stimulation .................................. .77
Figure 3. Assay of Mono Q fractions for ability to phosphorylate MBP .......................... .79
Figure 4. Immunoblot analysis of Mono Q fractions ....................................................... .80
Figure 5. Dose-response of Cay—stimulated MAPK activity ............................................ .82
Figure 6. Time course for Ca2+-stimulated MAPK activity ................................................ 84
CHAPTER III

Figure 1. Activation of MAPK by extracellular Ca2+ is partially inhibited by PMA ....... 101
Figure 2. Extracellular Ca2+ does not induce c-raf-l phosphorylation ............................ 103
Figure 3. Intracellular Ca2+ mobilization after Ca2+ stimulation ...................................... 105
Figure 4. Effect of thapsigargin, or PMA and thapsigargin together,

on Ca2+- or EGF-induced MAPK activation .................................................................... 108

Figure 5. Effect of Ca2+ channel blockers on Ca2+ or EGF-induced MAPK activation...110
Figure 6. Effect of calmodulin antagonist on Ca2+ or EGF-induced MAPK activation...1 13

Figure 7. Cytosolic level of 1P3 was increased after Ca2+ stimulation. ........................... 116
Figure 8 Effect of extracellular Ca2+ on PLCy phosphorylation ..................................... 117
Figure 9. Activation of MAPK by extracellular Ca2+ is partially inhibited

by pertussis toxin ............................................................................................................. 119
Figure 10. MAPK activation by extracellular Ca2+ and PBS in fibroblasts from mice that
differ in Cay-sensing receptor capability ........................................................................ 121

BSA
CaM l
CaMK
CAMP
cdks
CR1
CR2
CR3
DAG
EGF
EGFR
ER
FBS

F GF 3
GAP
GDP
GTP
G proteins
lGFs
1P3

1P4

ABBREVIATIONS

bovine serum albumin

calmodulin l

calmodulin dependent protein kinase
cyclic adenosine 3', 5'-phosphate
cyclin dependent kinases
conserved region 1 in raf kinase
conserved region 2 in raf kinase
conserved region 3 in raf kinase
diacylglycerol

epidermal growth factor

EGF receptor

endoplasmic reticulum

fetal bovine serum

fibroblast growth factors

GTPase activating protein
guanosine 5'-diphosphate
guanosine 5'-triphosphate

guanine nucleotide binding proteins
insulin-like growth factors

inositol (1, 4, 5)-tn'sphosphate

inositol (l , 3, 4, 5)—tetrakisphosphate

IRS
JAK
MAP-2
MAPK
MBP
MEK
NGF
NMDA
PDGF
PI3 kinase
PIP2
PLC
PKC
PMA
PTH
SDS-PAGE
SH2
SH3
STAT
TGFB
W-7

W-12

insulin receptor substrate

Janus kinase

microtubule-associated protein 2
mitogen-activated protein kinase

myelin basic protein

MAPK kinase

nerve growth factor

N-Methyl-D-aspartate

platelet-derived growth factor
phosphotidylinosite 3 kinase
phosphatidylinositol 4, 5-bisphosphate
phospholipase C

protein kinase C

phorbol 12-myristyl 13-acctate

parathyroid hormone

sodium dodecyl sulphate-polyacrylamide gel electrophoresis
src homology 2

src homology 3

signal transducer and activator of transcription
tumor growth factor [3
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCL

N-(4-aminobutyl)-2-naphthalenesulfonamide

xi

INTRODUCTION

It has been known since the 1970's that Ca2+ is required for normal mammalian cells
to grow in culture (1-4). The first evidence of Ca2+ acting as a growth promoter came from
a study with mouse 3T3 fibroblasts by Dulbecco and Belkington (5), in which they showed
that increasing the concentration of Ca2+ above the physiological level (1.8 mM) promoted
DNA synthesis.

Previous colleagues (6,7) in Carcinogenesis Laboratory at Michigan State University
have shown that human fibroblasts grow in a serum free medium supplemented with 1.0
mM Ca2+ as rapidly as in this medium with serum or growth factors when supplemented with
the serum replacements, and in the presence of insulin as the only protein growth factor.
Although normal fibroblast cells require 1-2 mM Call for their growth, studies (6-8) have
shown that fibroblasts transformed in culture or derived from fibrosarcomas are able to
replicate in culture in serum free medium containing 0.1 mM Ca2+ or at a lower
concentration, but no protein growth factors. This indicates that 1 these cells have some
alteration in the pathway by which Ca2+ drives growth. Since the mechanism of Cay-induced
cell growth is not understood, thus studying the mechanism of Cay-induced cell growth may
provide insight into the transformation process. Therefore, my studies concentrated on the
elucidation of the mechanism(s) of Cali-driven growth in normal human fibroblasts.

It is well known that protein growth factors,, such as epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), insulin, thrombin, nerve growth factor, and
bombesin, bind to specific receptors on the external cell surface, and initiate phosphorylation

cascades through the activation of either tyrosine kinase-linked or guanine nucleotide

l

2

binding protein (G protein)-coupled receptors, and one of the key events in these cascades
is the activation of mitogen-activated protein kinase (MAPK) by phosphorylation (9-15).
Several closely related MAPK isoforms, referred to by their molecular masses, i.e., p40,
p42, p44, and p54 (16), have been identified in various cell types. In human fibroblasts, it
has been shown that EGF (11), tumor necrosis factor (13,17,18) and interleukin (18) can
cause activation of p42 and/or p44 MAPKs. The activation of MAPK occurs when activated
MAPK kinase phosphorylates the tyrosine and serine/threonine residues of MAPK (19).
This phosphorylation can be detected because it results in a mobility shift of MAPK in
sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS-PAGE). The activated
MAPK can, in turn, phosphorylate microtubule-associated protein (MAP-2) (20), myelin
basic protein (MBP) (21), ribosomal S6 kinase (22), the EGF receptor (23), phospholipase
A2 (24), and the nuclear transcriptional factors c-myc (25,26), c-fos, and c-jun (23,27).

In mouse 3T3 fibroblasts in culture, it has been shown that extracelluar Ca2+
stimulates DNA synthesis (28-33) and c-fos expression (30-32,34) at a level similar to that
induced by PDGF (31,32). Furthermore, Epstein et al. (31,32) showed that both PDGF-
induced and Ca2 +-induced c-fos expression is dependent on protein kinase C (PKC).
However, unlike PDGF, Ca2+ did not induce the phosphorylation of the PDGF receptor or
the raf protein, indicating that the pathways which result in similar gene transcription are, at
least in part, independent.

The objectives of the present work were (1) to demonstrate that human fibroblasts
can grow in a medium containing 1.0 mM Ca2+ and serum replacements but no protein
growth factors and to determine whether Ca2+ stimulates the activation of MAPK; (2) to

investigate the involvement of various intracellular components in the pathway of Ca2+-

3

induced MAPK activation; (3) to investigate the involvement of certain membrane elements,
such as the Cay-sensing receptor of Brown et al. (35) in the pathway of Cay-induced MAPK
activation and cell growth.

Chapter 1 reviews the literature regarding extracellular Ca2+ as the first messenger,
growth factor pathways, and Ca2+ signaling in mammalian cells. Chapter II consists of a
manuscript published in the Biochemical Journal , 310, 881-885, 1995. It describes the
research I carried which demonstrates that in normal human fibroblasts extracellular Ca” can
stimulate the activation of MAPK and cell growth in the absence of protein growth factors,
and correlates the MAPK activation with the growth of these cells under such conditions;
Chapter III consists of a manuscript to be submitted to the Biochemical Journal. It
describes the research I carried which demonstrates the pathways involved in extracellular

Cap-induced MAPK activation.

4

LIST OF REFERENCES

l. Hazelton, B., Mitchell, B., and Tupper, J. Calcium, magnesium, and growth control in the
WI-38 human fibroblast cell. J. Cell Biol., 83: 487-498, 1979.

2. Balk, S.D., Polimeni, P.I., Hoon, B.S., LeStourgeon, D.N., and Mitchell, R.S.
Proliferation of Rous sarcoma virus-infected, but not of normal, chicken fibroblasts in a
medium of reduced calcium and magnesium concentration. Proc. Natl. Acad. Sci. U. S. A.,
76: 3913-3916, 1979.

3. Balk, S.D., Whitfield, J.F., Youdale, T., and Braun, A.C. Roles of calcium, serum,
plasma, and folic acid in the control of proliferation of normal and Rous sarcoma
virus-infected chicken fibroblasts. Proc. Natl. Acad. Sci. U. S. A., 70: 675-679, 1973.

4. Balk, S.D. Calcium as a regulator of the proliferation of normal, but not of transformed,
chicken fibroblasts in a plasma-containing medium. Proc. Natl. Acad. Sci. U. S. A., 68:
271-275, 1971.

5. Dulbecco, R. and Elkington, J. Induction of growth in resting fibroblastic cell cultures by
Ca++. Proc. Natl. Acad. Sci. U. S. A., 72: 1584-1588, 1975.

6. Wilson, D.M., Yang, D.J., Dillberger, J.E., Dietrich, S.E., Maher, V.M., and McCormick,
J.J. Malignant transformation of human fibroblasts by a transfected N-ras oncogene. Cancer.
Res., 50: 5587-5593, 1990.

7. Morgan, T.L., Yang, D.J., Fry, D.G., Hurlin, P.J., Kohler, S.K., Maher, V.M., and
McCormick, J.J. Characteristics of an infinite life span diploid human fibroblast cell strain
and a near-diploid strain arising from a clone of cells expressing a transfected v-myc
oncogene. Exp. Cell Res., 197: 125-136, 1991.

8. SchilL R.J. Expression of growth factor genes in transformed human fibroblasts and cells
derived from human fibrosarcomas: a possible mechanism for replication in the absence of
exogenous growth factors. Ph.D Dissertation. East Lansing: Michigan State University,
1 988.

9. Pelech, S.L. and Sanghera, J.S. MAP kinases: charting the regulatory pathways
[comment]. Science, 257: 1355-1356, 1992.

10. Burgering, B.M., de Vries Smits, A.M., Medema, R.H., van Weeren, P.C., Tertoolen,
LG, and Bos, J .L. Epidermal growth factor induces phosphorylation of extracellular
signal-regulated kinase 2 via multiple pathways. . Mol. Cell Biol., 13: 7248-7256, 1993.

11. Chao, T.S., Byron, K.L., Lee, K.M., Villereal, M., and Rosner, M.R. Activation of MAP

5

kinases by calcium-dependent and calcium-independent pathways. Stimulation by
thapsigargin and epidermal growth factor. J. Biol. Chem., 267: 19876-19883, 1992.

12. Nakielny, S., Cohen, P., Wu, J., and Sturgill, T. MAP kinase activator from
insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO. J., 11:
2123-2129, 1992.

13. Victor, 1., Schwenger, R, Li, W., Schlessinger, J., and Vilcek, J. Tumor necrosis
factor-induced activation and increased tyrosine phosphorylation of mitogen-activated
protein (MAP) kinase in human fibroblasts. J. Biol. Chem, 268: 18994-18999, 1993.

14. Pang, L., Decker, S.J., and Saltiel, AR Bombesin and epidermal growth factor stimulate
the mitogen-activated protein kinase through different pathways in Swiss 3T3 cells.
Biochem. J., 289: 283-287, 1993.

15. Lenorrnand, P., Sardet, C., Pages, G., L'Allemain, G., Brunet, A., and Pouyssegur, J.
Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but
not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J. Cell Biol., 122:
1079-1088, 1993.

16. Pelech, S.L. and Sanghera, J .S. Mitogen-activated protein kinases: versatile transducers
for cell signaling. Trends. Biochem. Sci., 1 7: 233-23 8, 1992.

17. Victor, 1. and Vilcek, J. Pathways of heat shock protein 28 phosphorylation by TNF in
human fibroblasts. . Lymphokine. Cytokine. Res., 13: 315-323, 1994.

18. Guy, G.R., Chua, S.P., Wong, N.S., Ng, SB, and Tan, Y.H. Interleukin 1 and tumor
necrosis factor activate common multiple protein kinases in human fibroblasts. J. Biol.
Chem, 266: 14343-14352, 1991.

19. Wu, J., Michel, H., Dent, P., Haystead, T., Hunt, DR, and Sturgill, T.W. Activation of
MAP kinase by a dual specificity Tyr/Thr kinase. Adv. Second. Messenger. Phosphoprotein.
Res., 28: 219-225, 1993.

20. Sillirnan, CC. and Sturgill, T.W. Phosphorylation of microtubule-associated protein 2
by MAP kinase primarily involves the projection domain. Biochem. Biophys. Res.
Commun., 160: 993-998, 1989.

21. Sanghera, J.S., Aebersold, R., Monison, H.D., Bures, E.J., and Pelech, S.L. Identification
of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein
kinase p44mpk. FEBS. Lett., 273: 223-226, 1990.

22. Sturgill, T.W., Ray, L.B., Erikson, E., and Maller, J .L. Insulin-stirnulated MAP-2 kinase
phosphorylates and activates ribosomal protein S6 kinase 11. Nature, 334: 715-718, 1988.

6

23. Davis, R.J. The mitogen-activated protein kinase signal transduction pathway. J. Biol.
Chem, 268: 14553-14556, 1993.

24. Lin, L.L., Wartmann, M., Lin, A.Y., Knopf, J.L., Seth, A., and Davis, R.J. cPLA2 is
phosphorylated and activated by MAP kinase. Cell, 72: 269-278, 1993.

25. Gupta, S. and Davis, R.J. MAP kinase binds to the NH2-terminal activation domain of
c-Myc. FEBS Lett., 353: 281-285, 1994.

26. Seth, A., Gonzalez, F.A., Gupta, S., Raden, D.L., and Davis, R.J. Signal transduction
within the nucleus by mitogen-activated protein kinase. J. Biol. Chem, 26 7: 24796-24804,
1992.

27. Davis, R.J. Transcriptional regulation by MAP kinases. Mol. Reprod. Dev., 42: 459-467,
1 995 .

28. Rubin, H. and Sanui, H. Complexes of inorganic pyrophosphate, orthophosphate, and
calcium as stimulants of 3T3 cell multiplication. Proc. Natl. Acad. Sci. U. S. A., 74:
5026-5030, 1977.

29. Bowen Pope, DP. and Rubin, H. Growth stimulatory precipitates of Ca2+ and
pyrophosphate. . J. Cell Physiol., 11 7: 51-61 , 1983.

30. Mitchell, P.G., Pledger, W.J., and Cheung, H.S. Molecular mechanism of basic calcium
phosphate crystal-induced mitogenesis. Role of protein kinase C. J. Biol. Chem, 264:
14071-14077, 1989.

31. Epstein, R.J., Druker, B.J., Irminger, J .C., Jones, S.D., Roberts, T.M., and Stiles, C.D.
Extracellular calcium mimics the actions of platelet-derived growth factor on mouse
fibroblasts. Cell Growth Differ, 3: 157-164, 1992.

32. Epstein, R. Calcium-inducible transmodulation of receptor tyrosine kinase activity. Cell
Signal., 7: 377-388, 1995.

33. Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., and Yuspa, S.H.
Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell,
19: 245-254, 1980.

34. Cheung, H.S., Mitchell, P.G., and Pledger, W.J. Induction of expression of c-fos and
c-myc protooncogenes by basic calcium phosphate crystal: effect of beta-interferon. Cancer
Res., 49: 134-138, 1989.

35. Brown, B.M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, 0., Sun, A.,
Hediger, M.A., Lytton, J., and Hebert, S.C. Cloning and characterization of an extracellular
Ca(2+)-sensing receptor fi'om bovine parathyroid. Nature, 366: 575-580, 1993.

CHAPTER I LITERATURE REVIEW

A. Extracellular Ca2+ as the first messenger

l. Extracellular Ca2+ and cell growth

In normal resting mammalian cells, the cytosolic free Ca2+ concentration is
maintained at 10-100 nM (1), in contrast, the extracellular free Ca2+ concentration in blood
and tissue culture medium, is between 1-2 mM. “The importance of intracellular Ca” in
regulating cell function is well recognized. Not less important, but less well understood, is
the fundamental role played by extracellular Ca2+ in the modulation of cell function ” (2).

Studies in the 19703’ have shown that there is a strict requirement of 1-2 mM extracellular
Ca2+ (physiological level) for the growth of chicken (3-5), mouse (6), and human (7)
fibroblasts in culture. However, the mechanism(s) which set this Ca2+ requirement for
normal fibroblasts to grow in culture is not clear yet.

A study by Dulbecco and Elkington (6) in 1975 showed that when added to resting
cultures of mouse 3T3 fibroblasts in serum-free medium, extracellular Ca2+ concentrations
above 1.8 mM induced DNA synthesis and cell growth. Additionally, they showed that the
growth rate in 5.4 mM Ca2+ was similar to that observed after the addition of 10% serum.

They also found that the effect of Ca2+ on cell growth was synergistic with that of serum
This was the first report showing that Ca2+ itself acted as a growth promoter. Several reports
(8-15) support and expand these findings and suggest some insight into the mechanisms by
which Ca2+-induced cell growth in 3T3 mouse fibroblasts. These will be discussed in detail

below.

8

Hazelton et al. (7) showed that physiological levels of extracellular Ca2+ were
required for human lung fibroblasts (WI-3 8) to enter 8 phase. When Ca” in the medium was
reduced to 0.06 mM or lower, a substantial fraction of WI-38 cells were prevented from
entering S phase. When the Ca2+ concentration in the medium was increased to 2 mM, the
cells entered S phase. Their study also showed that, in contrast to normal WI-38 cells, SV-
40 transformed WI-38 cells did not require Ca2+ to enter S phase. Since then, another group
(16) showed similar Ca2+ requirements (1-2 mM) for the growth of human lung fibroblasts
(WI-38 and IMR90) in medium containing serum pretreated with chelex to remove the Ca2+,

but not for that of their simian virus 40 (SV40) transformed counterparts. In 1986, Praeger
and Cristofalo (17,18) showed that WI-38 cells can grow in a serum free medium with Ca2+
(5 mM) or epidermal growth factor (EGF) in the presence of insulin, dexamethasone and
transferrin. They also showed that combining suboptimal doses of EGF and Ca2+ resulted
in an additive effect on cell growth.

