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This is to certify that the

thesis entitled

"Insqlin-Like Growth Factor Binding
Protein—2 Inhibits Proliferation in
MAC-T Cell Cultures"

presented by

Maria E. Zavala

has been accepted towards fulfillment
of the requirements for

M. S . degree in Animal Sciences

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Insulin-Like Growth Factor Binding Protein-2 Inhibits Proliferation in MAC-T Cell
Cultures

By

Maria E. Zavala

AN ABSTRACT OF A THESIS

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

MASTER OF SCIENCE
Department of Animal Science

2001

Dr. Michael VandeHaar

ABSTRACT

Insulin-Like Growth Factor Binding Protein-2 Inhibits Proliferation in MAC-T Cell
Cultures

By

Maria E. Zavala

Insulin-like growth factor-I (IGF-I) is a potent mitogen for mammary epithelial cells in
vitro. Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGFBP
synthesized by mammary epithelial cells in vitro and is thought to regulate IGF-I action.
To investigate the direct effects of IGFBP-2 and IGF-I on proliferation in mammary
epithelial cells, IGFBP-2 was added to cultures of MAC-T cells on collagen I with and
without IGF-I. Addition of IGFBP-2 inhibited the proliferation of MAC-T cells in the
presence and absence of IGF-I. This inhibition acted in a dose dependant manner where
100 ng/ml IGFBP-2 inhibited proliferation to a greater extent than 10 ng/ml IGFBP-2 (P
< 0.0001). However, the magnitude of the effect was greater in the presence of IGF-I (P
<0.001). For example, 100 ng/ml IGFBP-2 decreased proliferation by 40%. in the
absence of IGF-I, while 100 ng/ml IGFBP-2 decreased IGF-I-stimulated cell proliferation
by 51% in the presence of IGF-I. In conclusion, IGFBP-2 inhibits the proliferation of

MAC-T cells in vitro. This suggests IGFBP-2 may play a role in regulating mammary

epithelial cell proliferation in viva.

Cepyn'ght by
Maria E. Zavala
2001

For My Beloved Husband James

iv

ACKNOWLEDGMENTS

I would like to extend my thanks to Dr. VandeHaar for accepting me as his
graduate student and in doing so providing me with funding, lab space, and a project to
work on for my Master’s Thesis. I would also like to thank Drs. Haslam, Orth, Tucker,
and Wilson for monitoring my project’s development and serving as my committee
members at MSU. Of course this project would not have been possible if it were not for
the time and patience of Drs. Chelbek-Brown, F endrick, Weber, and Woodward who
provided me with protocols and advice on how to establish a cell culture system. Aside
from technical assistance I am also grateful for the friendship of my fellow graduate
students at MSU, especially Brian, Kristin, Nick, and Pasha whose support of my
progress has been invaluable. Lastly, I would like to thank Dr. Wendie Cohick and Dr.
Susan Fried for their discussions, advice on writing, and encouragement Without them

this thesis would have never been completed.

TABLE OF CONTENTS

Introduction ........................................................................................... 1
Review of Literature ................................................................................. 5
Insulin-like growth factor-I ................................................................. 5
Regulation of IGF-I production ............................................................ 6

IGF-I action ................................................................................... 8
IGFBPs ........................................................................................ 9
IGFBP production .......................................................................... 12
IGFBP action ............................................................................... 14

The role of the GH-IGF-I axis in bovine mammary gland growth and

development ................................................................................ 15
IGFBPs in bovine mammary tissue ...................................................... 19

The MAC-T cell line ....................................................................... 20
Summary .................................................................................... 22
Materials and Methods ............................................................................. 24
Materials .................................................................................... 24

Model Development ....................................................................... 25
Growing stock cultures ........................................................... 25

Cryogenic preservation ........................................................... 25

Plating cells ........................................................................ 25

Selection of culture substratum ................................................. 26

Serum-free medium ............................................................... 26

Thymidine incorporation assay ................................................. 27
Determination of plating efficiency ............................................. 28

Preparation of growth factor treatments ....................................... 29

Experimental design ............................................................... 29

Experiment 1 .............................................................. 29
Experiment 2 ............................................................ 30

Experiment 3 .............................................................. 30

Statistical analysis .......................................................................... 30
Results ................................................................................................ 31
IGF-I mitogenic activity .................................................................. 33

Des (1-3) IGF-I mitogenic activity ...................................................... 34
Inhibition of mitogenic activity .......................................................... 36
Discussion ........................................................................................... 38
lGF-I mitogenic activity ................................................................... 3 8

Des (1-3) IGF-I mitogenic activity ...................................................... 38
IGFBP-2 inhibition ...................................................................... 40
Summary ........................................................................................ 44
Bibliography45

vi

LIST OF TABLES

Table 1. A summary of the structural characteristics of human IGFBPs 1 through 6. . . ..10
Table 2. Comparison of plating efficiency in MAC-T cell cultures in SF M, and SFM
supplemented with 1% and 2% FBS .............................................................. 32

vii

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

LIST OF FIGURES

IGF-I may act in an endocrine, autocrine, and/or paracrine fashion ............... 6
GH binding to the liver and tissues stimulates the production of IGF-I .......... 7
The mitogenic effect of IGF-I in MAC-T cell cultures ............................ 34
The mitogenic effects of IGF-I vs. des (1-3) IGF-I in MAC-T cell cultures...35
The inhibition of MAC-T cell production by IGFBP-2 ............................ 37
The modulation of IGF-I action by IGFBPs ......................................... 42

viii

LIST OF ABBREVIATIONS

DMSO ...................................................................................................... Dimethy sulfoxide
DNA .................................................................................................. Deoxyribonucleic acid
df ............................................................................................................. degrees of fieedom
GH .............................................................................................................. Growth hormone
IGF ............................................................................................... Insulin-like growth factor
IGFBP ................................................................. Insulin-like growth factor binding protein
MAC-T ......................................................................... Mammary alveolar cells transfected
mRNA ....................................................................................... messenger Ribonucleic acid
MW ........................................................................................................... Molecular weight
SV 40 ........................................................................................................... Simian virus 40
RGD ......................................................................................... Arganine-Glycine-Aspartate

INTRODUCTION

In ruminants, the number of the secretory cells in the mammary gland is a major factor
that determines milk yield (Tucker, 1987). Efforts to increase this number have the
potential to increase milk yield and efficiency of production. Selective breeding of dairy
cows has led to great increases in secretory cell number and therefore milk yield

(F orsyth, 1996). A greater understanding of the physiological factors responsible for this
increase in cell number may lead to further improvements in milk production. One such
factor is growth hormone (GH). Studies of GH have revealed the existence of a potent
mitogen for the secretory cells of the bovine mammary gland. This mitogen is insulin-
like growth factor-I (IGF-I). Increasing our knowledge of these regulatory influences in
the IGF-I system may enable further increases in mammary secretory cell number and

thereby improve milk production.

Administration of exogenous GH to dairy cows has mitogenic and mammogenic effects
on the bovine mammary gland (Sejrsen et al., 1986; Peel and Bauman, 1987). Since GH
receptors have not been detected in bovine mammary tissue in vitro and GH has no effect
on proliferation of this tissue in vitro, GH is not believed to act directly on mammary
tissue in viva (Akers, 1985; Baumrucker and Stemburger, 1989). However, its actions
may be carried out indirectly through IGF-I. Insulin-like growth factor-I is a protein that
is predominantly produced by the liver of animals and humans (Daughaday and Rotwein,
1989). However, because IGF-I is produced by many tissues in addition to the liver

(D’Ercole et al., 1984), OH is considered to act by stimulating IGF-I in target tissues,

which then acts in an autocrine or paracrine manner on local cells (Holly and Wass,

1 989).

