help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sheffield, L. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sheffield, L. G.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*ESTRADIOL
*HYDROCORTISONE
Medline Plus Health Information
*Hormones
Endocrinology Vol. 139, No. 11 4568-4575
Copyright © 1998 by The Endocrine Society

ARTICLES

Hormonal Regulation of Epidermal Growth Factor Receptor Content and Signaling in Bovine Mammary Tissue1

Lewis G. Sheffield

Dairy Science Department, University of Wisconsin, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: Dr. Lewis G. Sheffield, Dairy Science Department, University of Wisconsin, 1675 Observatory Drive, Madison, Wisconsin 53706.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammary tissue from midpregnant heifers was cultured with epidermal growth factor (EGF) or transforming growth factor {alpha} for 1–3 days. After 1 day, 10 nM EGF or transforming growth factor {alpha} doubled DNA synthesis, whereas lower concentrations (0.1 or 1 nM) increased DNA synthesis 2- to 3-fold after 2–3 days in culture. In other studies, bovine mammary tissue was transplanted to ovariectomized athymic mice and treated for 10 days with saline, estradiol (1 µg/day), progesterone (1 mg/day), or estradiol + progesterone. Subsequent explant culture of the bovine tissue indicated that estradiol + progesterone augmented the ability of EGF to stimulate DNA synthesis. The increased response to EGF was associated with increased EGF binding and with increased EGF-induced tyrosine kinase that paralleled the increased EGF binding. In other studies, athymic mice bearing xenografted bovine mammary tissue were primed for 10 days with estradiol and progesterone, followed by 2-day treatment with saline (control), hydrocortisone (200 µg/day), PRL (1 mg/day), or hydrocortisone + PRL. Hydrocortisone and PRL alone decreased, and PRL + hydrocortisone eliminated, EGF-induced DNA synthesis. EGF receptor content was unaffected by hydrocortisone but was reduced by PRL or hydrocortisone + PRL. Furthermore, the ability of EGF to induce tyrosine kinase activity was decreased by PRL and by hydrocortisone + PRL. The decreased kinase activity was greater than the decrease in receptor binding, suggesting a specific modulation of EGF receptor kinase activity in response to lactogenic hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIDERMAL growth factor (EGF) has been shown to stimulate DNA synthesis and growth of a variety of tissues and cell types, including mammary gland (1, 2, 3, 4, 5). The homolog transforming growth factor-{alpha} (TGF-{alpha}), which binds to the same receptor as EGF (6, 7), also stimulates mammary epithelial proliferation (8, 9). Receptors for EGF are in the type I tyrosine kinase family (10) and have been identified in normal mammary tissue (11). Receptor population is dependent upon physiological state, being highest in peripuberal animals, decreasing in mature virgins, increasing again in early to midpregnancy, and decreasing during late pregnancy and lactation (12). In addition, mammary EGF receptor has been shown to increase in response to ovarian steroid hormones (estrogen and progesterone). EGF-induced proliferation of mammary epithelium has been shown to be increased by estrogen or estrogen + progesterone (13), and these hormones also increase EGF binding in mammary tissue (13, 14). However, the extent to which changes in EGF responses reflect changes in receptor binding, as opposed to changes in receptor tyrosine kinase activity or other factors, is unclear.

Currently, little is known concerning the mammogenic role of EGF and related factors in ruminants. EGF receptor is present in bovine mammary tissue (15). EGF and TGF-{alpha} have been shown to increase DNA synthesis in culture bovine mammary epithelial cells (16, 17). TGF-{alpha} messenger RNA (mRNA) has been demonstrated in bovine mammary tissue (18, 19). EGF mRNA has also been demonstrated in lactating bovine mammary tissue (20). However, the role that these factors play in bovine mammogenesis and how they interact with other factors remains unclear. Therefore, the objective of this study was to further examine the interactions between EGF receptor agonists and other hormones in inducing DNA synthesis and modulating EGF receptor number and function in bovine mammary tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue
All animal studies, including cow and mouse use, were approved by the local IACUC. Tissue was collected from midpregnant (4–6 months) Holstein heifers by surgical biopsy, as previously described (21). Briefly, heifers were sedated with Rompun and anesthetized with sodium thyamyl, the udder shaved, and an incision made about midway between teats and the base of the udder. Approximately 5 g of tissue was removed and placed in 100 ml HBSS containing penicillin (100 U/ml) and streptomycin (100 µg/ml). The resulting cavity was filled with absorbable gelatin, and the incision was sutured closed.