In 1977, Pledger et. al. (19) showed that serum contains two types of growth
promoting agents: competence factors, e. g., platelet-derived growth factor (PDGF), and
progression factors, e. g., insulin-like growth factors (IGFs). These factors function
synergistically to promote growth of BALB/c 3T3 mouse fibroblasts (19,20). Quiescent 3T3
cells exposed briefly to PDGF become “competent” to replicate their DNA and divide;
however, these “competent” cells did not progress efficiently into S phase unless they were
incubated throughout Go/G, with medium containing plasma which contains progression
factor (19). Studies by Rubin and Samui (9) and Bowen Pope and Rubin (10) suggest that
Ca2+ stimulated cell growth by forming precipitates with inorganic pyrophosphate or

orthophosphate. In their studies, they observed that Ca2+-stimulated DNA synthesis

9

correlated with the concentration of inorganic orthophosphate or pyrophosphate in the
medium, as well as the turbidity of the medium. They proposed that precipitates of Ca2+-
phosphate complexes acted in a manner similar to macromolecular stimulants such as
hormones and interacted with the plasma meinbrane. In 1979, Stiles et al. (20) found that
Ca3(PO4)2, like PDGF, acted early in the cell cycle as a competence factor, but did not act as
a progression factor. Mitchell et al. (11) and Cheung et al. (12) found that basic calcium
phosphate crystals, like PDGF, induced c-fos and c-myc gene expression. These above
studies were carried out in mouse 3T3 fibroblasts using DME medium with 5% platelet poor
plasma which contained the progression factors necessary for mouse fibroblasts to enter the
cell cycle after stimulation by competence growth factors, such as PDGF. The distinct role
of competence and progression factors has been demonstrated in mouse 3T3 fibroblasts
(19,20) and lymphocytes (21). Human fibroblasts appear to need only competence growth
factor such as PDGF or EGF for their growth. One explanation for this is that human
fibroblasts synthesize IGF-l (22) during cell proliferation, and this IGF-l may act as a
progression factor. It is also known that during proliferation, human fibroblasts secrete
fibroblast growth factor (F GF), and FGF itself is a mitogen for fibroblasts (23). It has been
suggested that FGF may act in a positive feedback loop to induce proliferation (23).
Sugimoto et al. (24) showed that in osteoblasts, cell growth is stimulated by high
concentration of Ca2+, and that was mediated by IGF -1.

In the Carcinogenesis Laboratory of Michigan State University, Morgan et al. (25)
showed that diploid human foreskin fibroblasts can replicate rapidly in serum-flee McM
medium, a modified version of MCDB 110 (26), if supplied with designated serum

replacements and with insulin as the only protein growth factor, instead of insulin and EGF

10

as described by Ryan et al. (27). In this medium, supplemented with serum replacements
factors normal fibroblasts could grow for their entire in vitro life time (McCormick et al.,
unpublished results). The McM medium used in these studies contained 1.0 mM Ca2+
(25,28). Morgan et al. (25) also showed that normal human fibroblasts did not replicate if the
Ca2+ concentration was reduced from 1 mM to 0.1 mM, suggesting that extracellular Ca2+

stimulates cell growth.

2. Extracellular Ca2+ and other functions

In addition to its involvement in cell proliferation, extracellular Ca2+ has also been
shown to function in hormonal secretion (29) and cell differentiation (30-35). Studies
(30,32-34) showed that mouse and human keratinocytes in culture proliferated in a medium
with a low Ca2+ concentration (0.05-0.1 mM), and when the Ca2+ concentration was increased
to 1.4-2.0 mM, these cells underwent terminal differentiation.

In parathyroid cells, the decrease of extracellular Ca2+ induces the secretion of
parathyroid hormone (PTH) (29,36). Extracellular Ca2+ also negatively regulates the growth
of parathyroid cells (36). Sakaguchi (36) found that the acidic fibroblast growth factor
autocrine system was a mediator of Ca2+-regulated parathyroid cell growth. Expression of
both the mRNA and peptide of acidic F GF was suppressed by increasing the extracellular
Ca” concentration.

Kidney cells, osteoclasts, and placental cells also have the ability to sense changes
in the extracellular Ca2+ concentration (29). Osteoclasts, the cells that resorb bone during the
skeletal remodeling cycle, recognize and respond to changes in the extracellular Ca2+

concentration in a manner similar to that of parathyroid cells. Increasing the concentration

ll

of extracellular Ca2+ reduces the expression of podosomes, which contribute to cellular
attachment to bone, and also decreases their bone resorptive activity (29,37). In placental
cells, high concentrations of extracellular Ca2+ inhibit the secretion of the polypeptide
hormone, placental lactogen, from placental fragments (38,39) as well as from dispersed or

enriched cultures of trophoblastic cells prepared from placenta.

B. Growth factor pathways

1. Growth factors and their receptors

Cells exchange growth and differentiation signals through grth factors and
hormones (40,41). Most growth factors and hormones are unable to pass the hydrophobic
cell membrane. Instead, they exert their effects by binding to specific receptor proteins on
the cell surface of the target cells (41). These cell surface receptors bind to the signal
molecule (e.g., a protein growth factor) and convert the extracellular signal into intracellular
events that affect the behavior of target cells. Cell surface receptor proteins are divided into
three classes: enzyme-linked receptors, GTP binding protein (G protein)-coupled receptors,
and ion channel-linked receptors (42). Ion channel-linked receptors, which are also known
as transmitter-gated ion channels, mediate their signals by neurotransmitters that are involved
in rapid synaptic signaling between electrically excited cells. Growth factors or hormones,
which promote cell growth in their target cells, exert their effects through the activation of
either enzyme-linked or G protein-coupled receptors (43).

With enzyme-linked receptors, the extracellular binding of a ligand causes the

12

activation of receptors that function directly as enzymes, such as tyrosine kinases, or are
associated with enzymes (42,44). Most of the receptors in this family have an extracellular
domain with ligand binding sites, a single transmembrane domain, and an intracellular
domain with catalytic sites (41,42). Growth factors that have this kind of receptor include
EGF, PDGF, FGF, and IGF, etc. (41). The majority of receptors of this type are protein
kinases, or are associated with protein kinases, each of which phosphorylates specific sets
of proteins in a target cell. Most of protein tyrosine kinase receptors undergo dimerization
upon ligand binding (41). After dimerization, these receptors undergo autophosphorylation,
and in turn, phosphorylate downstream kinases which are associated with the receptor
molecules by their src homology 2 (SH2) domain (45,46).

G protein-coupled receptors bind to their ligands, and transmit a signal through a
group of G proteins, which in turn regulate downstream effectors, such as enzymes and ion
channels (42,44). All G protein-coupled receptors have an extracellular domain which binds
to the ligand, a common seven transmembrane domain, which is structurally. distinct from
the single transmembrane domain of the enzyme-linked receptors, and an intracellular
domain which interacts with G proteins (42). Growth factors or hormones which bind to this
kind of receptor include bradykinin, thrombin, bombesin, interleukin 8, parathyroid
hormone, somatostatin, A2 adenosine, etc. (29,44,47). The G protein-coupled receptors
respond to the binding of a specific ligand by undergoing a conformational change. This
allows the intracellular domain of the receptor to interact with a specific type(s) of G
proteins. If a receptor can interact with only one subtype of G protein, it will activate
effectors only through this G protein, and the response will be relatively focused. On the

other hand, if a receptor interacts with several G-proteins, each of the G proteins will interact

13

with one or more effectors, and the response will be more diffirse since it will involve several

different pathways. The function of G proteins will be discussed in section 2.2.

2. Intracellular pathways

2.] Overview of the pathways upon growth factor stimulation

Growth factors or hormones promote cell proliferation by binding and activating
their specific receptors, mainly through either tyrosine kinase-linked receptors, or G protein-
coupled receptors, and the activation of these receptors initiates one or more phosphorylation

cascade(s) (48-54) (see figure on next page and detail description below).

Pathways involving the tyrosine kinase-linked receptors The activation of

 

receptor tyrosine kinases activates downstream effectors. As described above, the binding
of a ligand to a receptor induces dimerization and autophosphorylation of the receptor . The
individual phosphotyrosine residues located in the cytoplasmic domains of receptors serve
as highly selected binding sites that interact with specific cytoplasmic molecules (55). A
common feature of many signaling molecules that are known to bind to receptor tyrosine
kinases with high affinity is that they contain src homology 2 (SH2) domains, regions of
about 100 amino acids that have homology with the noncatalytic region present in the c-src
proto-oncoprotein (56-58). A large number of proteins containing SH2 domains, and the
specific sequences that these proteins recognize and bind to, have been identified (57,58).
The SH2 domain of the p85 subunit of phosphoinositide 3 kinase (PI3 kinase) has been

shown to associate with the human PDGF [3 receptor via tyrosines 740 and 751, which lie in

Growth Factor Pathways

 

~.~——y—.

 

 

 

 

<§ene Transcription

 

15

the kinase insert regions of this receptor (59), with the murine colony-stimulating factor-1
(CSF-l) receptor via tyrosine 721 (60), and with several phosphorylated tyrosines on the
insulin receptor substrate-1 (IRS-1) (61). All of these tyrosine residues lie within the
consensus sequence of tyrosine x x methionine (YxxM) (where x can be a wide range of
possible residues), which is important for PI3 kinase binding (62). Other SH2 domain-
binding sites have been identified on the human PDGF B-receptor. These include tyrosine
771 in the kinase insert region, the site for interaction with ras GTPase activating protein
(rasGAP) (63), tyrosine 1020 in the C-terminal tail for interaction with phospholipase C y
(PLCy) (64), tyrosine 1009, the binding site for the phosphotyrosine phosphatase, syp (65),
and tyrosine 579 and 581 in the juxarnembrane segment N-terminal to the catalytic domain,
as the binding site for src family protein tyrosine kinases (66). EGF receptors (EGFR) bind
PLCy with high affinity through the SH2 domain. It has been shown that the activated
EGF R associates with She protein in vivo, most likely through its SH2 domain (67). The
Shc protein serves as an adaptor protein which mediates the function of ras through other
proteins such as Grb2 and Son of sevenless (803) (62). GAP and its associated proteins p62
and p190 become tyrosine and serine phosphorylated following stimulation with EGF (68),
although a physical complex between GAP and the EGFR has not been identified. The in
vitro studies have detected complexes between isolated GAP SH2 domains and the C-tail
domain of the EGFR (69). Activation of PI3 kinase by the EGFR differs depending on the
cell type (55). In pheochromocytoma cell line PC12, PI3 kinase is activated by the EGFR
with kinetics comparable to those of other growth factor receptors (70). The EGFR has also
been shown to function through G proteins (71). In human epiderrnoid carcinoma A431

cells, growth is inhibited by EGF, and EGF treatment of these cells induces tyrosine

l6

phosphorylation and activation of signal transducers and activators of transcription (STATS)

(72), a family of proteins that once activated, translocate from the cytoplasm to the nucleus
and bind to DNA (73,74). The activation of STATS is mediated by another family of proteins
called Janus kinases (JAK) that have been shown to associate with the EGF R (75). Many
immediate early responsive genes including the cyclin-dependent kinase inhibitor p21 are
regulated by JAK-STATs (75).

The observation that a single receptor can interact with such a diverse range of
signaling molecules demonstrates the diversity of the pathways that ligand binding can
induce. For example, the association and regulation of PDGF receptor or EGF receptor with
PI3 kinase, rasGAP, src family, PLC, JAK-STATs, etc. leads to the involvement of many
pathways or components after ligand binding. The PI3 kinase has been shown to activate p70
ribosomal s6 kinase (p70rsk) (21). RasGAP is a regulator of the ras protein which interacts
with raf, and leads to MAPK activation which activates pp90rsk or directly regulates c-
jun/fos transcription (76), or the activation of ras can activate jun kinase (JNK) by some
unknown intermediators (76). The activation of the src/yes/fyn family leads to myc gene
transcription (77). The activation of PLCy hydrolyzes phophotidylinositol bisphosphate
(PIP2) to generate diacylglycerol (DAG) and inositol (1, 4, 5)-trisphosphate (1P3). DAG in
turn activates PKC and 1P3 induces intracellular Ca2+ release (21). Many of these pathways
crosstalk with each other through certain proteins. For example, the activation of PKC and
the elevation of intracellular Ca2+ lead to MAPK activation (50,53). The activation of P13
kinase has been shown to activate MAPK through the ras pathway (78). The activation of
src has been shown to activate the ras/MAPK pathway (78), and JAK-STATs are connected

with MAPK in lymphocytes (79).

Pathwag involvinLthe G protein-coupled receptors: The activated G protein-

 

coupled receptors interact with various types of G proteins (44), and G proteins, in turn,
interact with their specific downstream effectors (44,78,80-82), mainly through, PLC, cyclic
adenosine 3', 5'-phosphate (CAMP), ras/raf, and ion channels, depending on the type of G
proteins involved.

One important feature of the G protein-coupled receptors is that the same receptor
may couple to different G proteins in different cell types (80). For example, bradykinin-
activated PLC is fully inhibited by pertussis toxin in monkey renal cells (MDCK)(80,83,84),
is partially inhibited by pertussis toxin in mouse NIH3T3 fibroblasts (80,85,86), and is not
inhibited by pertussis toxin in human epidermoid carcinoma (A431) cells (80,87,88). The
types of G proteins involved could reflect the availability of specific G protein in the cell
type studied, or the G protein pools that can be accessed by the activated receptor in that cell
type upon stimulation (80). Also, multiple pathways can be generated by varying the
concentration of the ligand (89). For example, in pancreatic acinar tissue, Trimble et al. (90)
observed that a low concentration of secretin stimulated adenylate cyclase, but a high
concentration stimulated the activation of PLC to hydrolyze PIP2, leading to the generation
of 1P3 and induces Ca2+ mobilization. Chabre et al. (91) suggested that these two pathways
were mediated by different G proteins upon stimulation by different concentrations of the
ligand, i. e., that G, directly activates adenylate cyclase and most likely a member of the Gq
class regulates the stimulation of PLC. Alternatively, Bimbaumer (92) suggested that the
stimulation of PLC could also be caused by beta gamma subunits of G, , which generate an

additional signal but only at higher concentration of the ligands.

2.2 G proteins

G proteins consist of three polypeptides, an a subunit, a B subunit, and a 7 subunit
(44,93). The a subunit binds and hydrolyzes guanosine 5'-triphosphate (GTP) to guanosine
5'-diphosphate (GDP), and the B and 7 subunits form a dimer. When GDP is bound, the a
subunit associates with the By subunits to form an inactive heterotrimer that binds to the
receptor. Both a and By subunits can bind to the receptor (44). When a growth hormone or
growth factor binds to a receptor, the receptor becomes activated and undergoes a
conformational change. The GDP bound a subunit responds to this conformational change
in the receptor and also changes its conformation, which decreases the affinity of the a
subunit to GDP ; GDP then disassociates from this subunit, and is replaced with GTP. Once
GTP is bound, the a subunit assumes its active conformation and dissociates from the
receptor and the By subunit (44).

All isoforms of 0. subunits are GTPases. However the GTP hydrolysis rate differs
from one type of the a subunit to another (94,95). The free a and By subunits each activate
their target effectors. There are over 20 different G protein (1 subunits in mammalian cells
(44). The proteins are divided into four major classes (families) based on their amino acid
sequence similarity: as (1,, (1,, (1,, and o.l2 (44) . Each of these classes has several members,
for example, a, and a,lf are in the (its family, a“, (1,141,, 0., are in the o.i family, a, ,ctll ,uM, and
aware in the (1,, family, and an, (1B are in the al2 family (44). A specific G protein activates
its specific downstream effectors. Some types of G proteins or (1 subunits can be modified
by cholera toxin or pertussis toxin (44). The a, family, which activates adenylate cyclase and

regulates ion channels upon stimulation, can be modified by cholera toxin. The o.i family of

19

G proteins, which activates cyclic GTP, phosphodiesterase, or PLC (44,80), inhibits
adenylate cyclase, or regulates K+ and Ca2+ channels, can be modified by pertussis toxin. (1,,
and (1,, are insensitive to either of these two toxins. In general 01,, activates PLC, and r1l2
regulates NaVK+ exchange (81).

In addition to the signals generated by Go subunits, GBy subunits can also generate
signals (82,96-98). Ito et al. (99) showed that overexpression of GBy stimulated the
phosphorylation of MAPK and enhanced the kinase activity of c-raf-l , and coexpression of
dominant negative ras inhibited GBy-induced MAPK phosphorylation. This indicates that
ras/raf/MAPK pathway can be activated by GBy. It has been shown that adenylate cyclase
has a binding site for the GBy subunits (100). Hawes et al. (78) showed that GBy mediated
MAPK activation through a pathway involving PI3 kinase, and that the activation of P13
kinase in this pathway was upstream of Sos/ras. They also showed that GBy was able to
activate proteins of the src family, which in turn, activate She/Grb2 and Sos, and mediate the
activation of ras/ raf/MAPK pathway. Krapivinsky et al. (101) showed that GB'y regulated

the K+ channel by directly binding to it.

2.3 Phospholipase ‘C

As described above, both the tyrosine kinase-linked receptors and G protein-coupled
receptors can activate PLC. This enzyme is responsible for hydrolyzation of the inositol
lipid, such as PIP2 , and generation of two important intracellular second messengers
(43,102), DAG, and 1P3. DAG is responsible for activating the isoenzymes of PKC (103).
On the endoplasmic reticulum, 1P3 binds with the 1P3 receptor which releases Ca2+ from

intracellular stores. The release of intracellular Ca2+ is responsible for many cellular events.

20

The main isoforms of PLC are PLCB, PLCy, PLCS, and PLCs (80,104). Each one
of these represents a family of closely related molecules. Each of the subtypes is designated
by adding Arabic numerals after the Greek letters such as PLCyl and PLCyZ (104). PLCy
has an approximate molecular weight of 150 kDa, and PLCB and PLC 8 have a molecular
weight of 85 kDa. When assayed in vitro with pure substrate, PLCB, y, and 8 have been
shown to hydrolyze both phosphotidylinositide (P1) and PIP2 (80). The hydrolysis of P1P2
occurs at micromolar concentration of Ca2+, and that of PI occurs at millimolar Ca”
concentration (80). The PLCe forms have molecular weights of 85-88 kDa, and can only
hydrolyze PIP2 but not PI when assayed in vitro (105-107), but no sequence data has yet
been obtained on molecules of this family. The previously identified PLCa (80), which has
molecular weights of 57-70 kDa and shares no homology with other isoforms of PLC, is now
known to be a placental protein disulfide isomerase (108,109), and therefore is no longer
classified as a PLC. Although the overall amino acid sequence similarity between different
PLC isoforms is low, there is two regions of homology, region X, 150 amino acids, and
region Y, 240 amino acids, which are shared by PLCB, PLCy, and PLCB (80,104). Among
the three isoforms, there are about 60% identity in region X and 40% identity in region Y
(80,110). These regions seem likely to constitute the catalytic domain.