When IGF-I binds to membrane receptors on secretory cells in the mammary gland it
stimulates cell proliferation of these secretory or epithelial cells in vitro (Jones and
Clemmons, 1995; McGrath et al., 1991). Though the majority of IGF-I is produced in the
liver, it is also secreted locally by the stromal cells of the mammary gland (Campbell et
al., 1991). The localized production of IGF-I and its mitogenic effects in mammary
cultures suggest that IGF-I is a factor of great importance in stimulating mammary

epithelial cell proliferation in vivo.

Insulin-like growth factor binding proteins (IGF BPS) are a family of proteins that regulate
IGF-I activity. Insulin-like growth factor binding proteins have a higher affinity for IGF-
1 than does the membrane receptor for IGF-I and are thought to play a regulatory role in
IGF action (Jones and Clemmons, 1995). Insulin-like growth factor binding proteins are
synthesized in a wide variety of tissues in the body and may bind to IGF-I either in the
tissues or in the blood. In the mammary gland, epithelial cells secrete IGFBPs,
particularly IGFBP-2 (Campbell et al., 1991; Hodgkinson et al., 1991; and Collier et al.,

1993).

Serum IGFBP-2 concentration increases when ruminants enter a catabolic state as during
lactation, fasting, and mild insulinopenia (Sharma et al., 1994; Cohick et al., 1989; Ooi,

1990). In addition, serum concentrations of IGFBP-2 decrease while IGF-I mRNA and

serum IGF-I protein concentration both increase during treatment with GH (Sharma et al.,
1994; Cohick et al., 1989; Ooi, 1990). To investigate the direct effects of IGFBP-2 on

IGF-I on mammary epithelial cells, an in vitro model was developed.

Validation of several in vitro models of bovine mammary epithelial cells has already
been achieved (McGrath, 1987; Baumrucker and Stemburger, 1989; Peri et al., 1992; and
Huynh et al, 1991). One such model employs the MAC-T cell line. The MAC-T cells
are a nontransformed cell line derived from bovine mammary alveolar cells. These cells
proliferate in response to IGF-I treatment (Woodward et al., 1994) and have been shown
to bind IGF-I via cell surface receptors for the IGF-I ligand (Zhao et al., 1992). Primary
bovine mammary cells and tissue also bind and proliferate in response to IGF-I treatment
(Shamay et al., 1988; Baumrucker and Stemburger, 1989; and McGrath et al., 1991).
Thus, the MAC-T cells provide a useful model for studying the role of the IGF system in

mammary growth and development.

The objective of this thesis was to determine the effects of IGFBP-2 addition on IGF-I-
stimulated mitogenic activity in MAC-T cell cultures. A series of three experiments was
designed to determine the effects of IGF-I and to determine the effects of IGFBP-2
addition on MAC-T cell proliferation in my system. The specific objectives of

experiments were:

1. To determine the effects of exogenous IGF-I on MAC-T cell proliferation for

concentrations of IGF-I ranging from 0.001 rig/ml to 100ng/ml.

2. To determine the effects of exogenous des (1-3) IGF-I on MAC-T cell proliferation
for concentrations of des (1-3) IGF-I ranging from 0.01 11ng to 100 ng/ml.
3. To determine the effect of IGFBP-2 addition on IGF-I stimulated mitogenic activity

in MAC-T cell cultures.

Though the addition of IGFBPs to mammary cell cultures has been accomplished in
several laboratories (McGuire et al., 1992, Oh et al., 1993a, 1993c; Yee et al., 1994; and
Chen et al., 1994), the addition of IGFBP-2 to the MAC-T cells has never been reported.
Efforts to establish the role of the IGFBPs in mediating IGF-I mitogenic activity or in
actions independent of IGF-I may provide insight into the regulation of mammary growth
and development. Increasing our understanding of this regulation has potential to

increase the efficiency of milk production.

Review of Literature

Insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) is a 7.6 kDa protein composed of approximately 70
amino acids (Daughaday and Rotwein, 1989). The IGF-I molecule is composed of four
domains on a single peptide chain. These domains are designated A, B, C, and D; A and
B domains are homologous with A and B chains of the insulin molecule (Thissen et al.,
1994). The IGF-I molecule also shares structural homology across several species. The
IGF-I molecule has an identical amino acid sequence in the bovine, human, and pig,
differing from ovine IGF-I by one amino acid and from murine IGF-I by five amino acids

(Wong et al., 1989).

Insulin-like growth factor-I is found solubilized in serum, embedded in extracellular
matrix, and distributed throughout the extracellular spaces surrounding cells. The highest
concentration of IGF-I is found in blood (F urlanetto et al., 1977). The source of serum
IGF-I is believed to be the liver (D’Ercole et al., 1984). However, IGF-I is produced in
most organs (D’Ercole et al., 1984); such as the kidney, liver, heart and testes; and by
many cell types such as fibroblasts, smooth muscle, skeletal muscle, liver, granulosa,
Sertoli, and decidua cells (Sara and Hall, 1990). Production of IGF-I at multiple sites in
the body leads to the concept that IGF-I acts by endocrine, autocrine, and paracrine

mechanisms (Figure 1) (Schlecter et al., 1986; and Holly and Wass, 1989).

 

Target Tissues

Figure l. IGF-I may act in an endocrine, autocrine, and/or paracrine fashion. GH
can act upon the liver and/or the tissues stimulating IGF-I secretion into either the
blood or the extracellular space. The IGF-I secreted can act locally in an autocrine
or paracrine fashion, or can be carried by the blood to target tissues and act in an

endocrine fashion.

Regulation of IGF-I production

One of the principle hormonal stimuli for IGF-I production is growth hormone (GH)
(Copeland et al., 1980). Growth hormone binding to the GH receptor increases serum
IGF-I by inducing IGF-I synthesis in the liver (Bichell et al., 1992). Hypothalamic GH-
releasing hormone (GHRH) regulates the inductive effect of GH on IGF-I (Shibaski et

al., 1986), such that high levels of circulating IGF-I provide a negative feedback to the

hypothalamus that decreases the secretion of GHRH and GH (Ceda et al., 1987). This
negative feedback loop provides a mechanism for acute regulation of IGF-I production by

liver (Hill, 1989), as well as other tissues where IGF-I production is under control of GH

 

 

 

 

 

 

 

 

(Figure 2).
- IGF-I
] (-) ‘l
Hypothalamus
firs... ]
(4’) r—J—N
Liver
GHRH ____J &

GH __r?+-)'> Tissues

 

 

Figure 2. GH binding to the liver and tissues stimulates the production of IGF-I.
High levels of circulating IGF-l provide a negative feedback to the hypothalamus
that results in a decrease in the secretion of GHRH, a decrease in pituitary secretion

of GH, and thereby a decrease in the production of IGF-I.

In addition to hormonal stimulation, nutrition plays a primary role in regulation of IGF-I
production (Thissen et al., 1994). Suppression of serum IGF-I levels by energy/protein
deprivation has been well documented in many different species (Clemmons and
Underwood, 1991; McGuire et al., 1992; and Bauman and Vernon, 1995). Furthermore,
the decline of serum IGF-I during dietary restriction is independent of the increase in

pituitary GH secretion that occurs at the same time. This decline in serum IGF-1

concentration results from a decrease in hepatic IGF-I production and an increase in the
clearance of IGF-I from the blood (Clemmons and Underwood, 1991). Multi-level
regulation of growth factors like this link diet to growth in the body. Interestingly,
nutrient excess does little to raise the concentration of IGF-I in serum, meanwhile
nutritional deficiency reduces the concentration of IGF-I in serum (Thissen et al., 1994).
In addition, serum IGF-I in obese subjects are reported to be either low or high (Thissen
et al., 1994). Further discussion of the nutritional regulation of IGFs can be obtained in

the review by Thissen et a1. (1994).