The various isoforms of PLC are regulated differently. PLC'y is regulated by tyrosine
phosphorylation (80,111). It contains SH2 and SH3 domains. The SH2 domain targets the
molecule to tyrosine phosphorylated sequences present in other proteins such as tyrosine
kinase-linked receptors for PDGF, EGF, etc.. When activated, both PDGF and EGF
receptors have been shown to be associated with PLCy (45,46) by their SH2 domain and to

phosphorylate PLCyl on its tyrosine and serine residues (112-115). However, some other

21

receptors with tyrosine kinase activity, such as the insulin receptor and the CSF—l receptor,
do not phosphorylate PLCyl (46,116).

PLCB is regulated by G protein-coupled receptors (80). Evidence suggests that the
G proteins involved in regulating PLCB include two distinct types: pertussis toxin sensitive
and pertussis toxin insensitive (104). The (1 subunits of the Gq class of G proteins have been
demonstrated to be specific activators of the PLCB isoforrn (117). The specificity of the
interaction between different Ga subunits and PLC has been assessed by introducing cDNAs
corresponding to various Ga subunits into the monkey kidney cell line Cos-7 and measuring
the amount of inositol phosphates formed after stimulation with ALF; (104,118).
Transfection of cells with the Gaq or G01ll cDNA results in a marked increase in inositol
phosphate formation. Cotransfection of Gerq (or Gritll ) cDNA and PLC-B1 cDNA causes
even higher levels of inositol phosphate formation. The relative ability of members of the
Gq subfamily to activate PLCB isozymes has also been detennined. Membranes from Cos-7
cells transfected with cDNAs corresponding to Gaq , GetH , Getl4 , or Go“, have been
reconstituted with purified PLCB] or PLCB2 in the presence of GTPyS (104). All four
members of the Gaq subfamily were found to stimulate PLCB] , with Gaq or Gall being most
effective. Grit“5 was found to activate PLCB2 most effectively, while Gaq , Ga” , or Gritl4
showed much less stimulation. Therefore, there appears to be a specificity in the interaction
of different members for the Gq subfamily with different PLCB effectors. This specificity
may be important in generating tissue- or receptor- specific responses in vivo.

Other PLC regulators include Ca2+ and PKC. It has been shown that Ca2+ affects

PLC activation either directly or by modulating the receptor mediated responses (80). An

22

increase of intracellular Ca2+ appears to stimulate PLC activity (80). However, there is very
little information about which isoforms are Ca2+-regulated or whether there is a specific
isoform of PLC that is only sensitive to Ca“. PLCyl can be negatively regulated by PKC
(80,119). When Jurkat cells were treated with PMA, PLCy was shown to be phosphorylated
at Ser-1248. This phosphorylation did not allow the tyrosine phosphorylation of this protein,

and therefore, negatively regulated its activity (119).

2.4 Protein kinase C

PKC has been shown to be activated by various growth factors and hormones
(53,120). Multiple isoforms have been isolated, and different cell lines or tissues have been
found to express different isoforms (121). Furthermore, different stimuli or second
messengers may determine which PKC isoforms become activated (121). The PKC isoforms
are divided into three groups (121-123): conventional or classic PKC(cPKC) which includes
a, B1, B11, and 7, new PKC or novel PKC (nPKC) which includes 6, e, n, and 0, and atypical
PKCs(aPKC) which includes C and A. It has been shown that PKCa, BI, B11, 5, a, and l; are
ubiquitously distributed, e. g., in brain, lung, spleen, thymus and skin (121). PKC'y has been
found to be exclusively expressed in the central nervous system. And PKCn is strongly
expressed in skin and lung. PKCO is predominantly expressed in skeletal muscle. PKCa,
6, a, and C, have been shown to be expressed in both mouse NIH3T3 fibroblasts and rat6
fibroblasts (121,124,125).

PKCs have a protein kinase domain which is conserved in all PKC isoforms, and a
regulatory domain which shows different structures in different groups of PKCs (126). In

the regulatory domain, all PKCs have one (aPKC) or two (cPKC, nPKC) cysteine-rich

23

sequences called the C1 region. This region is the putative membrane binding region (122),
through which DAG activates PKC (121). The cPKC has a C2 region which contains many
acidic amino acids and is thought to participate in Ca2+ binding (121,122) even though there
is no sequence motif representing a known Ca2+ binding site (such as an E-F hand). The
nPKC and aPKC lack the C2 region. The activation of nPKCs is dependent on DAG but not
Ca2+. Phosphatidylcholine (PtdCho)/PLC or Phospholipase D hydrolysis of PtdCho leading
to the generation of DAG only will activate this group of PKCs (123). The activation of
aPKCs is not dependent on either Ca2+ or DAG. They can be regulated by the
Ptdlns(3,4,5)P3 pathway or the ceramide pathway (123). There is sufficient and convincing
evidence (121) showing that the activation of cPKC requires: the generation of DAG and 1P3
from plasma membrane-associated P1P2 by the activation of PLC, the release of Ca2+ from
the intracellular Ca2+ store by 1P3 stimulation, the binding of Ca2+ to the C2 region of PKC,
and subsequent translocation of PKC to the plasma membrane where it is activated by DAG
through its C1 region. The activation of cPKC also requires phosphatidylserine as a cofactor
which is constitutively present in the membrane (121).

Phorbol ester mimicks the action of DAG to activate PKC (121). Activation of
cPKC by phorbol ester does not require Ca2+, however the presence of Ca2+ will lower the
concentration of phorbol ester required for the full activation of PKC (121). Phorbol ester
treatment leads to the activation of PKC in various cells including Chinese hamster ovary
(CHO) cell line (103), mouse NIH 3T3 cell line (121), Swiss 3T3 cell line (53), and human
fibroblasts (127,128). In these cell lines, prolonged treatment with phorbol ester
downregulates the activation of many PKC isoforms (53,121). The same PKC isoform will

have a different PMA sensitivity in different cell lines. For example, PKCo. in most cell lines

24

is PMA sensitive, but in pituitary gland cells (GH4C1), this isoform is not downregulated
upon treatment with PMA (121,129). PKCB and 5 are PMA sensitive in most cell lines,
whereas the sensitivity of PKCe and PKCC to PMA is quite controversial (121). Different
PKC isoforms have been overexpressed in different cell lines (121). The overexpression of
PKC isoforms is correlated with an enhanced expression of the nuclear transcriptional factors
such as c-jun, c-fos, and c-myc, and leads to the abnormal growth of the cells (121). In rat
6 fibroblasts (130), the overexpression of PKCBI causes the cells to grow in a disorganized
manner and to be able to form colonies in soft agar. Rat 6 cells overexpressing PKCBI are
more susceptible to transformation by tranfecting an activated H-ras oncogene compared to
non-PKC expressing cells (131).

Activation of different isoforms of PKC can phosphorylate downstream effectors in
vivo. In CHO cells, overexpression of PKCa or PKC8 leads to MAPK activation (103). It
has also been shown that the activation of PKC mediates c-raf kinase activation through
direct phosphorylation (132,133). Studies with fibroblasts overexpressing PKC have
suggested that PKC functions in an agonist-stirnulating pathway leading to the activation of
phospholipase D or phospholipase A2 (122). The intracellular 85 kDa phospholipase A2
contains consensus phosphorylation sites for PKC and MAPK (122,134). However,
activation of PKC alone does not appear to be sufficient. Intermediary proteins such as

phospholipase A2-activating protein (135,136) may take part in activating this enzyme.

2.5 Raf
Growth factors, such as PDGF, activate downstream kinases, such as PKC, raf, and

MAPK, through phosphorylation (120,130,133). The viral-raf (v-raf) oncogene was

25

originally identified as the transforming gene of a murine sarcoma virus strain 3611 (137).
The protooncogenes c-raf (138) and A-raf were identified (139) by screening a mouse spleen
cDNA library with v-raf DNA as a probe. There are three active raf genes have been found
in human, mouse, and chicken, A-raf-l , B-raf, and c-raf-l (140). They are dispersed over
different chromosomes and have been mapped to sites that are frequently altered in human
tumors (141,142). The raf genes encode cytosolic phosphoproteins of similar size: c-raf-l,
74 kDa; A-raf-l , 68 kDa; B-raf, 73 kDa; and these raf proteins function as serine/threonine—
specific protein kinases (140,142). All raf proteins share three highly conserved regions
(CR1, CR2, CR3) embedded in variable sequences (143). The catalytic domain of the raf
kinases resides in CR3, whereas CR1 and CR2 fimction as the regulatory part of the raf
protein molecules (143-145). 'Ihese raf proteins are differentially expressed in tissues (142):
c-raf-l is ubiquitously expressed, A-raf-l is predominantly expressed in urogenital tissues
(139), and B-raf is most abundant in cerebrum and testis.

These kinases are inactive in quiescent cells, and can be activated by growth factors.
B-raf has been shown to be activated in mammalian pheochromocytoma PC12 cells after
stimulation by nerve growth factor (146,147). C-raf-l kinase is positively-regulated by
activated growth factor receptor kinases in a variety of fibroblastic (140,148), epithelial
(149), and lymphoid cells (150,151). Raf kinases are located in the cytosol of unstimulated
cells. Growth factors induce ras activation which causes binding to the raf protein (140).
Thus, binding by ras translocates raf to the plasma membrane which is necessary, but not
sufficient, for the activation of raf kinase activity (152). Activation of raf kinase activity
involves phosphorylation of serine/threonine residues on raf, and in the case of c-raf-l, of

additional tyrosine residues by non-receptor tyrosine kinases (152,153). In the case of B-raf,

26

but not c-raf-l, phosphorylation and activation can be observed in vitro using crude
preparations of the kinase and farnesylated ras (154). The identity of a specific kinase
activator of raf remains unknown (155). PKC has been suggested as a candidate, since
studies show that PKC mediates c-raf-l kinase activation by direct phosphorylation
( 132,133).

The activation of raf kinase leads to a characteristic shift in apparent molecular
weight upon phosphorylation (13,150). A-raf shares several functional properties with c-raf-
1 including transforming activity, stimulation of the raf/MAPK pathway, and the ability of
dominant negative versions to functionally block ras signaling (156). Once activated, raf
kinases phosphorylate the MAPK kinase (MEK) on its serine residues resulting in its
activation (155). Two serine residues of MEK at positions 218 and 222 become
phosphorylated, and either is sufficient for activation (157). MEK is a dual specificity
threonine/tyrosine kinase, which when catalytically active, phosphorylates and activates the
MAPK. Unlike raf and MEK, MAPKs have numerous substrates, including p90 ribosomal
S6 kinase, cytoplamic phospholipase A2, c-jun, c-foc, c-myc, etc. (155). Raf-1 may also
directly regulate the activation of the cell cycle through control of the mammalian Cdc25
phosphatase (158). The Cdc25 family of phosphatases activates many cyclin-dependent
kinases (cdks) by dephosphorylation of conserved threonine and tyrosine residues (155).
Active cdks control progression through each phase of the cell cycle. Raf-1 has been shown
to physically complex with human Cdc25 in vivo and in vitro (2296). Immunoprecipitates
of active, baculovirus-produced raf-l protein phosphorylate and activate the phosphatase
activity of recombinant CchS (158). However, it is not clear whether Cdc25 is

phosphorylated by raf-l or another associated kinase such as MEK or MAPK (155).

27

2.6. Intracellular Ca2+

The cytosolic concentrations of free Ca2+ in normal resting mammalian cells are very
low (0.1-1.0 uM) compared with the extracellular levels (1.0-2.0 mM) (29). Consequently,
the entry of a small quantity of Ca2+ into cells by Ca2+ channels in plasma membranes or the
release of Ca2+ from intracellular stores will result in a sharp increase in free cytosolic Ca2+
concentrations. The sudden rise in cytosolic Ca2+ is believed to play an important role in cell
proliferation (159). This idea is supported by the observation that mitogens, such as serum
(16,160), PDGF (159,161,162), bradykinin (163,164), and bombesin (165), induce an
intracellular Ca2+ increase (within seconds and which lasts for minutes) as well as cell
proliferation in normal cells such as fibroblasts and muscle cells. By blocking Ca2+ influx
with lanthanum, Estacion and Mordan (159) showed that the selective inhibition of the
sustained Ca2+ increase completely inhibited the progression of mouse fibroblasts into S
phase upon PDGF exposure. Direct evidence showing that intracellular Ca2+ is linked to cell
growth has been obtained in studies by Gill and colleagues (166,167). Their studies showed
that the intracellular Ca2+ pool depletion by thapsigargin arrested hamster smooth muscle
cells in culture in a Go-like quiescent state and blocked cell growth (see below for detail).

Ca2+ channels There are two types of Ca2+ channels in plasma membrane: voltage-

 

sensitive and voltage-insensitive (47). In general, in excitable cells, such as neuronal and
muscle cells, predominantly exhibit voltage sensitive calcium channels that open after
membrane depolarization (168,169). There are three types of voltage sensitive calcium
channels: L, T, and N type. N type Ca2+ channels are shown to be present exclusively in

neuronal cells. L and T type Ca2+ channels are shown to be present in other types of cells,

28

including mouse and human fibroblasts as demonstrated by the patch-clamp technique
which measures the conductance pattern of the cells (159,161,170). In nonexcitable cells,
such as fibroblasts and epithelial cells, Ca2+ not only passes through the L and T types of Ca2+
channels (159,161,170), but also a family of poorly characterized, voltage insensitive
calcium channels (169,171), which have been classified into three general groups: 1)
receptor-operated calcium channels, 2) second messenger-operated calcium channels, and
3) depletion-operated calcium channels. Regulation of the first group has been shown to be
independent of intracellular Ca2+ and some other second messengers (172). For example,
muscarinic receptors can regulate these Ca2+ channels (169), and the structural elements in
the receptor plays a role in its regulation (172). Stimulation of the second group of channels
can be directly regulated by intracellular calcium, 1P3, and possibly 1P4. The third group of
channels open after 1P3-mediated depletion of intracellular stores and provide a source of
calcium for refilling the stores. Regulation of calcium influx through this group of channels
is triggered by the depletion of intracellular calcium and may involve a calcium influx factor
released from the endoplasmic reticulum that stimulates the opening of low conductance
calcium channels (168,169)

It has been shown that Ca2+ entry through the different channels regulates gene
expression differently (173). Bading ct al. (173) demonstrated that N-Methyl-D-aspartate
(N MDA) receptors and L-type Ca2+ channels, the two major sites of Ca2+ entry into
hippocampal neuronal cells, transmit signals to the nucleus and regulate gene transcription
through two distinct Ca2+ signaling pathways. These investigators found that the L-type Ca2+
channels triggered Ca2+ entry and then initiated c-fos gene expression through the Ca2+

response element and/or the CAMP response element on the c-fos promoter region between

29

base pairs +1 and -222. Gene expression of c-fos was dependent on Ca2+-calmodulin-
dcpcndent protein kinase (CaMK) (173). On the other hand, NMDA receptor Ca2+ channels
induced the expression of c-fos through the serum response element of the c-fos promoter
region located on or near base pair -300. In this case, gene expression did not depend on
CaMK. These investigators also found that a specific L-type channel blocker or a NMDA

receptor antagonist completely abolished c-fos expression (173).

Intracellular Ca“ pools There are multiple intracellular Ca2+ pools present in

 

endoplasmic reticulum of various cell systems (174): the 1P3 sensitive pool and the 1P3
insensitive pool, both of which are sensitive to thapsigargin, and a third pool is unresponsive
to 1P3 and insensitive to thapsigargin. The thapsigargin sensitive pools (pools 1 and 2)
contain 75% of the total Ca2+ , and the third pool contains about 25% (174). Baurngarten et
al. (175) showed that human foreskin fibroblasts also has three different intracellular Ca2+
pools: an 1P3 sensitive, thapsigargin sensitive pool; an 1P3 insensitive, thapsigargin sensitive
pool; and an ionomycin sensitive pool which is not sensitive to either 1P3 or thapsigargin.

Studies by Gill and colleagues (166,167) have shown that thapsigargin depletes the
intracellular Ca2+ stores (pools) by specific inhibition of the endoplasmic reticulum (ER) Ca2+
-ATPasc, and in turn, will deplete the source of intracellular Ca2+ for these pools. They
found that the depletion of those Ca2+ pools resulted in the arrest of cells in G0 phase
(166,174), and thapsigargin suppressed the expression of the endoplasmic reticulum Ca2+

pump protein (176).

2.7 Calmodulin

30

The function of Ca2+ is mediated by its binding proteins. There are two different
kinds of Ca2+ binding proteins, trigger proteins and buffer proteins (168). Buffer proteins,
such as calsequestn'n, simply bind Ca2+ as its concentration increases within cells to buffer
or lower free Ca2+ levels since too high a free Ca2+ concentration is toxic to cells (168).
Trigger proteins, on another hand, change their conformation upon binding Ca2+ and
modulate effector molecules such as enzyme and ion channels. The trigger proteins include
calmodulin, calcineurin, phospholipase A2, PLC,, etc. (168). Calmodulin acts as the primary
mediator of Ca2+-dependent signaling in eukaryotic nonmuscle and smooth muscle cells by
serving as a high affinity intracellular receptor (177,178). It has been shown that calmodulin
is required for progression at specific points of the cell cycle in mammalian cells (179,180)
and that it affects cell proliferation in variety of cell types (1,181). For example, the selective
pharmacological inhibitors of calmodulin kinase, KN -62 and KN-93, have been shown to
prevent quiescent WS-l human fibroblasts from reaching S phase upon serum stimulation
(181,182). Takuwa et al. (16) showed that the potent calmodulin antagonists calmidazolium
and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide HCl (W-7) but not its inactive
analogue N-(4-aminobutyl)-2-naphthalenesulfonamide (W-12), strongly inhibit serum-
induced DNA synthesis in normal human fibroblasts (16) and human vascular cndothelial
cells (183). In neuronal cells, it has been shown (173) that the increase in intracellular Ca2+
caused by Ca2+ entry modulates the activation of calmodulin kinases (CaMK), such as
CaMK2 or CaMK 4, regulating the expression of their downstream genes. Calmodulin is
highly conserved throughout the eukaryotic kingdoms (184). It has a molecular weight of
approximately 17 kDa and a high percentage of acidic amino acids. A distinctive property

of calmodulin and of other related calcium-binding proteins is that they undergo a mobility

31

shift when run on a SDS-PAGE in the presence of Ca2+ (184). Calmodulin has been shown
to be encoded by several genes in vertebrates. Three calmodulin cDNAs (CaMl, CaM2, and
CaM3) have been cloned from rat and human, and multiple genes have been identified in
many other species (178,184,185). These cDNAs all encode identical calmodulin proteins.
Although all three mRN A transcripts are expressed in all mammalian tissues and cultured
cells, the expression level of each calmodulin gene has been shown to vary according to the
cell type and is modified by the concentration of extracellular growth factors, such as nerve
growth factor (186). In mouse C127 cells, it has been shown (178) that all three calmodulin
genes are expressed, but that only the CaM2 mRNA levels show significant changes as cells
progress through the cell cycle. These results indicate that the calmodulin genes may be
differentially regulated during the cell cycle.

In mammalian cells, the 148 amino acid calmodulin protein has a dumbbell-shape
structure with two Ca2+ binding sites in each half of the molecule (178). Except for the
budding yeast calmodulin which only binds three Ca2+ ions, calmodulins from all other
species have four highly conserved “BF-hand” Ca2+-binding sites, like those first described
in the crystal structure of parvaburnin (178,187). When intracellular Ca2+ increases, it binds
to calmodulin, and induces a conformational change in the molecule which exposes its
hydmphobic patches that are involved in interaction with and activation of its target enzymes
(177,184,188,189). Without the conformational alterations, calmodulin does not bind to
most of its various targets (184). More than 20 enzymes can be regulated by calmodulin
(178,188,190,191). These enzymes include cyclic 3', 5'-nucleotide phosphodiesterase
(178,192,193), adenylate cyclase (194,195), (Ca2+- Mg2+)ATPase (196,197), the cardiac

microsomal calcium transporter (197), 1P3 kinase (178,198,199), calmodulin-dependent

32

protein kinases, such as myosin light chain kinase (178,200) and the multifunctional
calmodulin-dependent protein kinase (201,202) as well as calmodulin-dependent protein
phosphatase (calcineurin) (203-205).

Ca2+ has been shown to be absolutely required for all enzyme activating functions
of vertebrate calmodulin (178). However, the Ca2+ requirement can be altered in vitro by
different concentrations of calmodulin. Increasing the calmodulin concentration can
decrease the amount of Ca2+ required to activate calmodulin-dependent enzymes. Also,
increasing the Ca2+ concentration can decrease the amount of calmodulin required for the
activation of calmodulin-dependent enzymes (178). These results indicate that in vitro Ca2+
and calmodulin cooperatively regulate the function of the target protein. Transformed cells
typically have elevated calmodulin levels and exhibit the ability to grow in Ca2+-deficient
medium which does not allow the growth of their nontransforrned counterparts (178).
Because it has not yet been possible to replace all three active endogenous calmodulin genes
with a single inducible calmodulin gene, the relationship between the calmodulin
concentration and the Ca2+ requirement for cell growth has remained unclear. It has been
shown that the growth of A. nidulans cells is dependent on the concentration of calmodulin
and Ca2+ as in mammalian cells (206). Using A. nidulans as a model system, it has been
shown (178) that increasing the calmodulin concentration allows these cells to grow at a very
low extracellular Ca2+ concentration (IO-fold lower than the noninduced or normal state).

These data indicate that a cooperative regulation exists inside cells between Ca2+ and
calmodulin, and this may provide a possible explanation for why cells that are transformed

and have elevated calmodulin levels can proliferate in a Ca2+-deficient medium (178).

33

2.8 MAPK

As described above, protein growth factors such as EGF, PDGF, insulin, thrombin,
nerve growth factor (N GF ), and bombesin, can initiate a phosphorylation cascade in various
cell types through the activation of either tyrosine kinase-linked or G protein-coupled
receptors, and one of the key events in this cascade is the activation of MAPK by
phosphorylation (49-54,207,208). Several closely related MAPK isoforms, usually referred
to by their molecular masses, i.e., p40, p42, p44, and p54 (209), have been identified in
various cell types. In human fibroblasts, it has been shown that EGF (50), tumor necrosis
factor (52,210,211) and interleukin (211) can cause activation of p42 and/or p44 MAPKs.

MAPK kinase activates MAPK by phosphorylation on its tyrosine and serine/threonine
residues (212), which results in a mobility shift of MAPK proteins when analyzed by SDS-
PAGE.

The activated MAPK can, in turn, phosphorylate microtubule-associated protein
(MAP-2) (213), myelin basic protein (MBP) (214), ribosomal S6 kinase (215), the EGF
receptor (216), phospholipase A2 (217), and the nuclear transcriptional factors c-myc
(218,219), and c-fos and c-jun (216,220). Pouysségur and his colleagues (221,222) showed
that in quiescent Chinese hamster fibroblast cell line CCL39, MAPK was activated by
thrombin or basic fibroblast growth factor and that the activation of MAPK (as detected by
an immunocomplex kinase assay using MBP as substrate) exhibited a biphasic pattern i.e.,
a rapid increase in activity followed by a rapid decrease and then a sustained level of lower
activity. They proposed that this sustained activity was required for the re-entry of these

cells from G0 into the cell cycle (222), i.e., for triggering the proliferative response, because

34

when they treated CCL39 cells with a thrombin analog instead of thrombin or with
carbachol, the second phase of MAPK activation was not observed and no DNA synthesis
took place.

Other investigators have demonstrated that the sustained activation of MAPK is
associated with proliferation in fibroblasts (223-225). In addition, Pages et al. (226) and
Frost et al. (227), using MAPK antisense RNA and/or MAPK kinase-deficient mutants to
suppress MAPK activation, showed that MAPK activation was required for activation of
transcriptional factors (227) and for rodent fibroblast proliferation (226).

In other cell systems, continuous MAPK activation could lead to the opposite cellular
behavior. In pheochromocytoma cell line PC12, cellular responses are determined by the
duration of MAPK activation. In these cells, it has been shown that sustained MAPK
activation is associated with differentiation, whereas transient MAPK activation is related
to proliferation (228). Continuous treatment of PC12 cells with FGF or NGF leads to
outgrowth of neurites and eventual cessation of cell division, whereas treatment with EGF
leads to a proliferative signal (229). Many signal transduction events have been found to be
shared between differentiation and proliferation in PC12 cells (230). However, notable
quantitative differences were found between differentiation and proliferative signals. In
PC12 cells, NGF stimulation results in a persistent elevation of rasGTP, whereas EGF
produces only a short lived rise in rasGTP (231). MAPK activation is sustained for several
hours following NGF stimulation, but it is short lived after EGF stimulation (229,232). In
PC12 cells, it has been found that sustained MAPK activation is always associated with
translocation of MAPK to the nucleus (229,232-234), whereas transient activation does not

lead to nuclear translocation. Transient activation will, therefore, have very different

35

consequences for gene expression compared with sustained activation (2314). In this way,
it is believed that cells can use transient and sustained activation of MAPK to determine a

variety of responses (228).

C. Ca2+ signaling in mammalian cells

1. Intracellular components involved in Ca2+ signaling and cell differentiation/hormonal
secretion

As described in “Section A”, extracellular Ca2+ can serve as the first messenger to
regulate cell differentiation, hormonal secretion, and cell proliferation in different cell
systems (29-3 5,23 5). The numerous intracellular components or pathways involved in these
processes have been found using different approaches (29-31,34,235)

In keratinocytes in culture, the addition of Ca2+ to the medium rapidly increases the
intracellular phosphoinositide turnover, intracellular Ca2+ levels (30,34), and tyrosine
phosphorylation of proteins (30,31). The rasGAP has been shown to be specifically affected
during Ca2 +-induced differentiation (31). Upon Ca2+ treatment, GAP associates with
tyrosine-phosphorylated proteins and translocates to the membrane (31). Also, a p62 protein,
which is associated with GAP, has been found to be heavily phosphorylated on tyrosine both
in membrane and cytosolic fractions (31). This protein is only phosphorylated by Ca2+
stimulation, and not via growth factor stimulation, such as EGF or TGF-B (30,31). Ca2 1 does
not induce the tyrosine phosphorylation of PLCy and PI3 kinase, which are activated upon
EGF stimulation in this cell type (31). Filvaroff ct al. (30) suggested that Ca2+-induced p62

phosphorylation, and possibly differentiation, was mediated by a Ca2+ receptor. Denning et

36

al. (235) showed that the induction of keratinocyte differentiation markers by Ca2+ was
mediated by specific protein isozymes. In the cultured murine cells they studied (235), they
detected PKC isoforms by immunoblotting (PKCu, 8, e, C). They observed that PKCa, 8,
and a were translocated from the soluble fraction to particulate fraction during Ca2+ -induced
differentiation, suggesting that the induction of keratinocyte differentiation by Ca2+ results
in the activation of specific PKC isoforms (235). In parathyroid cells, the increase of
extracellular Ca“ concentration decreases PTH release (29). Studies (236,237) showed that
stepwise increases in the extracellular Ca2+ concentration produced corresponding increases
in intracellular Ca2+ concentration in these cells. There was a close inverse relationship
between the high extracellular Ca2+-induced intracellular Ca2+ increase and the accompanying
decreases in PTH release (236,237), although it is not clear yet whether the intracellular Ca2+
change is the cause of the decrease in PTH release. However, other studies (29,23 8) showed
that increasing the intracellular Ca2+ directly by treating cells with Ca” ionophores, such as
A23 1 87, was associated with inhibition of PTH secretion. Extracellular Ca2+ also stimulates
the accumulation of inositol monophosphate, inositol bisphosphate, 1P3, and IP-4 (239). It
is believed that the increases of 1P3 mediates, at least partially, the intracellular Ca2+
elevation evoked by extracellular Ca2+ (240). Shoback et al. (239) showed that Ca2+
ionophore, ionomycin, which raises the intracellular Ca2+ directly to a level equivalent to that
observed at high extracellular Ca2+ concentration, had little or no effect on the accumulation
of inositol phosphates (29). And the addition of extracellular Ca2+ to parathyroid cells
increases 1P3 level, which has been shown to be through the activation of PLC, and is
mediated by a G protein-coupled receptor (29,241). In addition, extracellular Ca2+ inhibits

the receptor-mediated cAMP increase. The decrease of cAMP level upon Ca2+ treatment is

37

mediated by enhanced adenylate cyclase activity (2,29,242). In osteoclasts,
extracellular Ca2+ stimulates Ca2 1 release from Ca2 1 entry channels in the plasma membrane
which can be blocked by lanthanum (37) and from intracellular stores. A Ca2+ receptor is
suggested to be involved in the increase of intracellular Ca“ in this cell type (37). They
showed that in osteoclast cells, extracellular Ca2 1 induced intracellular Ca2+ elevation, which
leads to cytoskeletal changes affecting cell adhesion and decreasing the bone resorptive

activity (3 7).

2. Intracellular components involved in Ca2+ stimulated growth pathway

A few groups have studied the mechanism of Ca2 +-induced cell growth. Studies by
Rubin and Samui (9) and Bowen Pope and Rubin (10) showed that in mouse fibroblasts the
Ca2 + and phosphate precipitate can serve as the primary signal to promote cell growth. In
their studies, they observed that Ca2+-stimulated DNA synthesis was correlated with the
concentration of inorganic orthophosphate or pyrophosphate in the medium, as well as the
turbidity of the medium. They proposed that precipitates of Ca2+-phosphate complexes acted
in a manner similar to macromolecular stimulants (such as hormones) and interacted with
the plasma membrane. Most recently, Epstein et al. (13) and Epstein (14) showed that
calcium chloride mimicked PDGF in its ability to stimulate DNA synthesis and to induce c-
fos gene expression in mouse 3T3 fibroblasts. They showed that both PDGF-induced and
Ca2+-induced c-fos expression is dependent on PKC, but unlike PDGF, Ca2+ does not induce
the phosphorylation of the PDGF receptor and the raf protein, indicating that Ca2+ and PDGF

have distinct pathways resulting in similar gene transcription.

38

3. Ca2+ sensing receptor and its signaling

In 1993, Brown et al. (241) cloned a Ca2+-sensing receptor from bovine parathyroid,
and subsequently, the human Ca2+-sensing receptor was cloned (243). This receptor
possesses an extracellular domain of about 613 amino acids, a membrane domain which
contains seven membrane-spanning segments, and an intracellular domain of about 222
amino acids) (241,244). The seven membrane-spanning domain is the characteristic of the
superfamily of G-protein coupled receptors as described above in section B2.2. This protein
is about 120 kDa (241,244) and it has been shown to be expressed both in tissues known to
be involved in Ca2+ homeostasis (e.g., parathyroid, C-cells, and kidney) and in other cells that
do not directly regulate systemic Ca2+ (e.g., brain, neuronal, and smooth muscle cells) (2).
The Ca2+-sensing receptor has been shown to transmit signals from outside the cell to
induce cellular responses, such as to increase cytosolic Ca“ and [PB levels when expressed
in xenopus oocyte (241). The rise of intracellular Ca2+ is sustained by the Ca2+ influx through
Ca2+ channels in the plasma membrane (2). And also, the expression of this receptor cDNA
in xenopus oocyte has been shown to activate PLC (241,244,245) which will hydrolyze PIP2
to generate 1P3 and induce intracellular Ca2+ elevation. The activation is dependent on a G
protein (244,246) which is sensitive to pertussis toxin in xenopus oocyte (241). However,
the elevation of 1P3 and intracellular Ca2+ in parathyroid cells is not dependent on pertussis
toxin sensitive G protein (2,29), indicating that this receptor couples to a different subfamily
of G protein in these different cell types (241)

It has been shown that human diseases, such as familial hypocalciuric hypercalcemia
and neonatal severe hyperparathyroidism, result from mutations in this Ca2+-sensing receptor

(247-249). Various mutants of this receptor have been generated to study the function of this

39

protein (249-252). The results from these studies confirm that the mutation of this receptor
is responsible for various human diseases.

In parathyroid cells in culture, it has been shown that the reduction or loss of Ca2+
responsiveness is related to a marked reduction in expression of extracellular Ca2+-sensing
receptor (253,254). Recently, knockout mouse lacking Ca2+-sensing receptor (255) has been
generated and it has the characteristics similar to those of humans with a mutation in this
receptor. The heterozygous mice (CaR+/-) are analogous to humans with familial
hypocalciuric hypercalcemia and have modest elevations of serum calcium, magnesium and
parathyroid hormone levels, as well as hypocalciuria. The CaR-l- mice are analogous to
humans with neonatal severe hyperparathyroidism. They have markedly elevated serum
calcium and parathyroid hormone levels, parathyroid hyperplasia, bone abnormalities, as
well as retarded growth (255). This suggests that Ca2+ receptor mutations cause these human

disorders by reducing the number of firnctional receptor molecules on the cell surface (255).

4. Other possible Ca’I-sensing receptors

In parathyroid cells, extracellular Ca2+ not only induces the intracellular 1P3 and Ca2+
increase, but also inhibits receptor-mediated increases in CAMP (2,29). For example,
dopamine-stimulated CAMP is inhibited by extracellular Ca” (29). This inhibition can be
abolished by pertussis toxin in parathyroid cells (29), indicating that a pertussis toxin-
sensitive G protein is involved in this process (2,29). Such an inhibiting effect on CAMP is
also observed in the thick ascending limb cell from mouse kidney (256). The decrease of
CAMP level upon Ca2+ treatment is due to the enhanced adenylate cyclase activity (2,29,242),

which is activated via a pertussis toxin-sensitive G protein. This G protein apparently differs

40

from that which mediates the Ca2+-induced elevation of intracellular 1P3 and intracellular
Ca2+. The receptor coupled to this pertussis toxin-sensitive G protein in parathyroid cells is
either the same receptor Cloned by Brown et al.(2) which couples to two different G protein
subfamilies simultaneously (2), or could be another receptor which senses extracellular Ca2+
and mediates this action (29).
By using monoclonal antibodies, Juhlin et al. (257-259) identified an ~500 kDa
membrane protein on proximal tubular cells, cytotrophoblast cells of human placenta.
Exposure of these cells to extracellular Ca2+ (2) induces cellular responses, such as regulation
of l-hydroxylation of 25-hydroxyvitamin D in the proximal tubule and Changes in circulating
PTH levels. The fact that the Ca2+-sensing receptor cloned by Brown et al. from bovine
parathyroid has not been found to be expressed in these cells (2) suggests that the 500 kDa
protein is another type of Ca2+ sensing receptor. Recently, Lundgren et al. (260) isolated a
2.8 kb cDNA which contains an open reading fiame encoding 236 amino acids, and which
represents part of the gene form the above mentioned 500 kDa protein. Using this cDNA as
probe, a 15 kb transcript was identified in human kidney, placenta, and parathyroid (260).
Sequence analysis of the 2.8 kb cDNA showed that this protein is a member of the low-
density lipoprotein receptor superfamily and that it is Closely related to Heyman
nephritogenic protein, a kidney tubule glycoprotein, with Ca2+ binding activity (260,261).
The Heyman nephritogenic protein has been Cloned and sequenced recently (262). It is the
human variant of principal kidney autoantigen causing Heyman membranous
glomcrulonephritis in rats (262). The deduced 4655 amino acid residues give a calculated
molecular weight of 519.6 kDa for the mature protein. The primary structure reveals that it

contains an huge extracellular region (about 4400 amino acids), a single transmembrane-

4l

spanning domain of 23 amino acids, and an intracellular C-terminal domain of 209 amino
acids (262). This protein contains three types of cysteine-rich repeats Characteristic of the
low density lipoprotein receptor (LDLR) superfamily (262), and its intracellular domain
contains several SH3 recognition motifs, one SH2 recognition motif for the P85 regulatory
subunit of PI3 kinase, and additional recognition sites for PKC, casein kinase II, and
CAMP/CGMP dependent protein kinase (262). It is not yet known whether this protein,
Heyman nephritogenic protein, or the 500 kDa protein identified by Juhlin et al. (257-259),
function as Ca2+ sensors. Subsequent functional studies should resolve this question.