IGF-I action

Insulin-like growth factor-I action is mediated through the type-I IGF receptor. The type-
I IGF receptor is a transmembrane receptor that shares structural homology with the
insulin receptor (Hth et al., 1972). The IGF-I ligand initiates signal transduction by
binding to the cystine-rich regions of the alpha subunit’s extracellular domain. Tyrosine
kinase activity resides in the cytoplasmic beta domain, which causes auto-
phosphorylation of insulin receptor substrate-I and propagation of the cytoplasmic signal
by intracellular machinery (Jones and Clemmons, 1995). Insulin-like growth factor type-
1 receptors are present in a wide variety of cell types, and are critical in regulating the

effects of IGF-I at the cellular level (Sara and Hall, 1990).

The in vitro action of IGF-I has been established in a wide variety of cells. In general, the
effects of IGF-I can be separated into two categories: acute and chronic. The acute

effects of IGF-I include anabolic effects on protein and carbohydrate metabolism. The

chronic effects of IGF-I include effects on cell replication and differentiation. Jones and
Clemmons (1995) have reviewed the effects of the IGFs in multiple cell types and
determined that the end result of IGF action is dependent on cell type, cell environment,
and stage of cell differentiation. Other factors known to modulate the effect of IGF-I on

the target cells are the insulin-like growth factor binding proteins (IGFBPs).

IGFBPs

Insulin-like growth factor-I is present in circulation and extracellular space almost
entirely bound to IGFBPs (Jones and Clemmons, 1995). The high affinity of these
proteins for IGF-I suggests that the IGFBPs are involved in the mediation of IGF-I action
(Oh et al., 1993b; Rosenfeld et al., 1994). Furthermore, reversibility of this binding of
IGF-I with any one of the IGFBPs. provides modulation of the interactions between IGF-I

and its receptor.

Of all the serum protein fractions with high affinity for IGF-I, more than ten have been
identified (Rechler, 1997, Baumrucker and Erondu, 2000), and seven have been cloned
(Binoux, 1995; Oh et al., 1996). Of these binding proteins, six have been shown to bind
IGF-I with high affinity (IGFBPs 1-6), and nine have been shown to bind IGF-I with
much lower affinity (IGFBP related proteins 1-9) (Baumrucker and Erondu, 2000).
These binding proteins are commonly referred to as IGFBP-l , -2, -3, -4, -5, and —6.
Similar to IGF-I, the IGFBPs are synthesized ubiquitously, although their expression
varies with tissue origin, developmental stage, and environment (Rechler et al., 1997).

The IGFBPs-l through -6 are structurally related proteins, but are encoded by different

genes (Rechler et al., 1993). The six molecular species of IGFBPs have molecular

masses that range between 25 and 45 kDa and can be detected by western ligand and

immunoblotting techniques (Hossenlopp et al., 1986). The structural differences between

the six IGFBPs also confer their difference in affinities for IGF-I (Table 1) (Oh et al.,

1993b).

Table l. A summary of the structural characteristics of human IGFBPs —1 through -

6. Adapted from Rosenfeld et al. (1994); and Jones and Clemmons (1995).

 

 

 

 

 

 

 

 

 

Structural IGFBP-l IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6
Characteristic
MW 25,271 31,355 28,717 25,957 28,553 22,847
Number of 234 289 264 237 252 216
amino acids
RGD + + - - - -
Sequence
Glycosyla- - - + + + +
tion sites
Phos- + - + - + NA"
phorylation
Proteolysis - + + + + NA"

 

 

 

 

 

 

 

“Data for some characteristics are not yet available (NA).

10

 

Further analysis of the structures of the IGFBPs reveals the presence of an RGD
(Arginine-Glycine-Aspartate amino acid) sequence. This sequence is involved in ligand
recognition by integrin receptors and suggests that IGFBPs may associate with the cell
surface and carry out actions independent of IGF-I binding. The RGD sequences have
been identified on IGFBP-1 and —2 (Brewer et al., 1988). However, only IGFBP-3
appears capable of specific binding to cell membrane receptors (Oh et al., 1993 a, c),

even though this IGFBP lacks an RGD sequence.

Another theory regarding the ability of IGFBPs to act independently of IGF-I suggests
the involvement of postranslational processing. IGFBP-3 undergoes limited proteolysis
in serum (Hossenlop et al., 1990). The IGFBP-3 products of this proteolysis are two
different types of fragments with different activities. One has a weak affinity for the
IGFs and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs
but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is
independent of the IGFs (Lalou et al., 1996). Thus, not only are there at least six distinct
IGFBP genes producing distinct translational products, but in viva, some IGFBPs

undergo postranslational modifications that may alter their biological activity.

Other modifications to IGFBP structure include phosphorylation, glycosylation, and
changes in location such as cell surface association vs. embedding in the extracellular
matrix (Jones and Clemmons, 1995). These alterations add another dimension to the

regulation of IGF-I action by the IGFBPs. However, the effects of these alterations on

11

the affinity of the IGFBPs for IGF—I, and the physiological significance of these

alterations have not been determined.

IGFBP production

Most cells tested appear to secrete at least one form of IGFBP though the level of
secretion and profile of IGFBP produced can vary dependant on cell type, tissue origin,
developmental stage, hormonal and nutritional environment. Discussions of these factors
and details of their impact on IGFBP production can be found in reviews by Baxter and

Martin (1989), Rechler et a1. (1993), and Ferry et a1. (1999).

Though the relative amounts of each of the IGFBPs vary, factors associated with
fluctuations of IGF BPS-1 through -3 have been evaluated in serum. The major factors
affecting serum IGFBP levels are diet and serum GH. In general, dietary restriction
decreases IGFBP-3 concentrations while it increases serum IGFBP-1 and -2 (Thissen et
al., 1994). These changes in serum levels of each of the IGFBPs are parallel to changes
observed in abundance of their respective mRNA levels in the liver (Thissen et al., 1994).
Because IGF-I and insulin-like growth factor binding protein-3 concentrations in serum
increase during anabolic states and decrease during catabolic states, IGFBP-3 is thought
to enhance IGF-I action in target tissues (Thissen et al., 1994). In addition, because
IGFBP-2 increases and IGF-I decreases during catabolic states, IGFBP-2 is thought to

inhibit IGF-I action in target tissues (Thissen et al., 1994).

12

Aside from the influence of the diet on serum concentrations of IGFBPs, GH also
regulates IGFBP concentrations in serum. In humans, GH dependency of IGFBP-3 is
evident from the reduced serum concentrations of IGFBP-3 measured during states of GH
deficiency and insensitivity (Rosenfeld et al., 1994). In cattle, elevated concentrations of
IGFBP-3 in serum are observed during administration of GH (V icini et al., 1991; Cohick
et al., 1992; and VanderKooi et al., 1995). Similarly to IGFBP-3, IGFBP-2 also displays

a relationship with GH. When exogenous GB is administered to cattle, serum
concentrations of IGFBP-2 decrease Wicini et al., 1991; Cohick et al., 1992; McGuire et
al., 1992). The relationship between GH and IGFBP-2 and -3 are of interest because
IGFBP-2 and -3 are the predominant lGFBPs in serum; thus their concentrations are

relevant to the availability and action of serum IGF-I (Rosenfeld et al., 1994).

The effects of GH on the serum concentrations of the IGF BPS described above become
uncoupled by undemutrition (Clemmons and Underwood, 1991). This phenomenon is
evident as circulating concentrations of IGFBP-3 decrease rapidly upon fasting even
though GH levels are increased (Clemmons and Underwood, 1991). Insulin-like growth
factor binding protein-2 also becomes uncoupled during undemutrition as serum
concentrations of IGFBP-2 are highest when GH is high even though GH is known to
decrease serum levels of IGFBP-2 when applied exogenously (V icini et al., 1991). These

results demonstrate the complexity of nutritional status on the GH/IGF axis.

13

IGFBP action

There are several biological properties that are shared among the IGFBPs. Shared
properties have led to development of several theories describing the relationship
between the IGFBPs and IGF-1. Some of these are: l) IGFBPs regulate the transport of
IGF-I from the blood to the extravascular space; 2) IGFBPs prolong the half-life of IGF-I
and decrease its rate of clearance from blood; 3) IGFBPs target IGF-I to specific tissues
or cells; 4) IGFBPs directly modulate the interaction between IGF-I and the type-I IGF
receptor; and 5) IGFBPs have direct effects on cellular fimctions that are mediated by cell
surface IGFBP receptors (Jones and Clemmons, 1995). Though evidence in the literature
suggests the IGFBPs may possess many of these roles, the biological role of each specific

IGFBP is not well defined.