In addition to specific Ca2+ sensing receptors, other known growth factor receptors
could also mediate the function of extracellular Ca” and generate intracellular signals. For
example, it has been shown that the F GF receptor has a Ca2+ binding motif in its acid box,
which binds Ca2+ as shown by a Ca2+ blotting technique, and the Ca2+ binding is not observed
in a mutagenized form of the receptor that lacks the acidic box region (263). The
mechanism(s) by which Ca2+ directly modulates the activities of various cell types (2), such
as keratinocytes (15), cultured mammary cells (264), and intestinal goblet cells (265) still

needs to be identified.

42

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Bamed, N., McCredie, D., Powell, H., Kendall Taylor, R, Brown, E.M., and Thakker, R.V.
A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the

63

calcium-sensing receptor [see comments]. N. Engl. J. Med., 335: 1115-1122, 1996.

249. Chou, Y.H., Pollak, M.R., Brandi, M.L., Toss, G., Amqvist, H., Atkinson, A.B.,
Papapoulos, S.E., Marx, S., Brown, E.M., Seidman, J.G., and et a1 , Mutations in the human

Ca(2+)-sensing-receptor gene that cause familial hypocalciuric hypercalcemia Am. J. Hum.
Genet, 56: 1075-1079, 1995.

250. Pearce, S.H., Bai, M., Quinn, S.J., Kifor, 0., Brown, E.M., and Thakker, R.V.
Functional Characterization of calcium-sensing receptor mutations expressed in human
embryonic kidney cells. . J. Clin. Invest, 98: 1860-1866, 1996.

251. Bai, M., Quinn, 8., Trivedi, S., Kifor, 0., Pearce, S.H.S., Pollak, M.R., Krapcho, K.,
Hebert, SC, and Brown, E.M. Expression and characterization of inactivating and activating
mutations in the human Ca2+o-sensing receptor. J. Biol. Chem, 271: 19537-19545, 1996.

252. Pearce, SH. and Brown, E.M. Calcium-sensing receptor mutations: insights into a
structurally and functionally novel receptor [editorial; comment]. J. Clin. Endocrinol.
Metab., 81: 1309-1311, 1996.

253. Brown, A.J., Zhong, M., Ritter, C., Brown, E.M., and Slatopolsky, E. Loss of calcium
responsiveness in cultured bovine parathyroid cells is associated with decreased calcium
receptor expression. Biochem. Biophys. Res. Commun., 212: 861-867, 1995.

254. Mithal, A., Kifor, 0., Kifor, 1., Vassilev, P., Butters, R., Krapcho, K., Sirnin, R., Fuller,
F., Hebert, SC, and Brown, E.M. The reduced responsiveness of cultured bovine
parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression
of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein. .

Endocrinology, 1 3 6: 3087-3092, 1995.

255. Ho, C., Conner, D.A., Pollak, M.R., Ladd, D.J., Kifor, 0., Warren, H.B., Brown, E.M.,
Seidman, J.G., and Seidman, C.E. A mouse model of human familial hypocalciuric
hypercalcemia and neonatal severe hyperparathyroidism [see comments]. Nat. Genet, 11:
389-394, 1995.

256. Takaichi, K. and Kurokawa, K. Inhibitory guanosine triphosphate-binding
protein-mediated regulation of vasopressin action in isolated single medullary tubules of
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257. Juhlin, C., Lundgren, S., Johansson, H., Lorentzen, J ., Rask, L., Larsson, E., Rastad, J.,
Akerstrom, G., and Klareskog, L. SOO-Kilodalton calcium sensor regulating cytoplasmic
Ca2+ in cytotrophoblast cells of human placenta. J. Biol. Chem, 265 : 8275-8279, 1990.

258. Juhlin, C., Johansson, H., Holmdahl, R., Gylfe, E., Larsson, R., Rastad, J., Akerstrom,
G., and Klareskog, L. Monoclonal anti-parathyroid antibodies interfering with a Ca2+-sensor
of human parathyroid cells. Biochem. Biophys. Res. Commun., 143: 570-574, 1987.

64

259. Juhlin, C., Holmdahl, R., Johansson, H., Rastad, J ., Akerstrom, G., and Klareskog, L.
Monoclonal antibodies with exclusive reactivity against parathyroid cells and tubule cells
of the kidney. Proc. Natl. Acad. Sci. U. S. A., 84: 2990-2994, 1987.

260. Lundgren, S., Hjalm, G., Hellman, P., Ek, B., Juhlin, C., Rastad, J., Klareskog, L.,
Akerstrom, G., and Rask, L. A protein involved in calcium sensing of the human parathyroid

and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily. . Exp.
Cell Res., 212: 344-350, 1994.

261. Saito, A., Pietromonaco, 8., L00, A.K., and Farquhar, M.G. Complete Cloning and
sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein
receptor gene family. Proc. Natl. Acad. Sci. U. S. A., 91: 9725-9729, 1994.

262. Hjalm, G., Murray, E., Crumley, G., Harazim, W., Lundgren, S., Onyango, 1., Ek, B.,
Larsson, M., Juhlin, C., Hellman, R, Davis, H., Akerstrom, G., Rask, L., and Morse, B.
Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential
intracellular signaling properties. Eur. J. Biochem, 239: 132-137, 1996.

263. Patstone, G. and Maher, P. Copper and calcium binding motifs in the extracellular
domains of fibroblast growth factor receptors. J. Biol. Chem, 271: 3343-3 346, 1996.

264. McGrath, GM. and Soule, H.D. Calcium regulation of normal human mammary
epithelial cell growth in culture. In. Vitro., 20: 652-662, 1984.

265. Black, BL. and Smith, J.E. Regulation of goblet cell differentiation by calcium in
embryonic chick intestine. FASEB. J ., 3: 2653-2659, 1989.

CHAPTER II

Extracellular Ca2+ stimulates the activation of mitogen-activated protein kinase

and cell growth in human fibroblasts

Shixia Huang, Veronica M. Maher, and J. Justin McCormick"

Carcinogenesis Laboratory- Fee Hall
Department of Microbiology and Department of Biochemistry
The Cancer Center

Michigan State University, East Lansing, MI 48824

65

66
ABSTRACT

In serum-free medium containing serum replacements but totally lacking in protein
growth factors, diploid human fibroblasts remained quiescent if the extracellular Ca“
concentration was only 0.1 mM. However, when the Ca2+ concentration in this medium was
increased to 1 mM, the cells replicated as rapidly as they do in medium supplemented with
protein growth factors. When quiescent cells in medium with only 0.1 mM Ca2+ were
exposed to 1 mM or 10 mM Ca2+ or 100 ng/ml EGF, the 42 kDa and 44 kDa forms of MAPK
were rapidly activated, as demonstrated by a Characteristic electrophoretic mobility shift of
these proteins and by their enhanced ability to phosphorylate myelin basic protein (MBP).

Analysis of fractions from Mono Q anion-exchange Chromatography of lysates of cells
exposed to 10 mM Ca2+ or 100 ng/ml EGF revealed a peak of MBP phosphorylation activity
that coeluted with p42 and p44 MAPK as shown by immunoblot analysis. Activation of
MAPK by extracellular Ca2+ was dose dependent and biphasic, with a peak of activation at
5-10 min after exposure, followed by a period of sustained activation of MAPK at a lower
level. This pattern has been shown (Vouret-Craviari et al. 1993, Biochem J. 289, 209) to

correlate with the re-entry of mammalian cells into the cell cycle.

67

ABBREVIATIONS

EGF, epidermal growth factor; HRP, horseradish peroxidase; MAPK, mitogen-

activated protein kinase; MBP, myelin basic protein; SCS, supplemented calf serum.

Running Title: Extracellular Ca2+ stimulates MAPK and cell growth

68

INTRODUCTION

It is known that human fibroblasts in culture will replicate in medium containing
specific protein growth factors (1), and that Ca2+ is an important factor in cell metabolism
(2). Studies show that such protein growth factors as EGF, platelet-derived growth factor,
insulin, thrombin, nerve growth factor, tumor necrosis factor, and bombesin, can initiate a
phosphorylation cascade in various cell types, including human fibroblasts, and that one of
the key events of this cascade is the activation of MAPK by phosphorylation (3-9). Several
closely related MAPK isoforms, referred to by their molecular masses, i.e., p40, p42, p44,
and p54 (10), have been identified in various cell types. In human fibroblasts, EGF (5),
tumor necrosis factor (7,] l) and interleukin (1]) have been shown to cause activation of p42
and/or p44 MAPKs. Activated MAPK can, in turn, phosphorylate MBP, microtubule-
associated protein 2, the EGF receptor, phospholipase A2, and the nuclear transcriptional
factors c-myc, C-fos, C-jun (10, 12, 13).

Morgan et al. (14) in this laboratory showed that diploid human fibroblasts can
replicate rapidly in serum-free MCM medium (15), a modified version of MCDB 110 (1), if
supplied with the serum replacements, e.g., lipids, iron, attachment factors, ete, specified
by Ryan et al. (15) and with insulin as the only protein growth factor, instead of the two
growth factors specified by Ryan et al. (15), e.g., insulin and EGF. MCM medium, as
ordinarily prepared (15), contains 1 mM Ca2+. Morgan et al. (14) also showed that the cells

do not replicate if the Ca2+ concentration is reduced from 1 mM to 0.1 mM, suggesting that

69

extracellular Ca2+ stimulates cell growth. We tested this hypothesis by removing all protein
growth factors from the medium and seeing if Ca2+ alone could cause the cells to replicate
for an extended period. Since MAPK is known to integrate the signals from various growth
factor pathways, we also tested whether Ca2+ causes the activation of MAPK in these cells
as protein growth factors do. The answer to both questions was positive. In the total
absence of protein growth factors, extracellular Ca2+ supported growth of the cells during the
two weeks of the experiment. When quiescent cells were stimulated with Ca2+ or EGF,
MAPK activation was observed as demonstrated by a characteristic shift in the

electrophoretic mobility of MAPK and an enhanced ability of MAPK to phosphorylate MBP.

70

MATERIALS AND METHODS

Materials

MBP, EGF, and B-glycerophosphate were purchased from Sigma (St. Louis, MO).
Polyclonal anti-MAPK antibody Ab283 (from rabbit) was kindly provided by Dr. Marsha
R. Rosner of the University of Chicago. Monoclonal anti-MAPK antibodies were purchased
from Chemicon (Temecula, CA) and Zymed Laboratories (South San Francisco, CA). HRP-
linked goat anti-mouse IgG was obtained from BioRad Laboratories (Richmond, CA) and
HRP-conjugated goat anti-rabbit IgG was from Boehringer Mannheim Corp. (Indianapolis,

IN).

Cells and cell culture

Diploid human fibroblast cell lines LG] and SL80, derived in this laboratory fi'om
neonatal foreskins, were used at cell population doubling 15 to 28. The in vitro life span of
LG] cells is 40 population doublings, that of SL80 cells is 80 population doublings. Cells
were routinely grown in Eagle's minimum essential medium supplemented with 0.2 mM
aspartic acid, 1.0 mM sodium pyruvate, and 0.2 mM serine, 10% supplemented calf serum
(SCS) (Hyclone Laboratories, Logan UT) penicillin (100 units/ml), streptomycin (100
ug/ml), and hydrocortisone (1 ug/ml) (complete medium) at 37°C in humidified incubator

with 5% (30,.

Media for testing for growth stimulation

71

To assay for growth stimulation we used MCM medium (15), a modified version of
MCDB 110 (1) which was developed for the continuous proliferation of human fibroblasts.
We used the serum replacements specified by Ryan et al. (15) but omitting the protein
growth factors, i.e., EGF and insulin. The medium was prepared to contain 0.1 mM or 1

mM Ca2+ as indicated.

Preparation of cell lysates

Cells were grown in 100-mm diameter dishes in complete medium until subconfluent
or confluent as desired. To make the cells quiescent, the medium was replaced for 24to 30
h with MCM medium containing serum replacements but no protein growth factors and with
the Ca2+ concentration reduced to 0.1 mM or to zero where indicated. Unless otherwise
indicated, increased Ca2+ or EGF was then added directly to the medium on the cells with a
gentle swirling, and the incubation at 37°C was continued for the indicated time. To harvest
the cells, the medium was removed, the cell sheet was quickly washed twice with Ca2+-free,
Mg“-free PBS, and the cells were lysed in 0.3 to 0.5 ml of buffer composed of 20 mM Tris-
HCl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 1 mM
phenylrnethylsulphonylfluoride, 5 mM EDTA, 0.15 U/ml aprotinin, and 1 mM sodium
orthovanadate. The cells were collected with a rubber scraper and kept on ice for 20 min.
The lysates were centrifuged for 10 min at 4°C in a microfuge, and the protein concentration
of the supernatant was determined using a Bicinchroninic Acid Protein Assay kit (Pierce
Chemical Co., Rockford, IL) with BSA as the protein standard. The cell lysates were used

immediately after preparation or stored at -80°C until used.

72

Immunoblot Analysis of MAPK

Cell lysates containing 25 ug of protein were dissolved in buffer containing 0.05 M
Tris-HCI, pH 6.9, 0.72 M mercaptoethanol, 9% glycerol, 2.3% SDS and 0.1% Bromphenol
blue (sample buffer), and the proteins were separated by 10% SDS-PAGE, and transferred
from the gels onto Immobilon PVDF transfer membranes (Millipore Corp., Bedford, MA)
overnight at 35 mA, and the blots were blocked by incubating for 1 h at room temperature
in Tris-buffered saline, pH 7.6, (20 mM Tris-HCl, 137 mM NaCl) containing 0.1% Tween
20 (v/v) and 5% nonfat dry milk (w/v). To test for MAPK, the membranes were incubated
for l h with the indicated anti-MAPK antibodies in Tris-buffered saline containing 0.1%
Tween 20 and 5% nonfat dry milk, washed several times in this Tris-buffered saline, and
then incubated with goat anti-rabbit IgG or goat anti-mouse IgG as desired. Enhanced
chemiluminescence (Amersham, Arlington Heights, IL) was used as a detection system

according to the manufacturer's instructions.

MBP phosphorylation as an assay for MAPK activity

This was assayed essentially as described previously (16). Briefly, cell lysates
containing 2.5 ug of protein were incubated with MBP at 0.5 mg/ml for 10 min at room
temperature in a final volume of 20 ul containing 18 mM HEPES, pH 7.4, 10 mM
magnesium acetate, and 2 uCi [7-32P]ATP (2-10 Ci/mmol) (DuPont, New England Nuclear

Research Products, Boston, MA). The reaction was stopped by the addition of 5X sample

73

buffer. After 5 min at 95°C, proteins were separated on a 10% SDS-PAGE. The dried gel
was used to expose Kodak X-Omat film and a phosphor screen. The phosphor screen was
scanned and the amount of MBP phosphorylation was quantified using a Phosphorlmager
(Model 400B, Molecular Dynamics, Sunnyvale, CA) according to the manufacturer's

instructions.

Purification of MAPK by mono Q column chromatography

Cell lysates from Ca2+-stimulated or EGF-stimulated cells or untreated cells were
prepared and normalized for protein concentration. MAPK was purified by Mono Q anion-
exchange Chromatography as described (17). Before purification, lysates were thawed and
centrifuged for 5 min in a microfuge, and the supernatant containing 1.5 to 2 mg of protein
was diluted 2-fold in column buffer, which contains 50 mM B-glycerophosphate, pH 7.2, 100
M sodium orthovanadate, 1 mM EGTA, and 1 mM dithiothreitol. The diluted sample was
filtered through a 0.2 pm filter and loaded onto a Mono Q HRS/5 column FPLC (Pharmacia,
Piscataway, NJ) (1.5 ml bed volume) at a flow rate of 0.4 ml/min. Using a linear gradient
of 0 to 0.4 M NaCl to elute, 1 ml fiactions were collected. Aliquots of 10.5 it] were assayed
for MAPK activity using the MBP phosphorylation assay, and aliquots of 110 pl were

assayed by immunoblot analysis, as described above.

74

RESULTS AND DISCUSSION

Evidence that extracellular Ca2+ stimulates cell growth

To determine if extracellular Ca2+ could stimulate diploid human fibroblasts to
replicate in the total absence of protein growth factors, we plated LG] and SL80 cells in
MCM medium containing 0.1 mM Ca2+ and 1% SCS. The next day, the number of attached
cells was determined, and the medium was replaced with MCM medium containing the serum
replacements of Ryan et al. (15) but minus the protein growth factors. The rate of replication
of the cells in this medium supplemented with Ca2+ or EGF or 10% SCS is shown in Figure
1. Under the most stringent condition, 0.1 mM Ca2+, the number of cells increased only
slightly or at most doubled. In the presence of 1 mM Ca2+, the LG] cells doubled every 48
h, and the SL80 cells every 36 h. This rate of growth was as fast or faster than the cells' rate
of growth in medium containing EGF (10 ng/ml), and in the case of SL80 cells, almost as
fast as their rate with 10% SCS. Higher concentrations of EGF do not increase the growth
rate, these results indicate that 1 mM Ca2+ stimulates human fibroblasts to proliferate as they

do in response to protein growth factors.

MAPK activation as a result of Ca2+ or EGF stimulation

To determine whether MAPK is involved in activation of the signal pathway induced
by extracellular Ca2+ in human fibroblasts, we treated LG] and SL80 cells for 5 min with 1
mM Ca”, 10 mM Ca2+, or 100 ng/ml EGF, and assayed the cell lysates for evidence of

MAPK activation using immunoblot analysis. Protein growth factors are typically used at

75

 

 

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Figure 1 Growth stimulating activity of Ca’+

Cells were plated into 60 mm-diameter dishes in MCM medium containing 0.1 mM Ca" and
1% SCS. The next day (day 1), the number of attached cells was determined, the medium was
replaced with MCM medium containing the serum replacements of Ryan et al. (15) minus
insulin and EGF, but modified as follows: 0.1 mM Ca2+ (O); 1 mM Ca” (0); 0.1 mM Ca”
with 10 ng/ml EGF (V); 0.1 mM Ca” with 10% SCS (V). The cells were refed with
appropriate medium on day 4, 7, and 10, and the number of cells in four dishes each time for
each condition was determined on day 4, 7, 10 and 14. Similar results were obtained in two

separate experiments.

76

~10 higher concentrations in MAPK assays than in growth studies. We used anti-MAPK
polyclonal antibody Ab283 which was previously shown to detect both the 42 and 44 kDa
isoforms of MAPK (5). With the cell lysates prepared from untreated cells, this antibody
revealed two bands with molecular masses of approximately 42 and 44 kDa (lane 1 of Figure
2A and ZB). With cell lysates prepared from cells exposed for 5 min to 1 mM or 10 mM
Ca2+ or to 100 ng/ml EGF (lanes 2, 3, and 4 of Figure 2A and 2B), slower moving bands
were observed, one just above the 42 kDa band and one just above the 44 kDa band. This
molecular weight shift is Characteristic of the phosphorylated form of the MAPK isoforms
(18, 19). The percentage of each MAPK isoform that traveled more slowly was greater with
lysate from cells exposed to 10 mM Ca2+ or IOOng/ml EGF than from cells exposed to 1 mM
Ca2+. We also stripped the membrane using 62.5 mM Tris-HCI, pH 6.9, 2% SDS, and 0.1
M mercaptoethanol for 30 min at 50°C and reprobed it with monoclonal antibodies that
recognize only the 42 kDa MAPK in human cell lines. The results indicated that the lower
bands are the p42 form of MAPK (data not shown).

To determine whether this phosphorylation of MAPK resulted in a molecule with
kinase activity, the cell lysates were also tested as described for their ability to phosphorylate
MBP . As shown in Figure 2C and 2D, Ca2+ or EGF increased the degree of MBP
phosphorylation. The degree of MBP phosphorylation produced by lysates from cells
stimulated with 10 mM Ca2+ was much greater than that from cells given 1 mM Ca2+, and
approximately equal to that from the cells treated with lOOng/ml EGF.

The activation of MAPK by Ca“ or lOOng/ml EGF was also evaluated on Mono Q

column-purified MAPK from the cell lysates. The lysates from untreated cells or from cells

77

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exposed to 10 mM Ca2+ or EGF were fractionated by chromatography, and aliquots of
fractions 8-20 were assayed for the ability to phosphorylate MBP (Figure 3). For LG] cells,
stimulated with either Ca2+ or EGF, high activity was observed in fractions 12 and 13, but
not in the corresponding fractions from untreated cells. For SL80 cells, high activity was
found in fractions 13 and 14 with EGF, and fraction 14 with Ca”. The presence of MAPK
in those specific fractions and its absence from other nearby fractions was demonstrated by
immunoblotting and probing with the polyclonal anti-MAPK antibody that recognizes both
isoforms (Figure 4). The two forms coeluted. Note that in the fractions from the Ca2+-
stimulated or EGF-stimulated cells, there was a shift in the electrophoretic mobility of the
MAPK isoforms. Coelution of these two isoforms on Mono Q column has been previously
reported (20) in a study using a similar volume of cluent. When a much larger volume of the
cluent was used, the 42 kDa and 44 kDa isoforms showed a better separation, although some
fractions still contained both isoforms (2]). As shown in Figure 3, the control sample from
SL80 cells showed a small peak of phosphorylated MBP in fraction 10. However, the
immunoblot analysis showed no detectable MAPK in that fraction (data not shown), and use
of a second assay for MAPK activity (microtube-associated protein 2 phosphorylation) also
showed no such activity in this fraction (data not shown).

To test whether albumin or other factors used as the serum replacements played a
role in the activation of MAPK, we also assayed for Ca2+-induced MAPK activation in
confluent cells that had been incubated ovemight in MCM medium containing 0.1 mM Ca2+,
but no serum replacements, as well as in confluent cells similarly incubated overnight but

then incubated for two additional hours in phosphate-buffered saline. Under both

79

 

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Figure 3 Assay of mono Q fractions for ability to phosphorylate MBP

Cell lysates were prepared as described from cells exposed to 10 mM Ca2+ (O ) or 100 rig/ml
EGF (V) for 5 min or left untreated (O). Lysate protein (1.5 to 2 mg) was diluted by column
bufl‘er and loaded onto a Mono Q column. The protein was eluted by a linear NaCl gradient,
and 1 ml fractions were collected. Aliquots of fraction 8 through 20 were assayed for MBP
phosphorylation. The salt gradient is denoted by the broken line. Similar results were

obtained in two experiments.

80

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circumstances, when 10 mM Ca2+ was added to the solution on the cells, MAPK activation
was observed (data not shown).

To determine if there was a dose-response relationship for Ca2+- induced MAPK
activation, quiescent LGl and SL80 cells in McM medium lacking Ca2+ and supplemented
with serum replacements, but without protein growth factors, were exposed for 5 min to
various concentrations of Ca”. Cell lysates were prepared and analyzed for their ability to
phosphorylate MBP. A dose response was observed with both cell lines (Figure 5). At high
concentrations of Ca2+, MAPK activation would be expected to exhibit saturation; however,
at concentrations of 50 mM Ca2+ or higher, a precipitate formed, making it impossible to

extend the curve.

Time course for Ca2+-induced MAPK activation

Pouysse'gur and his colleagues showed that in quiescent Chinese hamster fibroblast
(cell line CCL39), MAPK is activated by thrombin or basic fibroblast growth factor and that
this activation exhibits a biphasic pattern i.e., a rapid increase in activity followed by a rapid
decrease and then a sustained level of lower activity (22, 23). They proposed that this
sustained activity is required for the re-entry of these cells from G0 into the cell cycle (23),
i.e., for triggering the proliferative response, because when they treated CCL39 cells with
a thrombin analog instead of thrombin or with carbachol, the second phase of MAPK
activation was not observed and no DNA synthesis took place. To see if Ca2+-stimulated
MAPK activation in human fibroblasts also exhibits such a biphasic pattern, we carried out

a time course for Ca2+ stimulation, using phosphorylation of MBP as a measure of MAPK

82

 

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Figure 5 Dose-response of Ca2+-stimulated MAPK activity

Cells were grown to confluenceand the medium was changed to McM medium with serum
replacements of Ryan et al. (15) but no protein growth factors, and modified to be without
Ca”. After 24 h, the indicated concentration of Ca2+ was applied. After 5 min, cell lysates
were prepared and assayed for MBP phosphorylation. Similar results were obtained in two

separate experiments.

83

activation. The results are shown in Figure 6. Exposure of LG] and SL80 cells to 10 mM
Ca2+ rapidly activated MAPK, with a maximum peak of activation occurring at 5 to 10 min.
Afier the initial burst of activation, a second wave of sustained activation was observed in
both cell lines. The level of activation of MAPK did not come down to the basal level in
either cell line during the 180 min of stimulation. This sustained activation, along with our
finding that in the total absence of protein growth factors, Ca2+ induces growth of human
fibroblasts (Figure 1), suggests that Ca2+ stimulates cell proliferation by activating MAPK
in a biphasic manner. Recently, Frost et al. (24) and Pages et al. (25), using MAPK antisense
RNA and/or MAPK kinase-deficient mutants to suppress MAPK activation, showed that
MAPK activation is required for activation of transcriptional factors (24) and for rodent
fibroblast proliferation (25).

In summary, as far as we can determine, our study is the first to show that in the total
absence of protein growth factors extracellular Ca2+ can stimulate sustained growth of
diploid human fibroblasts and can induce the activation of MAPK. In the absence of protein
growth factors, the 42 kDa and 44 kDa isoforms of MAPK were both activated, as they are
by protein growth factors. A study by Chao et al (5) indicates that various pathways can
lead to MAPK activation in human fibroblasts. For example, EGF-mediated activation does
not require intracellular Ca2+, whereas thapsigargin-mediated activation depends on
intracellular Ca2+. We intend to determine whether extracellular Ca2+ stimulates MAPK

activation by one of these pathways or by some alternative mechanism.

84

800

 

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600

400

200 \

MBP phosphorylation
(percent of controD

 

O I I I I I r I I
O 50 100 150 200
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Figure 6 Time course for Ca’*-induced MAPK activity

The medium on cells in exponential growth was changed to the MCM medium with serum
replacements of Ryan et al. (15) but no protein growth factors and only 0.1 mM Ca”. Afier
24 to 30 h, the Ca2+ concentration was increased to 10 mM for the indicated time. Cell
lysates were prepared and assayed for MBP phosphorylation. Results are expressed as
means 2’: S.E.M. of triplicate determinations. Similar results were obtained in two separate

experiments.

85

ACKNOWLEDGEMENTS

We thank Dr. Marsha. R. Rosner for kindly supplying us with the anti-MAPK
antibody and Dr. Robert P. Hausinger of Michigan State University for his advice on Mono
Q purification of MAPK. This research was supported in part by DHHS Grant AG11026

from the National Institute on Aging and DOE Grant ER60524.

10.

11.

12.

13.

14.

15.

16.

17.

86

REFERENCES

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Sci. USA78, 5588-592

Brown, E. M. (1991) Physiol. Rev. 71, 371-411

Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G.,
(1991) J. Biol. Chem. 266, 4220-4227

Nakielny, S., Cohen, P., Wu, J ., and Sturgill, T. (1992) EMBO J. 11, 2123-2129

Chao, T. 0., Byron, K. L., Lee, K. M, Villereal, M., and Rosner, M, R. (1992) J. Biol.
Chem. 267, 19876-19883

Pelech, S. L., and Sanghera, J. S. (1992) Science 257, 1355-1356

Victor, 1., Schwenger, R, Li, W., Schlessinger, J., and Vilcek, J. (1993) J. Biol. Chem.
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Pang, L., Decker, s. J., and Saltiel, A. R. (1993) Biochem. J. 289, 283-287

Burgering, B. M. T., de Vries-Smits, A. M. M., Medema, R. H., van Weeren, P. C.,
Tertoolen, L. G. J., and Bos, J. L. (1993) Mol. Cell. Biol. 13, 7248-7256

Pelech, S. L., and Sanghera, J. S. (1992) TIBS 17, 233-238

Guy, G. R., Chua, S. P., Wong, N. S., Ng, S. B., and Tan, Y. H. (1991) J. Biol Chem.
266, 14343-14352

Sturgill, T. W., Ray, L. B., Erikson, E., and Maller, J. L. (1988) Nature 334, 715-718
Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556

Morgan, T. L., Yang, D., Fry, D. G., Hurlin, P. J., Kohler, S. K., Maher, V. M., and
McCormick, J. J . (1991) Exp. Cell Res. 197, 125-136.

Ryan, P. A., McCormick, J. J., and Maher, V. M. (1987) Exp. Cell Res. 172, 318-328
Zheng, C. F., and Guan, K. L. (1993) J. Biol. Chem. 268, 11435-11439

Gupta, S. K., Gallego, C., Johnson, G. L., and Heasley, LE. (1992) J. Biol. Chem 267,

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Offermanns, S., Jones, 8. V. P., Bombien, E., and Schultz, G. (1994) J. Immunol. 152,
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Boulton, T. G., Nye, S. H., Robbins, D. J ., Ip, N. Y., Radziejewska, E., Morgenbesser,
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Pouyssegur, J. (1993) Proc. Natl. Acad. Sci. USA 90, 8319-8323

CHAPTER III

Characterization of the second messenger pathway of extracellular Ca2+-induced

mitogen-activated protein kinase activation in human fibroblasts

Shixia Huang, Veronica M. Maher, and J. Justin McCormick“

Carcinogenesis Laboratory- Fee Hall
Department of Microbiology and Department of Biochemistry
The Cancer Center

Michigan State University, East Lansing, MI 48824

88

89

ABSTRACT

Human fibroblasts in culture will grow in serum free medium containing serum
replacement factors but without protein growth factors as long as the Ca2+ level is 1.0-2.0
mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling but can be reinduced to
cycle by raising the Ca2+ level. Mitogen-activated protein kinase (MAPK) is activated in this
process just as it is by protein growth factors (Huang et al. [1995] Biochemical J. 310, 881-
885). We now report that the exposure of human fibroblasts to extracellular Ca” increased
cytosolic inositol (1, 4, 5)-trisphosphate level and caused a transient rise in the concentration
of intracellular Ca2+. Ca2+-induced MAPK activation was partially abolished by treatment
of the cells with pertussis toxin which inhibits certain species of G proteins. It was also
decreased by treatment with thapsigargin which depletes intracellular Ca2+ stores, by
exposure to phorbol 12-myristyl l3-acetate that downregulates protein kinase C (PKC), by
treatment with calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-
naphthalenesulfonamide HCL and calmidazolium, as well as by exposure of the cells to
lanthanum, a Ca2+ channel inhibitor. We found that the c-raf-l protein was not
phosphorylated after Ca2+ stimulation, and the Ca2+ sensing receptor (Brown et al. [1993]
Nature 366, 575-5 80) was not involved in mediating MAPK activation or cell growth. These
results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s)
involving a pertussis toxin sensitive G protein, phospholipase C, intracellular Ca2+,

calmodulin, and PKC.

90

ABBREVIATIONS

DAG, diacylglycerol; EGF, epidermal growth factor; F BS, fetal bovine serum; 1P3,
inositol (1, 4, 5)-trisphosphate; MAP-2, microtubule-associated protein 2; MAPK, mitogen-
activated protein kinase; PDGF, platelet-derived grth factor; PIP2, phosphatidylinositol
bisphosphate; PLC, phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristyl
13-acetate; SCS, supplemented calf serum; W-7, N-(6-aminohexyl)-5-chloro-l-

naphthalenesulfonamide HCL; W-12, N-(4-aminobutyl)-2-naphthalenesulfonamide.

91

INTRODUCTION

It is well known that the extracellular Ca2+ concentration can regulate hormonal
secretion [1] and cell differentiation [2-4] in some cell types. For example, mouse and
human keratinocyte cells in culture [3-5] proliferate in a medium containing Ca2+ which is
5-40 times (0.05-0.1 mM) lower than the physiological level (1 .0-2.0 mM), but when the
Ca2+ concentration of the medium is increase to the physiological range, these cells undergo
terminal differentiation. Exposure of these cells to medium with physiological levels of Ca2+
causes a rapid increase in phosphoinositide turnover and intracellular Ca2+ levels [2,3] and
the phosphorylation of tyrosine in various proteins [4].

Mesenchymal cells exhibit the opposite response. Normal human [6], mouse [7], and
chicken [8-10] fibroblasts in culture stop proliferating when placed in a medium with
reduced Ca2+ (0.01-O.1 mM). However, when the Ca2+ concentration is increased to the
physiological level, the cells proliferate. We recently showed [11] that when one exposes
human fibroblasts to medium containing Ca2+ at physiological levels of or higher but lacking
protein growth factors, mitogen-activated protein kinase (MAPK) activation is induced and
this correlates with cell growth.

In mouse 3T3 fibroblasts in culture, it has been shown that extracelluar Ca2+
stimulates DNA synthesis [12-17] and c-fos expression [14-16,18] similar to that induced
by platelet-derived growth factor (PDGF) [15,16]. Furthermore, Epstein et al [15,16]
showed that both PDGF-induced and Ca2 + -induced c-fos expression is dependent on protein
kinase C (PKC). However, Ca2+ does not induce the phosphorylation of the PDGF receptor

and the raf protein as PDGF does, indicating that the pathways which result in similar gene

92

transcription are at least in part independent.

As is well known, protein growth factors, such as epidermal growth factor (EGF),
PDGF, insulin, thrombin, nerve growth factor, and bombesin, bind to specific receptors on
the external cell surface, and initiate a phosphorylation cascade through the activation of
either tyrosine kinase-linked or G protein-coupled receptors [19-25]. One of the key events
in this cascade is the activation of MAPK by phosphorylation. Two of the well understood
pathways leading to MAPK activation from various growth factor stimulation are the ras/raf
pathway [19], and phospholipase C (PLC) pathway [26,27]. The activation of PLC, by either
type of activated receptor, causes hydrolysis of the inositol lipid-phosphatidylinositol
bisphosphate (PIP2), and generates the intracellular second messengers [26,27],
diacylglycerol (DAG), and inositol (1, 4, 5)-trisphosphate (1P3). DAG is responsible for
activating PKC isoenzymes [28], which leads to the activation of many other kinases
including c-raf [29,30] and MAPK [31]. 1P3 binds to the 1P3 receptor on the endoplasmic
reticulum to release Ca2+ fi'om intracellular stores [27], and the free Ca2+ is responsible for
the induction of many cellular events, including MAPK activation [21]. Intracellular Ca2+
exerts its effects by binding to proteins such as calmodulin [32].

How human fibroblasts “sense” the concentration of extracellular Ca2+ is unknown.

One possibility is that there is some type of receptor on the external cell membrane
analogous to the receptors for protein growth factors. Recently, Brown et al. [33] cloned a
Ca2+-sensing receptor from bovine parathyroid [33], and subsequently, the human homolog
of that receptor was cloned [34]. This receptor, which couples to G protein (s), has been
shown to mediate the release of the extracellular Ca2+-regulating parathyroid hormone. Once

the receptor is stimulated by extracellular Ca”, it increases the intracellular level of Ca2+ and

93

1P3 [33]. A knockout mouse lacking this receptor has recently been generated by Ho et al
[35]. Homozygous Ca2+ receptor-deficient mice exhibit severe neonatal
hyperparathyroidism, markedly elevated serum levels of Ca2+ and parathyroid hormone,
parathyroid hyperplasia, bone abnormalities, and retarded growth [35].

Our interest was in determining how exposure to physiological levels of Ca2+ is able
to cause the growth of human fibroblasts in culture. This is of particular importance since,
unlike their normal counterparts, human fibrosarcoma-derived fibroblasts [36] and human
fibroblasts malignantly transformed in culture [37,3 8] are able to grow in medium with a
reduced Ca2+ concentration, even in the absence of protein growth factors. This suggests that
some aberration in this pathway plays a causal role in the transformation of these cells.