The IGFBPs have multiple modes of action: endocrine, paracrine and autocrine. Soluble
IGFBPs are believed to attenuate IGF-I action (Elgin et al., 1987). Cell-associated
IGFBPs may either attenuate or enhance IGF-I action (Jones and Clemmons, 1995).
While the physical properties of the IGFBPs have been the target of extensive
investigations during the last decade, the diversity among the IGFBPs and their effects on
different target cell systems prevents the formation of a common mechanism of action

that would explain all of their functions.

14

The role of the GH-IGF-I axis in bovine mammary gland growth and lactation
Mammary Growth

During puberty, bovine mammary tissue enters a phase of allometric growth. The
number of mammary secretory cells that deve10ps during this period may play an
important role in determining future milk yield (Tucker, 1987). Several studies have
examined the relationship between GH and pubertal bovine mammary growth. One such
study has shown that circulating GH is positively correlated with mammary growth in
pen-pubertal heifers (Sejrsen et al., 1983). In addition, several studies have shown that
GH administration to pubertal heifers increases mammary parenchyma (Sejrsen et al,
1986; Sejrsen et al., 1999). In contrast, overfeeding heifers results in a decrease in
endogenous GH and decreased mammary development (Sejrsen et al., 1983). Thus GR is

believed to play an important role in promoting pubertal mammary growth.

During the dry period, the bovine mammary gland undergoes substantial growth and
development in preparation for lactation. Due to a dearth of data on the effect of GH on
mammogenesis during the dry period, much of the support for a stimulatory effect of GH
on mammogenesis during this period stems from data using other ruminant models. For
example, studies conducted in pregnant ewes and goats suggest that GH-stimulates an
increase in mammary parenchyma (Stelwagen et al., 1993; Knight et al., 1994). The
stimulatory effect of GH on mammogenesis in these ruminants during the dry period
suggests that GH may act as a bovine mammary mitogen. However, since GH does not
directly bind bovine mammary tissue; the effect of GH on mammogenesis is likely

mediated by other factors. One potential factor believed to mediate the mammogenic

15

effects of GH on the bovine mammary gland is IGF-I. Further evidence of the role of
IGF-I in the regulation of mammogenesis comes from studies employing the use of tissue

and cell culture systems as described below.

The ability of IGF-I to stimulate proliferation of bovine mammary tissue in vitra is well
established. Culture systems using bovine mammary tissue slices, mammary organoids,
mammary epithelial cells, and mammary epithelial cell lines have all shown that IGF-I is
a potent mitogen of bovine mammary tissue in vitra (Shamay and Gertler, 1986; Shamay
et al., 1988; Baumrucker and Stemburger, 1989; McGrath et al., 1991; and Woodward et
al., 1994; Woodward et al., 1996). This information is consistent with the idea that IGF-l

mediates the mammogenic effects of GH on the mammary gland.

Lactation

Treatment with exogenous GH stimulates milk production in the dairy cow (Peel and
Bauman, 1987). Two potential mechanisms for GH-stimulated increases in milk
production are an increase in secretory cell number or in the synthetic capacity of each
cell of the bovine mammary gland. During lactation, administration of GH stimulates an
increase in milk production that takes place without significant changes in the ratio of
milk yield to feed intake (Tyrrell et al.1982). These data suggest that GH can coordinate
more efficient use of nutrient stores and protein synthesis. Several studies have lent
support to this hypothesis by demonstrating that GH treatment increases total RNA per
gland, as well as key lactogenic enzymes such as acetyl CoA carboxylase, acetyl CoA

synthetase, and fatty acid synthase (Baldwin, 1990; Bauman and Vernon, 1995). This

16

GH-stimulated increase in the synthetic capacity of the gland is coupled to increases in
nutrient availability and mammary blood flow (Davis et al., 1988; Fullerton et al., 1989;
Peel and Bauman, 1989). Thus during lactation, GH has been shown to play a lactogenic
role in the bovine mammary gland. However, because GH does not bind to bovine
mammary tissues (Akers, 1985; Baumrucker and Stemburger, 1989), the mechanisms
whereby GH mediates these increases in the synthetic capacity of the bovine mammary

gland are unknown.

One of the possible mechanisms by which GH may exert its effects on milk production
by the bovine mammary gland is through the actions of IGF-I. As discussed in a
previous section, GH stimulates the production of IGF-1 (Copeland et al., 1980).
Furthermore, administration of exogenous GH to lactating cows causes an increase in
IGF-I concentrations in the blood and milk (Cohick et al., 1989; Prosser et al., 1989).
These data show that GH stimulates an increase in IGF-I in the blood and may also
stimulate the production of IGF-I locally by mammary stromal tissue (Campbell et al.,
1991). Thus GH-stimulated IGF-I may affect mammary tissue in an endocrine and
paracrine fashion. Regardless of its source, IGF-I can act directly on mammary tissue
because type-I IGF receptors that have been detected in the bovine mammary gland

(Dehoff et al., 1988).

The effect of IGF-I on milk production by the bovine mammary gland has been examined

using several approaches. For example, Presser and coworkers (1990) used close-arterial

infusion of IGF-I into the mammary gland of goats to show that the infusion of IGF-I

17

increased both milk yield and mammary blood flow. However, because the relationship
between milk yield and mammary blood flow was not determined, the mechanism for the
effect of IGF-I on milk yield in this study cannot be resolved. Another study conducted
by Shamay and coworkers used an in vitra system to examine the effects of IGF-I on
fatty acid synthesis and alpha lactalbumin synthesis in bovine mammary tissues (Shamay
et al., 1988). The results of this study suggest that IGF-I is not lactogenic in the bovine
(Shamay et al., 1988), therefore the lactogenic effects of GH administration may be

mediated by factors other than IGF-I.

A second mechanism that could be involved in the effect of GH on milk production is an
increase in the number of secretory cells in the gland. However, in contrast to the
mammogenic effects of GH/IGF-I during puberty and the dry period, treatment of cows

in mid-lactation with GH has no effect on total mammary DNA (Baldwin, 1990). Thus
the effect of GH on the bovine mammary gland during lactation may largely involve a
sustainable increase in the synthetic capacity of the gland in contrast to an increase in
secretory cell number. However, due to the limited data available it is difficult to
determine whether there is truly a change in secretory cell number during this
physiological stage. It must be noted that data on the effect of GH on the amount of
secretory cells is limited and ofien employs the use of rudimentary techniques. In
addition, studies supporting a role for the GH/IGF-I axis as a mechanism mediating
bovine mammogenesis draw their support from loosely based assumptions regarding the
mechanisms mediating the effects of the GH/IGF-l axis. Thus to better evaluate the role

of OH on mammogenesis during lactation, future work is needed to characterize the

18

population and regulatory mechanisms controlling secretory cell numbers in the bovine

mammary gland.

In conclusion, these data show that the mechanism involved in the GH-stimulated
increase in milk yield involves an increase in the synthetic capacity of the bovine
mammary gland; whether or not an increase in secretory cell number occurs is not clearly
known. Furthermore, the effects of GH-administration on milk yield and/or secretory cell
number appear to be dependent on the physiological stage of GH administration. Future
studies employing the use of more precise techniques and/or that target the mechanism
that mediates the effects of GH/IGF-I axis will greatly enhance our understanding of

growth and lactation in the bovine mammary gland.