Since exposure of mouse fibroblasts to high concentrations of extracellular Ca”
stimulates c-fos transcription via PKC [15], we first examined the possible involvement of
PKC in Ca2+ -induced MAPK activation in human fibroblasts. Intracellular Ca2 *
measurements were also carried out because studies showed that extracellular Ca2+ regulates
intracellular Ca2+ elevation in other cell system [1-3]. Our finding evidence of the
involvement of PKC and intracellular Ca2+ led us to examine their downstream effectors c-
raf-l and calmodulin respectively, and their upstream activators, PLC, G protein, and the
possible involvement of the Ca2+ sensing receptor. The results showed that the addition of
extracellular Ca” activated MAPK through a pertussis toxin-sensitive G protein, and led to
the elevation of intracellular 1P3 and Ca2+. The activation of MAPK by extracellular Ca2+
is dependent on PKC, intracellular Ca2+, and calmodulin, but is not dependent on c-raf-l , or

the Ca2+-sensing receptor we examined.

94

MATERIALS AND METHODS

Materials

EGF, PDGF-BB, lys-bradykinin, thapsigargin, N-(6-aminohexyl)-5-chloro-1-
naphthalenesulfonamide HCL (W-7), N-(4-aminobutyl)—2-naphthalenesulfonamide (W-12),
calmidazolium (compound R24571), nifedipine, verapamil, lanthanum chloride were
purchased from Sigma (St. Louis, MO, USA). Pertussis toxin was from Calbiolchem (La
Jolla, CA, USA). Phorbol 12-myristyl l3-acetate (PMA) (from Calbiolchem) was a gift
from Dr. David DeWitt of Michigan State University. Indo-l-Am, cell permeate, and indo-l,
cell irnperrneate, were from Molecular Probe (Eugene, OR, USA). Ionomycin (fi'ee acid) and
the 1P3 Biotrak Radioirnmunoassay System Kit (TRK 1000) were from Amersham Life
Science Inc. (Arlington Heights, IL, USA). Lab-tek, 4-well chambered coverglass
(Nuct#l36420) was from VWR Scientific (Chicago, IL, USA). Monoclonal anti-MAPK
antibodies were purchased from Chemicon(Temecu1a, CA, USA) and Zymed Laboratories
(South San Francisco, CA, USA). Monoclonal anti-c-raf-l antibody was from Transduction
Laboratories (Lexington, KY, USA). Horseradish peroxidase-linked goat anti-mouse IgG
was obtained from Bio-Rad Laboratories (Richmond, CA, USA). Supplemented calf serum

(SCS) and fetal bovine serum (FBS) were from Hyclone Laboratories (Logan, UT, USA).

Cells and cell culture
Diploid human fibroblast cell line LGl, derived in this laboratory from neonatal
foreskin, was used in this study. Cells were routinely grown in Eagle's minimum essential

medium supplemented with 0.2 mM aspartic acid, 1.0 mM sodium pyruvate, and 0.2 mM

95

serine, 10% supplemented calf serum (SCS), penicillin (100 units/ml), and streptomycin (100
ug/ml) (complete medium) at 37°C in an humidified incubator containing 5% C02. For
studies on the effects of various growth conditions, medium on confluent or subconfluent
cells was changed to modified McM medium, a derivative of MCDB-110 medium [39],
made with 0.1 mM Ca2+ and supplemented with serum replacements [40] but without any

protein growth factors [1 1]. This medium will be referred as serum free medium.

Isolation and culture of mouse skin fibroblasts

Pregnant female mice (with Ca2+-sensing receptor status of +/- that had previously
been mated with +/- male mice) were kindly provided by Dr. Christine Seidman of Harvard
Medical School [3 5]. When the litter was born, newborn to 4 day-old mice were sacrificed
with CO2 and washed with 70% ethanol. The skin was removed to a 60 mm dish and washed
twice in PBS, then transferred to a 50 ml sterile glass flask and minced finely with scissors.
Ten ml of 0.24% trypsin was added to the flask containing the mouse skin, and the tissue
was incubated at 37°C for 30 min to 1 hour, and shaken every 5 min. The suspension was
then centrifuged at 400xg for 4 min to remove tissue fragments, and the turbid supernatant
containing single cells was transferred to a 15 ml tube containing 4 ml Eagle’s medium with
20% SCS and centrifuged at 2000xg for 10 min. The pellet was suspended in 3 ml Eagles
medium containing 20% F BS, penicillin (600 units/ml), and streptomycin (600 ug/ml). To
prevent possible bacteria contamination, a concentration of penicillin and streptomycin six
times higher than that normally used in cell culture medium was used. Cells were counted
and plated at 4 x 105 to 8 x 105 per 100 mm dish in the same medium, and incubated at 37°C

in a humidified incubator containing 5% C02. The next day, medium in each dish was

96

removed, and cells were washed with warm Eagles medium once, and fresh medium, i. e.,
Eagles medium containing 20% FBS and the noted high concentration of antibiotics, was
added into each dish. About 5 days later, confluent cells in each dish were trypsinized and
expanded in Eagles medium with 10% FBS, and regular penicillin and streptomycin

concentration (100 ug/ml).

Media for testing for growth stimulation of mouse fibroblasts

The same serum free medium that we employed previously for studying growth
stimulation in human fibroblasts [11], was used for mouse skin fibroblasts. With growth
stimulation assays, serum free medium was used with no additives, i. e., at 0.1 mM Ca2+, or

with 1 mM Ca2+, or supplemented with 10% FBS.

Preparation of cell lysates

Cells were grown in 60 or 100 mm-diameter dishes in complete medium until
confluent. Then the medium was replaced for 24-30 hours with serum free medium. Unless
otherwise indicated, increased Ca2+ or EGF or other reagents were added directly to the
medium on the cells. Cells treated with different reagents or left unheated were washed twice

in cold Ca2+ -free, Mg2+ -fiee PBS, and the cell lysates were prepared as described previously

[11].

Immunoblot analysis of MAPK (Western Blot)
Cell lysates containing 25 ug of protein were dissolved in sample buffer as

previously described [1 1], proteins were separated by 10% SDS/PAGE, and then transferred

97

to Immobilon transfer membrane (Millipore Corp., Bedford, MA, USA). Immunoblot
analysis was carried out as described [1 1]. Briefly, the blots were blocked by incubating for
1 h at room temperature in Tris-buffered saline, pH 7.6, (20 mM Tris-HCL 137 mM NaCl)
containing 0.1% Tween 20 (v/v) and 5% nonfat dry milk (w/v). To test for MAPK, the
membranes were incubated for 1 h with the indicated anti-MAPK antibodies in Tris-buffered
saline containing 0.1% Tween 20 and 5% nonfat dry milk, washed several times in this Tris-
buffered saline, and then incubated with goat anti-mouse IgG as desired. Enhanced
chemiluminescence (Amersham, Arlington Heights, IL) was used as a detection system

according to the manufacturer's instructions.

Microtubule-associated protein 2 (MAP-2) phosphorylation assay (MAPK activity
assay)

MAP-2 was purified from pig brain essentially as described [24]. The activity was
assayed as described [11] but using MAP-2 as substrate instead of myelin basic protein .
Briefly, cell lysates containing 2.5 pg proteins were incubated with MAP-2 (0.5 mg/ml), and
[7-32P]ATP for 10 min at room temperature, the reaction was stopped by the addition of 5X
sample buffer (1x sample bufier contains 0.05 M Tris-HCL pH6.9, 0.72 M mercaptoethanol,
9% glycerol, 2.3% SDS and 0.1% Bromphenol blue). The proteins in the reaction were
separated on a 7.5% SDS-PAGE, and gels were dried and exposed to Kodak X-Omat film

to visualize the amount of MAP-2 phosphorylation.

Intracellular Ca2+ measurement

The intracellular Ca2+ concentration was measured as described [41] both in the

98

resting cells and cells incubated with extracellular Ca2+ , lys-bradykinin, ionomycin,
thapsigargin or EGF. Briefly, cells were cultured in Lab-Tek coverslip chambers and
incubated in serum free medium (with 0.1 Ca”) for 24 hours. The cells were then loaded
with a Ca2+ sensitive fluorescent dye, indo-l-AM (2 uM) in the same medium by incubating
them for 1 hour at 37°C. To remove the extracellular dye, cells were then washed three times
with McM medium (0.1 mM Ca2+) modified to contain 20 mM Hepes but without phenol
red. They were then incubated in this medium until the beginning of the measurement
(within 2 hours). The concentration of free Ca2+ in the cells loaded with indo-l-AM was
calculated from data obtained with an ACAS 570 Interactive Laser Cytometer (Meridian
Instruments, Okemos, MI, USA). An argon ion laser beam was led to the specimen on an
inverted microscope, and emission from the illuminated spot on the sample was directed to
a sensitive photomultiplier tube. The image pairs were captured at 8-second intervals. All
image analysis experiments were conducted at room temperature. The ratio of the intensities
of fluorescent emission at 405 nm and 530 nm (since the UV/Blue cube was used) with
excitation at 355 nm was measured. The free Ca2+ concentrations in each single cell was
calculated by comparing the ratio of the emissions to that generated in a standard curve. The
standard curve was produced by mixing the indo-l , cell impermeate, with varying amounts

of added Ca“ in a physiological buffer containing EGTA as described [42].

Cytosolic 1P3 measurement
Cytosolic 1P3 was measured by using 1P3 binding protein [43] from 1P3 Biotrak
Radioimmunoassay System Kit (TRK 1000) (Amersham Life Science Inc.). Samples were

prepared essentially as described in the manufacturer’s instruction booklet with some

99

modifications. Briefly, cells were grown to confluence in 60 mm dishes in complete
medium, and then incubated in serum free medium for overnight, treated with 10 mM Ca2+
for 1 min, or with 50 ng/ml bradykinin for 0.5 or 1 min, or left untreated. At the end of
treatment, medium was aspirated and 0.5 ml of 10% cold trichloroacetic acid (v/v) was added
to each dish, and samples in each dish were scraped by rubber policeman and transferred to
an eppendorf tube. The precipitates were sedimented by centrifugation at 2100xg in
microcentrifuge for 15 min at 4° C. The supernatant containing 1P3 was transferred to a 15
ml falcon blue cap tube and extracted three times with 10 volumes of water-saturated diethyl
ether. 1P3 extracts were neutralized by titration to pH7.0-8.0 using NaHCO3. 1P3
concentrations were measured and calculated from the parallel standard curve made

according to the manufacturer’s instructions.

100

RESULTS AND DISCUSSION

Evidence that activation of MAPK by extracellular Ca2+ is partially inhibited by PMA
Many protein growth factors stimulate the activation of PKC [24,44], which leads to
the activation of downstream kinases, including MAPK [31]. Since in mouse fibroblasts,
extracellular Ca2+ induces c-fos expression in a PKC-dependent manner [15], we first tested
the involvement of PKC in the Ca2+ -induced activation of MAPK in human fibroblasts.
Exposure human fibroblasts, as well as other types of cells, to PMA for a few
minutes has been shown to activate PKC [45], and a long exposure to PMA downregulates
PKC [20,24,45,46]. To determine whether PKC mediates the Ca2+-induced MAPK
activation pathway in human fibroblasts, confluent LG] cells were incubated in medium
overnight with or without 100 nM PMA , then treated for 5 min with Ca”, EGF, or fresh
PMA, or left untreated. Cell lysates were assayed for MAPK activation. Incubation of cells
with PMA for 5 min induced MAPK activation, as shown by the mobility shift of the p42
MAPK(Figure 1) and detected by an activity assay using MAP-2 as the substrate for
phosphorylation by MAPK (data not shown). These data indicated that in human fibroblasts,
MAPK activation is mediated by PKC. Down-regulation of the PMA-sensitive PKC by
overnight exposure of the cells to with PMA completely abolished the effect of subsequent
PMA treatment (Figure 1), indicating that PKC is completely downregulated by PMA .
Overnight treatment of cells with PMA partially abolished Ca2+ -induced MAPK activation,
but had no effect on EGF-induced MAPK activation, as shown by the p42 MAPK mobility
shift (Figure 1) and detected by MAP-2 phosphorylation assay (data not shown). These

results indicated that in human fibroblasts extracellular Ca2+-induced MAPK activation is

101

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102

partially dependent on PKC activation, whereas, EGF-induced MAPK activation is
independent of PKC activation. Since PKC is completely downregulated by the overnight
PMA treatment, these data suggest that there are at least two independent pathways for Ca”
-induced MAPK activation, one dependent on a PMA-sensitive PKC, and one independent

of this PKC.

Evidence that extracellular Ca2+ does not induce c-raf-l phosphorylation

Growth factors such as PDGF activate downstream kinases, such as PKC, raf-l , and
MAPK, by phosphorylation [30,44,47]. It has been shown that the activation of PKC
mediates c-raf-l kinase activation by direct phosphorylation [29,30]. The activated form can
be detected after the separation of cellular proteins by SDS-PAGE since the phosphorylated
form migrates more slowly [30]. To determine whether c-raf-l is in the pathway of
extracellular Ca2+ -induced MAPK activation, we determined the phosphorylation status of
c-raf-l in human fibroblasts before and after stimulation with Ca”, EGF, or PDGF. A slower
moving form of c-raf-1(Figure 2) was observed in lysates from cells treated with PDGF,
when compared with that from untreated cells (Figure 2), indicating that in human fibroblasts
phosphorylation of c-raf-l is induced by PDGF. No mobility shift of c-raf-l was observed
in lysates from cells treated with extracellular Ca2+ or EGF, indicating that extracellular Ca2+
does not act via a pathway involving c-raf-l. This conclusion agrees with that of Epstein
[15], who showed that in mouse 3T3 cells, c-raf-l is not activated by extracellular Ca2+ but

is activated by PDGF.

103

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Evidence that extracellular Ca2+ induces intracellular Ca2+ mobilization in human
fibroblasts

Intracellular Ca2+ has been shown to be an important second messenger in cell
proliferation driven by protein growth factors and growth hormones [48-50]. Gill and
colleagues [49,50] have shown that intracellular Ca2+ pools are directly linked to cell growth
in hamster smooth muscle cells since when these investigators depleted the intracellular Ca2+
pools, the cells were arrested in G0 phase. To determine whether intracellular Ca2+ is
mobilized as a result of incubating human fibroblasts with extracellular Ca”, the intracellular
Ca2+ concentration was measured in resting cells and cells incubated with Ca“, lys-
bradykinin, ionomycin, or EGF, using indo-l microflurometry with an ACAS fluorescent
microscopy. Ionomycin, a calcium ionophore, and bradykinin, a protein mitogen which
mediates its signal by binding to a receptor, were used as positive controls since it has been
shown that the treatment of cells with these agents causes a transient increase in the free
intracellular Ca2+ concentration [21] . As shown in Figure 3, cells treated with bradykinin
(B) and ionomycin (C) exhibited a transient increase in the concentration of intracellular
Ca2+, which lasted about 60 seconds. Afier the addition of Ca2+, intracellular Ca2+
concentration was increased quickly (Figure 3, D) to a level similar to that caused by
ionomycin and bradykinin, and the transient lasted for about 180 seconds. No obvious
intracellular Ca2+ transient was observed after EGF stimulation (Figure 3, E). This result is
in agreement with a previous study in human fibroblasts that showed that when cells were
pretreated with EGTA to chelate the extracellular Ca2+ in the medium [51] there was no

noticeable elevation of the concentration of intracellular Ca2+ upon EGF stimulation.

105

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Figure 3 Intracellular Ca2+ mobilization after Ca2+ stimulation. Subconfluent cells incubated in
serum free medium overnight were preloaded with indo-l-Am for 1 hour, washed and placed in McM
medium containing 0.1 mM Ca2+ without phenol red but with Hepes. lndo-l microflurometry were
carried out usingACAS fluorescent microscope as described in “Material and method”. Intracellular Ca2+
concentrations were measured immediately following the addition of 0.1 mM Ca“ medium as control
(A), 50 ng/ml lys-bradykinin (B), 1.5 ug/ml ionomycin (C), 10 mM Ca“(D), 100 ng/ml EGF (E), or 1
uyml thapsigargin (F), or measured after cells were pretreated with l ug/ml thapsigargin for 24-30 hours,
then treated with l ug/ml thapsigargin (G), or 10 mM Ca’*(H ). Bach curve is the data taken from a single
cell of3-5 cells measured in each experiment, and each experiment was repeated two tothree times with

similar results.

106

Evidence that the mobilization of intracellular Ca2+ transient by extracellular Ca2+ is
partially abolished by thapsigargin

Because Gill and colleagues [50] have found that in mouse muscle cells, thapsigargin
depletes the intracellular Ca2+ stores (pools) by specific inhibition of the endoplasmic
reticulum (ER) Ca2+ -ATPase, and that this results in the arrest of the cells in G0 phase
[49,50], we examined the effect of thapsigargin treatment on Ca2+-induced intracellular Ca2+
mobilization in human fibroblasts. We first measured the intracellular Ca2+ concentration
in cells after 1 ug/ml thapsigargin treatment. To test whether prolonged treatment with
thapsigargin depletes intracellular Ca2+ store, cells were incubated in medium with
thapsigargin for 24-30 hours, and intracellular Ca2+ concentration was measured after the
addition of thapsigargin. The addition of thapsigargin to human fibroblasts increased the
intracellular Ca2+ concentration (Figure 3, F). The profile we obtained was similar to that
showed by Chao et al. using human fibroblasts [21]. Prolonged treatment with thapsigargin
totally blocked the thapsigargin-increased intracellular Ca2+ concentration (Figure 3, G).
These data indicated that at the concentration used for the pretreatment, 1 ug/ml, the
thapsigargin-sensitive Ca2+ pools are completely depleted.