IGFBPs in bovine mammary tissue

Insulin-like growth factor binding proteins are synthesized in bovine mammary tissue.
Immuno—histochemical staining has determined that IGFBP-2 is localized to the luminal
surface of epithelial cells of bovine mammary tissue slices (Collier et al., 1993). Insulin-
like growth factor binding protein-2 is also the predominant form of IGFBP secreted by
primary bovine mammary epithelial cells when cultured in serum-free media (McGrath et
al., 1991; and Woodward et al., 1996). These cultures also produce IGFBP-3 especially
when IGF-I is added to the media (McGrath et al., 1991). Insulin-like growth factor
binding protein-l and -4 were also detected in the mRN A analysis of bovine mammary
tissue (Sharma et al., 1994), but the presence of these proteins in mammary tissue or

primary cell cultures has not been reported.

19

 

Determining the role of the IGFBPs in mammary cell growth and development is
complicated by difficulties in maintaining bovine mammary cell cultures. Methods for
culturing bovine mammary epithelial cells were developed several years ago (Shamay
and Gertler, 1986). However, use of these techniques has not been wide spread.
Underdevelopment of primary bovine mammary epithelial cell culture techniques is
likely due to the problems associated with biological variability and cell recoveries that
are associated with its use (Forsyth, 1989; and Akers, 1990). The development of bovine
mammary epithelial cell lines provides an alternative to using primary cells thereby
easing some of the difficulties involved with studying growth and development of the

mammary gland at the cellular level.

The MAC-T cell line

The MAC-T cell line is a well-characterized mammary epithelial cell line that was
derived from primary bovine mammary alveolar cells (Huynh et al., 1991). The MAC-T
cells were immortalized by stabile transfection with the SV-40 large-T antigen but not
transformed. The MAC-T cells exhibit epithelial morphology and stain positively for
cytokeratin and negatively for virnentin, which indicates that these cells are of epithelial
origin. The MAC-T cells are also able to differentiate when induced by addition of
extracellular matrix and prolactin. The differentiated phenotype was characterized to
include 1) the ability to form secretory domes with a lumen from a pavement of columnar
cells; 2) increased casein mRNA abundance; 3) increased alpha S and beta casein

secretion; 4) increased number and size of casein secretory vesicles; and 5) increased

20

lactose synthesis and secretion (Huynh et al., 1991). Thus, the MAC-T cell line provides

a useful model for the study of bovine mammary epithelial cells in vitra.

As discussed above, studying the role of IGF system at the cellular level is complicated
by endogenous production of IGFBPs by many cell types. The MAC-T cells secrete
IGFBP-l, -2, -3, -4, and -6 in serum-free media (Cohick and Turner, 1998). The
predominant IGFBP produced by MAC-Ts in serum-free media is IGFBP—2, which is
also the predominant IGFBP produced by primary bovine mammary epithelial cells in

serum-free culture (McGrath et al., 1991; and Woodward et al., 1996).

The MAC-T cells also secrete low levels of IGF-I (Romagnolo et al., 1992).
Approximately 22 ng IGF-I /106 cells was measured in conditioned media from MAC-T
cells after 72 h of culture (Romagnolo et al., 1992). Conditioned media containing
endogenous IGF-I also stimulates proliferation in MAC-T cell cultures (Romagnolo et
al., 1992). Because MAC-T cells both secrete and respond to IGF-I in culture, IGF-I is

thought to act in an autocrine and/or paracrine fashion.

Proliferation stimulated by IGF-I is likely mediated by the IGF type I receptor in MAC-T
cell cultures. Since IGF type I receptors are expressed by most if not all tissues in the
body including the bovine mammary gland (Nissley et al., 1985; Dehoff et al., 1988),
they are likely expressed by MAC-T cells. Using Scatchard analysis, the binding of IGF-I
to MAC-T cell surfaces was shown to be specific, saturable, and time dependent (Zhao et

al., 1992). Though these data support the existence of type-I IGF receptors on MAC-T

21

 

cell surfaces, additional experiments verifying the presence of the type-1 receptor and its

mediation of IGF-I action in MAC-T cell cultures have not been reported.

Summary

The effect of nutrition and GH on milk production and the mammary gland are well
documented. The effects of these influences on mammary growth and development are
likely mediated at least in part by the IGF system. Insulin-like growth factor—I is a potent
mitogen in the bovine mammary gland. Systemic and local production and local binding
indicate that IGF-I regulates mammary cell growth in an endocrine, autocrine, and/or

paracrine fashion.

A family of high affinity IGF binding proteins further regulates IGF-l action. These
proteins are also produced in the bovine mammary gland. Insulin-like growth factor
binding protein-2 is the predominant form of IGFBP secreted by bovine mammary tissue
in vitra and has been localized to the luminal surfaces of mammary epithelial cells in
bovine mammary tissue slices (McGrath et al., 1991; Collier et al., 1993). The mRNA
for this IGFBP is also detected in bovine mammary tissue (Shanna et al., 1994). These
findings suggest IGFBP-2 plays a role in mediating the effects of IGF-I on mammary
growth and development. However, due to the complexity of the IGF system and the
difficulty in studying its local effects on the mammary gland in viva, the precise role of

IGFBP-2 in the mammary gland is not known.

The use of in vitra systems has aided in the study of the effects of the IGF system at the

cellular level. Using in vitra systems has enabled mammary physiologists to examine the

22

direct effects of the production and action of the IGF system on bovine mammary
epithelial cells. My study focuses on the effect of IGFBP-2 on IGF-I stimulated
mitogenic activity in mammary epithelial cell cultures. This model employs the MAC-T
mammary epithelial cell line, which is a well-characterized cell line, derived from bovine
mammary alveoli. Information gathered in these experiments may increase our
understanding of the effects of the IGF system on mammary epithelial cell growth.
Efforts to enhance understanding of the IGF system and mammary growth and
development have potential for economic impact in the dairy industry. Manipulation of
such factors could increase the number of secretory epithelial cells and thereby milk
production by the bovine mammary gland. Increasing our knowledge of factors that

regulate mammary growth can foster increases in milk production and productive

efficiency.

23

Materials & Methods

Materials

Receptor grade recombinant human insulin-like growth factor-I (IGF-I) (MW = 7649),
receptor grade recombinant analog of human insulin-like growth factor-I (des (1-3) IGF-
I) (MW = 7366), and recombinant bovine insulin-like growth factor binding protein-2
(IGFBP-2) (MW = 30776) were purchased from GroPep (Adelaide, Australia).
Dulbeco’s modified eagle medium (DMEM), HEPES buffer, Dulbecco’s phosphate
buffered saline (DPBS) (with and without calcium and magnesium), and antibiotic-
antimycotic solution (100X) containing pennicillin (10,000 TU/ml), streptomycin (10
mg/ml), and amphotericin B (25 ug/ml), were obtained from GibcoBRL (Grand Island,
NY). Fetal Bovine Serum (F BS), trypsin-EDTA solution (10X), Hank’s Buffered Saline
Solution (HBSS), gentamycin solution, bovine insulin, glutathione, soybean trypsin
inhibitor, sodium selenite, and apo-transferrin were obtained from Sigma Chemical
Company (St. Louis, MO). [Methyl-3H] thymidine (specific activity 50 Ci/mmol) was
purchased from ICN Pharmaceuticals, Inc. (Irvine, CA). Rat tail collagen I and Matrigel
were purchased from Collaborative Biomedical Products (Bedford, MA). Twenty-four
well cell culture plates and 75 cm2 cell culture flasks were purchased from Corning

(Coming, NY). All other reagents used were of analytical grade.

24

Model development

Growing stock cultures

The MAC-T cell line is an established bovine mammary epithelial cell line (Huynh et a1,
1992). The MAC-T line was routinely cultured as described by Huynh et a1. (1992).
Briefly, MAC-T cells were cultured in 75 cm tissue culture flasks with DMEM F -12
medium supplemented with 10% F BS, antibiotic-antimycotic solution (10%), and
gentamycin (1 %). Growing cultures were fed every 48 h and kept in a humidified, 37°C,
5% C02 incubator. Stock cultures were harvested with a trypsin-EDTA solution (0.10%

w/v) and cryogenically preserved.

Cryogenic preservation
Cells were preserved by methods described by Weber et a1. (1999) where preserved cells
were kept in fieezing medium composed of serum-free medium supplemented with F BS

(44%), and DMSO (6%) and stored in liquid nitrogen until use.

Plating cells

In preparation for proliferation experiments, cells were processed and plated by methods
originally described by Woodward et a1. (1994) where aliquots of MAC-T cells were
thawed and washed in DMEM with antibiotic-antimycotic solution (10%) and
gentamycin (1%). The MAC-Ts were counted using a hemocytometer and plated at a

density of2 x 104 cells/ml.

25

Selection of culture substratum

To fulfill the requirement of MAC-T cells for a substratum (Hyunh et al., 1992), growth
of MAC-T cells was tested on various substrate. Matrigel was the first extracellular
matrix (ECM) attempted. Matrigel is composed of larninin, collagen IV, fibronectin,
enactin, and various proteoglycans (Becton Dickenson Labware, 1998). Matrigel is a
reconstituted basement membrane isolated from the Engelbreth-Holm-Swarm tumor cell
line. Matrigel was chosen because it more closely resembles the composition of the
basement membrane in the mammary gland than other ECMs available. However,
because my thymidine incorporation assay requires adherence of cells to the plate that
could not be achieved with Matrigel, the cultures grown on the Matrigel could not be

assayed, and use of the Matrigel substratum was discontinued.

The next substratum attempted was collagen I. Collagen I was chosen because studies
comparing attachment of murine mammary epithelial cells on various ECMs
demonstrated epithelial cells grown on collagen I had the strongest attachment efficiency
compared to the other ECMs tested (Xie and Haslam, 1997). MAC-T cells cultured on
collagen I were adherent during the thymidine incorporation assay and thus collagen I

was employed as the substratum for this model system.

Serum-free medium
Serum may contain factors that influence cell growth such as IGF-I. Serum may also
contain factors that interact with exogenous growth factors such as IGFBPs. Therefore,

serum-flee medium was chosen for these experiments because including serum in media

26

may not only confound our studies of the IGF system on MAC-T cell proliferation, but
serum has been shown to cause variations in growth rates and chemosensitivities in

human mammary cells (Emennan et al., 1987).

Serum-free medium was prepared essentially according to Shamay and Gertler (1986).
The recipe was modified as follows. The media DMEM F-12 was used instead of Media
199 because it is more nutrient dense. Albumin was deleted from the media because
albumin may contain fatty acids that can modulate growth (Nilausen, 1996).
Furthermore, albumin may contain other proteins such as IGF BPS. Insulin was added at
10 ng/ml rather than at lOOng/ml because insulin concentrations greater than 10 ng/ml
cause stimulation of the type I IGF receptor (Winder et al., 1989; Bauman and Vernon,
1993). The contents of our culture medium were DMEM F -12 (GibcoBRL, Grand
Island, NY), HEPES buffer (7.4 ug/ml), antibiotic-antimycotic solution (10%),
gentamicin (50 mg/ml), glutathione (l rig/ml), soybean trypsin inhibitor (1 rig/ml),

sodium selenite (1 ng/ml), insulin (10 ng/ml), and apo-transferrin (5 pg/ml).

Thymidine incorporation assay

Incorporation of [methyl- 3H] thymidine into DNA was assayed to evaluate the
proliferative responses of MAC-T cells to growth factor treatment. The MAC-T cells
were originally plated and incubated for 48 or 72 h prior to assay. This incubation time
allowed cells to attach to the substratum and synchronize in the GO/Gl phase of the cell

cycle (Zavizion et al., 1993).

27

After the 1 or 2 d incubation, the cultures were washed with DPBS, and fresh media
containing growth factor treatments was applied. The thymidine incorporation assay was
performed according to Xie and Haslam (1997). After treatment for 16 h, 5 uCi of
[methyl-3 H] thymidine was added to each well. The cultures were incubated for 2 h
more, and then assayed for [methyl-3 H] thymidine incorporation into DNA that occurs
only during the S phase of the cell cycle. To assay for incorporated [methyl-3H]
thymidine, cells were washed with ice cold HBSS, 10% trichloroaceitic acid, and 90%
ethanol. Afier the last wash, lysis buffer containing 0.5 M NaOH and 0.1% Triton X-100
(v/v) was added and the solution containing the cell lysates was neutralized with 5 mM

hydrochloric acid. Radioactivity was measured using a liquid scintillation counter.

Determination of plating efficiency

To determine whether the method used for plating cells was distributing equal amounts of
cells per well, the amount of DNA per well was measured in MAC-T cell cultures. The
method fiom West et a1. (1985) was used to determine total DNA per well for MAC-T
cell cultures. Briefly, MAC-T cells were cultured as above, except that the media used in
this experiment were serum-flee medium (SFM), SF M + 1% fetal bovine serum (F BS),
and SFM + 2% FBS. MAC-T cell cultures were maintained for 66 h before they were
assayed for total DNA per well. This 66 h time point was chosen because it is at this
point and afterwards that the mitogenic activity of IGF-I was measured by the thymidine

incorporation assay.

28

After 66 h of culture, the medium was aspirated and cells were washed with ice cold
DPBS. Cultures were then incubated with 10 mM EDTA (pH 12.3) until all cells were
lysed. The EDTA solution was then neutralized with l M KH2P04 and sonicated.
Aliquots of each well were added to a microtiter plate and 33258 Hoescht dye dissolved
in 100 mM NaCl and 10 mM Tris (pH 7.0) was added to each aliquot. Aliquots were read
on a fluorescent plate reader. Total DNA per well was determined using a standard curve

made from calf thymus DNA.

Preparation of growth factor treatments

Treatments containing growth factors or binding proteins were prepared 30 min prior to
use. Lypohilized growth factors were solubilized in 10 mM hydrochloric acid and diluted
with serum-free medium to the desired concentration. Medium containing growth factor

treatments was applied to cells directly after dilution.

Experimental design

Experiment 1: The objective of this experiment was to measure the response of MAC-T
cell cultures to different levels of IGF-I. The levels of IGF—I chosen were 0 ng/ml, 0.001
ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml. The MAC-T cell’s
response to these levels of IGF-I was measured in cpm [methyl-3 H] thymidine
incorporated/well. The responses from nine wells per treatment were assayed. This

experiment was repeated three times.

29

Experiment 2: The objective of this experiment was to assess the impact of endogenous
lGFBPs on the mitogenic response of MAC-T cell cultures to IGF-I-stimulation. Des (l -
3) IGF-l molecule is a truncated IGF analog with decreased affinity for the IGF BPS (Ross
et al., 1989). The use of des (1 -3) IGF-I provided an indirect assessment of the effect of

IGFBP on the mitogenic activity of IGF-I.

To measure the effects of IGF-I vs. des (1-3) IGF-I on MAC-T cell proliferation, IGF-1
and des (1-3) IGF-I were added to MAC-T cell cultures. Each of the IGFs was added at
the following concentrations: 0.1ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml. The MAC-T
cell’s response to these levels of IGF-1 was measured in cpm [methyl-3 H] thymidine
incorporated/well. The responses from nine wells per treatment were assayed. This

experiment was repeated five times.

Experiment 3: The objective of this experiment was to measure the effect of IGFBP-2 on
MAC-T cell cultures with and without a stimulatory concentration of IGF-I. The
concentrations of IGF-I were 1 ng/ml and 10 ng/ml. These concentrations were chosen
because the response to 1 ng/ml was half-maximal, and 10 ng/ml was maximal in this
system. The concentrations of IGFBP-2 that were chosen were 10 ng/ml and 100 ng/ml.
These concentrations were chosen because the addition of 100 ng/ml IGFBP-2 (3.2 nM
IGFBP-2) was in molar excess of the concentrations of IGF-I tested, and 10 ng/ml
IGFBP-2 (0.32 nM IGFBP-2) was in molar excess of 1 ng/ml IGF-I (0.13 nM IGF-I) but
not in excess of 10 ng/ml of IGF-1 (1.3 nM IGF-I). Thus the two concentrations of
IGFBP-2 were chosen to enable us to determine the effects of IGFBP-2 in excess of

concentrations of IGF-I at the concentrations of half-maximal and maximal response.