We then tested whether Ca2 + fiom the thapsigargin-sensitive stores is responsible for
the intracellular Ca2+ increase after stimulation with extracellular Ca”. Cells were pretreated
with thapsigargin for 24-30 hours, and intracellular Ca2 + concentration was measured afier
the addition of extracellular Ca2 i. We found that thapsigargin pretreatment partially
abolished the transient increase of intracellular Ca2 * concentration (Figure 3, H) that was
normally seen after stimulation with extracellular Ca2+ (Figure 3, D) This indicated that the

thapsigargin-sensitive Ca2 i stores are partially responsible for the Ca2 + increase after Ca2 *

107

stimulation.
Previous reports suggest that the depletion of intracellular Ca2+ pools triggers Ca2+
entry across the plasma membrane by a process known as “capacitative Ca2+ entry” [3 2,52].
It is the Ca2+ from both the intracellular Ca2+ stores and from the Ca2+ entry channel [32] that
accounts for the longer lasting increase in intracellular Ca2+ concentration after stimulation
of the cells by various growth factors including PDGF [32,48]. The fact that we observed
a longer lasting transient increase in intracellular Ca2+ concentration afier extracellular Ca2+
stimulation (Figure 3, D) than we saw in cells treated with ionomycin (Figure 3 C) or
bradykinin (Figure 3, B) in low Ca2+ medium, and that this Ca2+ increase can be abolished
only partially by thapsigargin pretreatment (Figure 3, H), suggests that, after extracellular
Ca2+ stimulation, in addition to that Ca2+ from thapsigargin-sensitive intracellular Ca2+ stores,
Ca2+ either from channels or from thapsigargin-insensitive stores, contributes to the transient
intracellular Ca2+ mobilization we observed. It is known that there is more than one type of
intracellular Ca2+ store in the endoplasm reticulum membrane [53,54] and that the

thapsigargin-sensitive stores account for only 75% of the stored Ca2+ [53].

Evidence that activation of MAPK by extracellular Ca2+ is partially inhibited by
thapsigargin

To test whether in human fibroblasts MAPK activation is mediated by intracellular
Ca2+ from the Ca2+ stores, we tested the effect of thapsigargin treatment on Ca2 +-induced
MAPK activation. Cells were treated with thapsigargin for 24-30 hours, and then stimulated
with Ca“, or with EGF, or were left untreated. Cell lysates were assayed for MAPK

activation by immunoblot analysis. We found that thapsigargin partially inhibited the p42

108

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MAPK mobility shifi induced by extracellular Ca2+ , but did not inhibit the mobility shift
induced by EGF (Figure 4). These data indicate that intracellular Ca2+ mobilization from the
thapsigargin-sensitive Ca2+ stores lies, at least in part, in the pathway between extracellular
Ca2+ and MAPK activation, but not in the pathway between EGF and MAPK activation.
To determine whether the PMA-sensitive PKC and intracellular Ca2+ from the '
thapsigargin-sensitive stores are in the same or a different pathway for extracellular Ca2+ -
induced MAPK activation, cells were pretreated with PMA and thapsigargin together and
then stimulated with Ca2+ for 5 min and lysates were subjected to immunoblot analysis using
anti p42 MAPK antibody. The pretreatment with PMA and thapsigargin resulted in an
almost complete inhibition of Ca2+-induced MAPK mobility shift (Figure 4), the inhibition
was greater than by either of the inhibitors given alone. This indicates that PMA-sensitive
PKC and thapsigargin-sensitive intracellular Ca2+ stores lie, at least partially, in two distinct
pathways. EGF-induced MAPK activation is not affected by either PMA or thapsigargin,
indicating that EGF does not act through PKC activation or mobilization of the thapsigargin-

sensitive intracellular Ca2+ stores.

Evidence that activation of MAPK by extracellular Ca“ is abolished by the channel
blocker lanthanum, but not by the channel blockers verapamil or nifedipine

Growth factors such as PDGF evoke transient increase in intracellular Ca2+
concentration through mobilization of intracellular Ca2 + stores and Ca2 + entry by channels
[48]. Ca” channel antagonists have been shown to decrease the rate of growth of many cell
types [48,55]. Estacion and Mordan [48] showed that in mouse fibroblasts the selective

inhibition of the sustained increase in Ca2+ concentration b blockin Ca2+ influx with
Y g

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lanthanum completely inhibits progression of the cells into S phase upon exposure to PDGF.
To test whether Ca2+ entry through Ca” channels plays a role in the Ca2+ -induced MAPK
activation, we pretreated cells with Ca“ channel blockers nifedipine, which blocks L type
voltage sensitive Ca2+ channels, verapamil, which blocks voltage sensitive channels
nonspecifically, or lanthanum, a nonspecific channel blocker [56], for 30-60 min. The three
populations of cells were then exposed to Ca2+ or EGF for 5 min, or were lefi untreated. Cell
lysates were assayed for MAPK mobility shift by immunoblot analysis. The results (Figure
5) show that Ca2+-induced MAPK mobility shift was partially inhibited by lanthanum , but
that there was no obvious effect as a result of verapamil or nifedipine treatment. The
inhibition effect by lanthanum suggests that Ca2+ entry through Ca2+ channels plays a role in
extracellular Ca2+-induced MAPK activation pathway. Although the mechanism by which
lanthanum acts as a channel blocker is as yet not clear, it has been suggested that as a poly
cation, it may bind to the channels, preventing Ca2+ entry [57].

EGF-induced MAPK activation was not abolished by lanthanum or nifidipine, but
surprisingly, it was abolished by verapamil (Figure 5). Since no changes in intracellular Ca2+
concentration were observed upon EGF stimulation (Figure 3, E), this effect was not
expected. In human meningioma derived cells, it has been shown that Ca2+ channel blockers,
such as verapamil, block cell growth driven by growth factors such as EGF and PDGF
[55,58], although no intracellular Ca2+ change is observed upon stimulation by growth factors
[55]. Verapamil has been shown to bind to muscarinic receptors or bind and inhibit the
ryanodine receptor channel of the sarcoplasmic reticulum [59]. Therefore, the effect of
verapamil on EGF-induced MAPK activation pathway might result from its interference with

other signal transduction components.

112

Evidence that activation of MAPK by extracellular Ca2+ is partially abolished by
calmodulin antagonists

Since we found that the increase of intracellular Ca2+ concentration appears to play
a role in Ca2+-induced MAPK activation pathway, we examined possible downstream
effectors of the intracellular Ca2+. The increased concentration of intracellular Ca2+ binds to
Ca2+ binding proteins to exert its effect. These proteins include calmodulin, calcineurin, and
phospholipase A2, etc. [32]. Calmodulin has been shown to affect cell replication in variety
of cell types [60,61]. For example, the selective pharmacological inhibitor of calmodulin
kinase, KN -62, prevents quiescent WS-l human fibroblasts from reaching S phase upon
serum stimulation [61,62]. Takuwa et al. [63] have shown that the potent calmodulin
antagonists calmidazolium and W-7, but not its inactive analogue W-12, strongly inhibit
serum-induced DNA synthesis in normal human fibroblasts [63] and human vascular
endothelial cells [64]. Furthermore, a study on neuronal cells [65] showed that the transient
increase of intracellular Ca2+ concentration via Ca2+ entry channels modulates the activation
of calmodulin kinases (CaMK2 or CaMK 4) to regulate the expression of their downstream
genes.

Using calmodulin antagonist W-7 to inhibit the effect of calmodulin, we tested the
role of calmodulin in the Ca2+ - or EGF-induced MAPK activation pathway. Cells were
pretreated with W-7 at 30 uM, a concentration which has been shown to be effective in
human fibroblasts [63], and then stimulated with Ca“ or EGF, and the p42 MAPK activation
was analyzed by immunoblot analysis. W-7 partially abolished both the Ca2+ - and the EGF-
induced MAPK mobility shift (Figure 6). To determinewhether the effect of W-7 is specific

for calmodulin, we also pretreated cells with calmidazolium or W-12, and then exposed them

113

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to Ca” or EGF. We found that, like W-7, calmidazolium (15 uM) partially abolished both
Ca2+ and EGF-induced MAPK shifi (data not shown), whereas W-12 (9O uM), the inactive
analogue of W—7, had no obvious effect on Ca2*- or EGF-induced MAPK activation (data not
shown).

Since two different calmodulin antagonists show a similar effect, and W-12, the
inactive analog of W—7, which has approximately 10 times lower affinity for calmodulin than
W-7 [63,64,66,67], shows no obvious effect on MAPK activation, we conclude with Takuwa
et al. [63,64] that the effect of calmodulin antagonists is very likely based on their
antagonism against calmodulin. Lu and Means conclude in a review [68] that Ca2+ is an
absolute requirement for all enzyme activating functions of vertebrate calmodulin. This
supports our suggestion that calmodulin mediates the function of intracellular Ca2+ afier
extracellular Ca2+ stimulation. We have no ready explanation for the fact that EGF-induced
MAPK activation is also abolished by calmodulin antagonists, although no obvious
intracellular Ca2+ elevation was observed after EGF stimulation (Figure 3). It is also possible
that calmodulin antagonists have effects on other components in the signal transduction

pathway, although such a mechanism is not supported by W-12 data.

The role of PLC

In mammalian cells, protein growth factors activate G protein-coupled receptors or
tyrosine kinase-linked receptors, which stimulate the production of DAG and 1P3 through
the activation of PLC [69]. DAG activates PKC, whereas 1P3 increases cytosolic Ca2+ level
by triggering the release of Ca2+ from intracellular stores. These two receptor-dependent

mechanisms are coupled to different isoforms of the PLC family. G protein-coupled

”5

receptors activate PLCB [69-72]. The tyrosine kinase-linked receptors act through PLCyl
by tyrosine phosphorylation [70,73]. In human and mouse keratinocytes and bovine
parathyroid cells, extracellular Ca2+ has been shown to activate PLC and produce DAG and
IP3, which in turn, activate PKC and elevate intracellular Ca2+ levels [2,3]. The fact that our
results show that both PKC activation and intracellular Ca2+ elevation from intracellular Ca2+
stores are involved in the extracelluar Ca2+-induced MAPK activation pathway, suggests that
PLC is involved in the activation of PKC and elevation of intracellular Ca2+. To test
this hypothesis, we measured PLC activity by the accumulation of IP3 in cells [74], using
an IP3 binding protein [33]. Figure 7 shows that IP-3 level was elevated 8-10-fold after 1
min of Ca2+ stimulation, and 5-fold after bradykinin stimulation for 30 seconds, and 3-fold
after 1 min stimulation. The increased level found after bradykinin treatment is similar to
that shown in other studies with human fibroblasts [75,76]. Bradykinin is known to function
through the G protein-coupled bradykinin receptor and activate PLCB to generate IP3 and
DAG [72,77]. The elevation of IP3 afier Ca2+ stimulation suggests that PLC is involved.
Since PLCy has been shown to be phosphorylated on its tyrosine residue upon
activation [70,73], we tested its involvement by immunoprecipitation followed by
immunoblot analysis. Human fibroblast LGl cells were treated with Ca2+ (Figure 8, lane 2),
or EGF (Figure 8, lane 3), or lefi untreated (Figure 8, lane 1), and the cell lysates were
immunoprecipitated with anti-PLC'y antibody as described [78]. Proteins were subjected to
SDS-PAGE, transferred to a membrane, and probed with anti-phosphotyrosine antibody for
immunoblot analysis. Human epidermoid carcinoma A431 cells which are known to exhibit
PLCy activity when treated with EGF [78] were used as a positive control. Our results show

that EGF induced PLCy tyrosine phosphorylation in A431 cells (Figure 8, lane 5). No

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mM Ca2+ for l min (2), or 50 ng/ml bradykinin for 0.5 min (3) or 1 min (4). Values are means

:hS.E.M. of four observations.

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tyrosine phosphorylation of this protein was observed upon Ca2+ (Figure 8, lane 2) or EGF
(Figure 8, lane 3) stimulation in human fibroblasts. This indicates that PLCy is not involved
in Ca2+ -induced MAPK activation pathway and that some other PLC isoform, such as PLCB,

is involved.

Evidence that MAPK activation by extracellular Ca“ is partially inhibited by pertussis
toxin

Because of the increase of cytosolic level of IP3 afler Ca2+stimulation, which
suggests the involvement of PLC, we hypothesized that PLC might be activated by a G
protein. Based on their sensitivity to pertussis toxin, G proteins are divided into two
categories [70]: pertussis toxin-sensitive and -insensitive G-protein. Therefore, we tested the
effect of pretreatment with pertussis toxin on Ca2+-induced MAPK activation. Cells were
pretreated with 0.5 ug/ml pertussis toxin overnight, and then stimulated with Ca2+ or EGF,
and their lysates were assayed for MAPK activation. Pertussis toxin partially abolished the
MAPK mobility shift caused by extracellular Ca“, but had no effect upon EGF stimulation
(Figure 9). This suggests that Ca2+ -induced MAPK activation is at least partially dependent
on a pertussis toxin sensitive G-protein, whereas EGF-induced MAPK activation is
independent of this type of G-protein. These data indicating the involvement of a pertussis
toxin sensitive G protein, which has been shown to be one of the major activators of PLC,
and the data provided above indicating the involvement of PKC, which can be activated by
DAG, along with the IP3 elevation after Ca2+ stimulation, strongly suggest that PLC is

involved.

119

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Evidence that the Ca2+ sensing receptor of Brown et al. [33] is not involved in
extracelluar Ca2+-induced cell growth or MAPK activation in mouse skin fibroblasts

How the fibroblasts sense the extracellular Ca“ concentration is unknown. It could
be that Ca2+ channels open when the extracellular Ca2+ reaches a critical threshold, as
suggested by the inhibition effect of lanthanum. On the other hand, the involvement of a G
protein suggests that there may be Ca2+ specific receptors on the external cell surface,
analogous to protein growth factor receptors, and that these are involved in this process. Just
such a Ca2+-sensing receptor has been cloned from bovine parathyroid cells and has been
shown to mediate the elevation of intracellular Ca2+ and IP3 [33]. Mice lacking this receptor
show a dramatically retarded growth [35], which suggests that this receptor may play a role
in growth control. Therefore, we tested the possible involvement of this receptor in
regulating cell growth and MAPK activation by using knockout mice lacking this Ca2+
sensing receptor [3 5]. After determining that mouse skin fibroblasts exhibit the same ability
as human skin fibroblasts to grow in serum free medium with 1 mM Ca2+ and to induce
MAPK activation after Ca2+ stimulation (data not shown), we tested whether skin fibroblasts
from mice lacking this receptor exhibit any deficiency in growth or MAPK activation after
Ca2+ stimulation, when compared with their wild type or heterozygous counterparts. We
found that all mouse skin fibroblasts, regardless of their receptor status (+/+, +/-, or -/-), had
the same ability to grow in the medium containing 1 mM Ca2+ or supplemented with 10%
FBS (data not shown). Also 10 mM extracellular Ca2+ or 10%FBS induced a MAPK
mobility shift in these mouse fibroblasts to the same degree, regardless of the status of the
Ca2+ sensing receptor (Figure 10). These results indicate that this Ca2+ sensing receptor is

not involved in Ca2+-induced cell growth or MAPK activation in mouse fibroblasts. Since

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the mouse fibroblasts exhibit the same response to extracellular Ca” as human fibroblasts
as judged by growth and MAPK activation assays, we conclude that it is unlikely that this
Ca2+ receptor to be involved in Ca2+-induced cell growth or MAPK activation in human
fibroblasts.

Based on the results we obtained, we propose that in human fibroblasts extracellular
Ca“, through a mechanism as yet unknown, activates a G protein and this in turn activates
PLC, generating DAG and IP3, which in turn activates PKC and transiently increases the
intracellular Ca2+ levels. The intracellular Ca2+ binds to calmodulin and, in turn, exerts its
function on MAPK directly or indirectly. We realize that cellular responses are complicated,
and that multiple pathways exist, so that components we found to be involved in human
fibroblasts might function in a different sequence, e.g., intracellular Ca2+ might activate PLC
or PKC, as reported for other systems [70]. Cross-talk between pathways could further
complicate the issue. However, the most interesting question is how the extracellular Ca2+
is able to generate a signal within the cells. Although we found that the Ca2+-sensing
receptor cloned by Brown et el. [33], which mediates the intracellular IP3 and intracellular
Ca2+ elevation in parathyroid cells, and does so using a pertussis toxin-insensitive G protein
[1], is not involved in this process in skin fibroblasts, the probable involvement of a pertussis
toxin sensitive G protein suggests that there is another receptor that mediates the Ca2+-
induced cellular responses in human fibroblasts. One candidate is the F GF receptor, since
it has been shown to have a Ca2+ binding motif in its acid box [79], and it is present in human
fibroblasts [80,81]. Furthermore, in neuronal cells, the F GF receptor mediated signals have
been shown to be dependent on a pertussis toxin sensitive G proteins [82]. In parathyroid

cells, an increase in extracellular Ca2+ also inhibits receptor-mediated increases in CAMP

l23

[1,83,84] which can be abolished by pertussis toxin [1], indicating that a pertussis toxin
sensitive G protein is involved in this process [1,84]. This G protein apparently differs from
that which mediates the Ca2+-induced intracellular IP3 and intracellular Ca2+ elevation, as the
latter responses have been shown to be pertussis toxin-insensitive in parathyroid cells [1,83].
The receptor that couples to a pertussis toxin-sensitive G protein in parathyroid cells could
be the receptor cloned by Brown et al. [33], if, as suggested, it simultaneously couples to
two different G protein subfamilies [84] or it could be another, as yet unknown, receptor
which senses the extracellular Ca2+ concentration and mediates a signal [1]. It has been
shown that exposure of kidney proximal tubular cells to extracellular Ca“ [84] induces
cellular responses, such as regulation of the concentration of l-hydroxylation of 25-
hydroxyvitamin D in the proximal tubule, and changes in circulating PTH levels. The Ca”-
sensing receptor cloned from bovine parathyroid has not been found to be expressed in these
kidney cells [84]. However, Juhlin et al. [85-87], using monoclonal antibodies have
identified an ~500 kDa membrane protein in kidney proximal tubular cells and
cytotrophoblasts. Sequence analysis of a 2.8 kb cDNA, isolated by Lundgren et al.[88], that
represents a fragment of the gene for the 500 kDa protein indicates that the protein is a
member of the low-density lipoprotein receptor superfamily and is closely related to Heyman
nephritogenic protein, a kidney tubule glycoprotein with Ca2+-binding activity [88-90]. It
is not yet known whether this protein functions as a Ca” sensor. Functional studies of this
protein should resolve this question [84] .

Although a membrane-bound Ca2+ receptor could function as a sensor of the external
Ca2+ concentration, alternative mechanisms cannot be excluded. For example, the inhibition

effect by lanthanum suggests that extracellular Ca2+ may directly operates a channel which

l24

senses the extracellular Ca2+ concentration. Nevertheless, the present study provides
information on some of the mechanisms which must be integrated into our understanding of
how extracelluar Ca2+ acts as a growth promoter, and suggests a number of specific

hypothesis that can be tested.

125

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