30

 

Insulin-like growth factor-1 and IGFBP-2 treatments were added to MAC-T cell cultures
in combination and alone. The MAC-T cell’s response to these levels of IGF-I and
IGFBP-2 was measured in cpm [methyl-3 H] thymidine incorporated/well. The responses

from nine wells per treatment were assayed. This experiment was repeated three times.

Statistical analysis

The general linear model procedure (PROC GLM) of SAS (SAS Institute Inc., 1998) was
used in all statistical analyses performed. Data from experiments 1 and 2 were analyzed
using Dunnett’s T-test, comparing IGF treatments to serum-free medium as a control.
Comparisons among treatments in experiment 3 were analyzed as non-orthogonal
contrasts. All data are presented as least square means (LSMEAN S) plus or minus the

standard error of the mean (SEM).

31

Results

Plating efficiency

MAC-T cell cultures were plated in SFM, SFM supplemented with 1% FBS (v/v), and
SFM supplemented with 2% FBS. Plating efficiency for MAC- T cells in these media
was tested by measuring total DNA in ug DNA/well for nine wells per media treatment.
The average, standard error of the mean, and coefficient of variation for each medium are
reported in Table 2. The coefficient of variation between wells (n=9) was less than 10%
for each type of media tested. This demonstrates that the plating techniques used were

distributing cells evenly across culture wells in my experiments.

Table 2. Comparison of plating efficiency in MAC-T cell cultures in serum free
media (SFM), and SFM supplemented with 1% and 2% fetal bovine serum (FBS).
After 66 h of culture in each media tested, the DNA/well was measured. The
average DNA/well, standard error of the mean (SEM), and coefficient of variation

(C.V.) are reported for culture wells (n=9) grown in the presence of each media.

 

 

Media Average pg DNA SEM C.V.
lwell

SFM 2.65 0.01 8%

1% FBS 2.76 0.01 7%

2% FBS 2.76 0.01 7%

 

 

32

Experiment 1: IGF-I Mitogenic Activity

The effect of adding IGF-I to MAC-T cell cultures in concentrations is shown in Figure
3. Concentrations of IGF-I within the range of 0.01 ng/ml to 100 ng/ml stimulated
proliferation in a dose dependent manner when compared with serum-free media alone (P
< 0.05). Maximal stimulation of proliferation occurred at 10 ng/ml in these experiments.
Concentrations of 1 ng/ml stimulated proliferation at approximately half the maximal

rate.

33

22000

 

 

18000 -
16000 —
14000 «
12000 — c
10000 -

cpmlwell

 

 

     

8000 .
4000 - ' '._’"* if};
2000 - as
o i ’ “ es;

0 0.1 1 10 100

IGF-l (nglml)

Figure 3. The mitogenic effect of IGF-I in MAC-T cell cultures. Concentrations of
IGF-I within the range or 0 to 100 ng/ml were applied to MAC-T cell cultures (n=9)
for 18 h. The mitogenic effect of IGF-I was determined by [methyl- ’H] thymidine
incorporation into DNA during the last 2 h of treatment. The (*) indicates
concentrations of IGF-I that stimulated proliferation significantly when compared

with SFM (p < 0.05).

Experiment 2: Des (1-3) IGF-I Mitogenic Activity

The mitogenic activity of des (1-3) IGF-I, a recombinant analog of IGF-I with a reduced
affinity for IGFBPs (Ballard et al., 1987), was also measured in MAC-T cell cultures
(Figure 4). Des (1-3) IGF-I in concentrations ranging fiom 0.1 ng/ml to 100 ng/ml
stimulated proliferation in a dose dependent manner compared with serum-flee media

alone (P < 0.05). Maximal stimulation of proliferation was observed with 10 ng/ml of

des (1-3) IGF-I. The mitogenic activity of des (1-3) IGF-I was not different fiom that of

IGF-I at any concentration tested (P > 0.05).

25000

 

20000--

15000

10000

cpmlwell

5000 ~

 

 

 

0 OJ 1 10 100
Treatments (nglml)

Figure 4. The mitogenic effects of IGF-I vs. des (1-3) IGF-I in MAC-T cell cultures.
Concentrations of insulin-like growth factor-I (IGF-I) and des (1-3) IGF-I within the
range of 0 to 100 ng/ml were applied to MAC-T cell cultures (n=9) for 18 h. The
mitogenic effects of IGF-I and des (1-3) IGF-I were determined by measuring
[methyl- 3H] thymidine incorporation into DNA during the last 2 h of treatment.
The (*) indicates IGF-I and ties (1-3) IGF-I concentrations that stimulated
Proliferation significantly when compared with serum free media (SFM) (P < 0.05).
The mitogenic activity of des (1-3) IGF-I was not different from that of IGF-I at any

concentration tested (1’ > 0.05).

35

Experiment 3: Inhibition of Mitogenic Activity by IGFBP-2

Effects of IGFBP-2 addition on IGF-I stimulated nritogenic activity in MAC-T cell
cultures are shown in Figure 5. Addition of 10 and 100 ng/ml IGFBP—2 inhibited MAC-T
cell proliferation in the presence and absence of IGF-I (P < 0.0001). This inhibition acted
in a dose dependent manner where 100 ng/ml IGFBP-2 inhibited proliferation to a greater
extent than 10 ng/ml IGFBP-2 (P < 0.0001 ). However, the magnitude of the effect was
greater in the presence of IGF-I (P <0.001). For example, in the absence of IGF-1, 100
ng/ml IGFBP-2 decreased basal cell proliferation by 40% or 6600 cpm/well. Meanwhile,
100 ng/ml IGFBP-2 decreased proliferation of cells treated with 10 ng/ml IGF-I by 51%

or by 17300 cpm/well.

36

40000
350001

 

25°00 ‘ a Idng/TnIEFBP-ZT
20000 J :6 a as; an I 10 nglml IGFBP-2
15000 ~ 3 ‘1 919°39’31'5'53P31

cpmlwell

10000 -
5000 ~ é‘.

 

 

 

 

0 1 10
IGF-l (nglml)

Figure 5. The inhibition of MAC-T cell proliferation by IGFBP-2. Insulin-like
growth factor binding protein -2 (IGFBP-2) was added to MAC-T cell cultures
(n=9) for 18 h with and without stimulatory concentrations of IGF-I. The effect of
IGFBP-2 on MAC-T cell proliferation was determined by [methyl- 3it] thymidine
incorporation into DNA during the last 2 h of treatment. The (a) indicates addition
of IGFBP-2 inhibited cell proliferation (P < 0.0001). The (b) indicates 100 ng/ml

IGFBP-2 inhibited proliferation to a greater extent than 10 ng/ml IGFBP-2 (P <
0.001).

37

Discussion

IGF-I mitogenic activity

Insulin-like growth factor-I is a potent mitogen in MAC-T cell cultures. Insulin-like
growth factor-I stimulates proliferation of MAC-T cells in a dose—dependent fashion.
These results are in agreement with prior studies using MAC-T cells (Woodward et al.,
1994, 1996; Zhao et al., 1992), primary bovine mammary epithelial cells, and mammary
tissue explants (Shamay et al., 1988; Baumrucker and Stemburger, 1989; McGrath et al.,
1991; and Peri et al., 1992). A comparison of these in vitro studies to that of my own
demonstrates my MAC-T model parallels other bovine mammary in vitro models with
respect to the IGF system. Furthermore, use of the MAC-T cell model may aid in
increasing our understanding of the role of the IGF system in bovine mammary growth

and development.

Des (1-3) IGF-I mitogenic activity

Des (1-3) IGF-I is a truncated IGF-l analog characterized by its increased mitogenic
activity when compared to intact IGF-I. Des (1-3) IGF-1’s increased mitogenic activity is
attributed a decreased affinity for IGFBPs (Ross et al., 1989). Evidence in the literature
shows truncated forms of IGF-I stimulate mammary epithelial cell proliferation to either
a similar or greater extent when compared with intact IGF-l (Peri et al., 1992; McGrath et

al., 1991; and Romagnolo et al., 1994). My data show no difference between des (1-3)

38

IGF-I and IGF-1 mitogenic activity in MAC-T cell cultures at the concentrations of IGF

tested.

One possible explanation for the similarity between IGF-I and des (1-3) IGF-I mitogenic
activities is that these lGFs were added at concentrations that were significantly greater
than the concentrations of endogenous IGFBPs. If the [CPS were added at molar
concentrations that were higher than the molar concentrations of the endogenous IGFBPs
then the IGFBPs would likely have a negligible impact on IGF-stimulated proliferation.
This idea is supported by data from Ross et a]. (1989) where higher concentrations of
exogenous IGFs were required to exert biological effects in cultures that secreted
IGFBPs. Therefore, if the endogenous IGFBPs had no effect on IGF action in my
culture system, then addition of IGF-I or des (1-3) IGF-I would produce similar

mitogenic responses as is shown by my data.

Another explanation for the similarities between IGF-I and des (1-3) IGF-l mitogenic
activities is that the IGFBPs produced by the MAC-T cells in this study are not inhibitory
to IGF-stimulated mitogenic activity. The MAC-T cells secrete IGFBP-l, -2, -3, -4, and -
6 in serum-flee media (Cohick and Turner, 1998). However, the effect of the majority of

these IGF BPS on IGF action in MAC-T cell cultures is not known.

39

IGFBP-2 inhibition

My data show for the first time that IGFBP-2 inhibits proliferation of MAC-T cell
cultures. These findings are consistent with other cell culture systems in which
exogenous IGFBPs inhibit IGF-I action (Gopinath et al., 1988; Rivitos et al., 1988; and
McCusker et al., 1991). The inhibitory effect of IGFBP-2 is of interest because IGFBP-2
is synthesized and secreted by bovine mammary tissue and thus may play a role in the
regulation of mammary growth and development (McGrath et al, 1991; Collier et al.,

1993).

My data show that IGFBP-2 inhibits MAC-T cell proliferation both in the presence and
absence of exogenous IGF-I. Though these data might suggest that IGFBP-2 inhibits
MAC-T cell proliferation in a manner that is independent of the presence of IGF-I, the
mechanism involved in IGFBP-2 inhibition, as well as the role of IGF-I in IGFBP-2
inhibition, remain unclear because MAC—T cells secrete low levels of IGF-I (Romagnolo,
1994). Although IGF-I accumulation in conditioned medium was not measured in my
MAC-T cell culture system, Romagnolo measured 21.7 ng IGF-I secreted per 106 cells
after 72 h of culture in a similar MAC-T cell culture system (1994). These data suggest
that the 2 x 104 MAC-T cells/ml in my cultures would have secreted 0.4 ng IGF-I/ml of
media after 72 h of culture. Because my cultures were assayed after only 17 h of culture,
they could have accumulated up to 0.1 ng IGF-I/ml of media. This concentration of IGF-
I has been shown to stimulate cell proliferation in this assay system (see Results of
Experiment 1). Therefore it is possible that in the absence of exogenous IGF-I, addition

of IGFBP-2 inhibited proliferation stimulated by endogenous IGF-I in these MAC-T cell

40

cultures. To determine the mechanism behind IGFBP-2 inhibition of MAC-T cell
cultures, mechanistic studies are required. Furthermore, studies that address the
mechanisms behind IGFBP-2 inhibition of MAC-T cell proliferation will increase our
understanding of the role of IGFBP-2 in the regulation of growth, development, and the

IGF system in the bovine mammary gland.

In our studies, the inhibitory effect of IGFBP-2 on IGF-I-stimulated cell proliferation in
the MAC-T cell assay system may be mediated directly or indirectly (Figure 6). For
example, the binding of IGFBP-2 to IGF-I would inhibit proliferation of MAC-T cells
indirectly because it involves the binding and sequestering of IGF-I (Figure 6a-b).
Meanwhile the binding of IGFBP-2 to a cell surface receptor may directly inhibit
proliferation (Figure 6c). Although our studies did not test the mechanisms behind the
inhibitory effect of IGFBP-2 on IGF-I-stimulated mitogenic activity, several other studies
have investigated the inhibitory effects of exogenous IGFBPs on IGF-I action in vitro.

The results of these studies are discussed below.

41

nunmary Epithelial
Cell

 

 

 

 

 

 

 

 

 

 

KEY:

- IGF-l

IGFBP

¢ IGF type I receptor

‘ IGFBP-2 receptor

 

 

 

 

 

 

 

 

 

Figure 6. The modulation of IGF-I action by IGFBPs. In (A) free IGF-I binds to
the type I IGF receptor on the mammary epithelial cell. In (B) IGFBP binds IGF-I
and decreases the amount of IGF-I binding to the Type I receptor on the mammary
epithelial cell. In (C) IGFBP binds an IGFBP receptor thereby inhibiting IGF

action. Thus, the addition of IGFBPs inhibits IGF-I-stimulated cell proliferation.

42

Several studies have demonstrated that the addition of IGFBPs and IGF-1 to cultured cells
results in decreased IGF-I action due the sequestering of IGF-I by IGFBPs (Gopinath et
al., 1988; Rivitos et al., 1988; and McCusker et al., 1991). In these studies, exogenous
IGFBPs were shown to limit the availability of labeled IGF-I for binding to its cell-
surface receptor. Thus the inhibition of IGF-I action in these cultures was attributed to
binding of IGF-I to IGFBPs in the culture medium which indirectly inhibited IGF-I

action in the target cells.

Another possible mechanism for the inhibitory effects of IGFBPs in cell cultures is the
direct effect of IGFBP-2 on target cells. Oh and coworkers (1993a) demonstrated that
IGFBP-3 inhibited proliferation in cultures of human breast cancer cells by binding to
cell-surface proteins. Furthermore, the addition of IGF-I to these cultures attenuated the
inhibitory effect of IGFBP-3 by forming IGF-I-IGFBP-3 complexes, thereby preventing
cell surface binding of IGFBP-3 (Oh et al., 1993b). This finding implies that in some
cases, IGF BPS act independently of IGF-IGFBP binding and can trigger their own effects

independent of IGF-I on target cells.

Though the ability of IGFBP-2 to bind to cell surfaces is unknown, the genes for IGFBP-
2 does encode an RGD sequence which is suggestive of cell surface binding (Brewer et
al., 1988). Therefore the ability of IGFBP-2 to bind to MAC-T cell surfaces and act
independently of IGF-I binding to inhibit cell proliferation is a potential mechanism for

IGFBP-2 inhibitory action in MAC-T cell cultures.

43

Summary

My hypothesis was that IGFBP-2 would inhibit IGF-I-stimulated proliferation of MAC-T
cells in culture. I found that IGFBP-2 inhibited IGF-I-stirnulated MAC-T cell
proliferation as measured by thymidine incorporation. Addition of IGFBP-2 also
inhibited basal proliferation of MAC-T cells in serum-free media The mechanism by
which IGFBP-‘2 inhibits MAC-T cell proliferation in this target cell system remains
unknown however it may involve IGF-I-dependant or independent mechanisms as

described above.

Future research to elucidate the mechanism behind IGFBP-2’s inhibition of mammary
epithelial cell proliferation may provide insight on the role of IGFBP-2 in the regulation
of mammary growth in the bovine. Increasing our understanding of factors such as
IGFBP-2 that limit mammary epithelial cell proliferation will enable researchers to
develop safe methods to increase proliferation and thereby increase the efficiency and

production of milk.

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45

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