Explant culture
Tissue was transported to the laboratory (about 5 min) and cut into 0.5-mm-thick slices with a Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro, NJ). Connective tissue and fat were dissected away and the remaining tissue cut into pieces approximately 3 mm2. Tissue was placed on Dacron rafts and placed in Eagle’s MEM supplemented with nonessential amino acids, penicillin (100 U/ml) and streptomycin (100 µg/ml). Tissue was cultured at 37 C in an atmosphere of 5% CO2-95% air with 100% humidity. Tissue was treated with EGF (Harlan Bioproducts, Madison, WI) or TGF-{alpha} (UBI, Lake Placid, NY) (0, 0.1, 1, or 10 nM), and DNA synthesis was estimated, as described below.

DNA synthesis
To assess DNA synthesis, tissue was incubated, for the final 6 h of culture, with media containing 1 µCi/ml 3H-thymidine (DuPont, Boston, MA). Tissue was homogenized, and proteins and nucleic acids were precipitated with 10% (final concentration) trichloroacetic acid. Lipids were extracted by sequential washes with methanol:chloroform (2:1; 3 times) and ethanol (2 times). Tissue was then digested with 5% perchloric acid (70 C, 45 min), an aliquot neutralized, and counted by liquid scintillation. The remainder was used for DNA assay (22).

To determine cell types responding to EGF, autoradiography was performed. Tissue was incubated with 3H-thymidine, as described above; fixed in phosphate buffered formalin; embedded in paraffin; cut into 5-µm sections; and affixed to slides. Slides were dipped in photographic emulsion (Ilford K-5, Polysciences, Warington, PA), exposed at 5 C for 2 weeks, developed with Kodak D-19 (Eastman Kodak Co., Rochester, NY), and counterstained with hematoxylin. Every sixth section was evaluated under high magnification, and the entire section was examined for labeling. Cells with 10 or more silver grains over the nucleus were considered labeled, and percentage of epithelial cells labeled was determined as an index of DNA synthesis (labeling index).

Athymic nude mouse studies
Athymic nude mice (Harlan-Sprague-Dawley) were maintained under aseptic conditions in a small animal isolator (CCI Equipment, Mattion, OH). All food, water, bedding, and cages were autoclaved before use. At approximately 5 weeks of age, mice were ovariectomized; and 10 pieces of bovine mammary tissue (0.5 x 3–5 mm2) were placed sc via the same dorsal incision used for ovariectomy (24–32 mice per heifer). Mice were allowed 2 weeks recovery before beginning experiments described below.

For mammogenesis studies, mice were injected once daily for 10 days with either saline (150 mM NaCl containing 1 mg/ml gum arabic), estradiol (1 µg/day), progesterone (1 mg/day), or estradiol + progesterone (3–4 mice per cow per treatment group). After the last injection, mice were euthanized by pentobarbitol overdose, and xenografted bovine mammary tissue was removed. Tissue from one mouse was placed in culture with 0, 0.1, or 1 nM EGF for 2 days, and DNA synthesis was estimated as described above. Remaining tissue was pooled and used to assess EGF binding and EGF-induced tyrosine kinase activity, as described below.

For lactogenesis studies, mice were injected for 10 days with estradiol + progesterone, followed by 2 days injection (2 injections per day) with saline, hydrocortisone (100 µg/injection, 2 injections/day), PRL (500 µg/injection, 2 injections/day), or hydrocortisone + PRL (3–4 mice per cow per treatment group). At the end of the injection period, mice were killed, and bovine tissue was removed. Tissue from one mouse was placed in tissue culture with 0, 0.1, or 1 nM EGF for 2 days, and DNA synthesis was estimated as described above. Remaining tissue was used to estimate EGF binding and receptor kinase activity, as described below.

EGF binding
Tissues were homogenized in lysis buffer (50 mM HEPES, pH 7.5, containing 10 mM MgCl2, 0.25 M sucrose, 1 mM Na3VO4, 2 mM MnCl2, 50 mM NaF, 20 mM sodium pyrophosphate, and 1 mM phenylmethlysulfonylflouride) and were centrifuged at 2,000 x g to remove nuclei and intact cells. Supernatant was then centrifuged at 50,000 x g for 1 h, and membranes were dissolved in lysis buffer containing 0.1% Triton X-100. Protein content was determined by the method of Bradford (23). Proteins (100 µg) were added to microcentrifuge tubes along with various concentrations of 125I-EGF. Receptor grade EGF (UBI) was labeled with 125I (DuPont) using the Iodogen method (Pierce Chemical Co., Rockford, IL). After 4 h incubation at 37 C (shown optimum in initial studies), receptors were precipitated by adding 2 mg/ml BSA and 150 mg/ml polyethylene glycol 6000, incubating on ice for 5 min and centrifuging (15,000 x g for 5 min). Resulting pellets were counted and receptor numbers and affinities determined using the Ligand program (Biosoft, Fergeson, MO). In initial studies using tissue from midpregnant heifers, linearity over amount of membrane material used and optimum time of binding was determined. In these studies, ligand binding was determined using EGF concentrations (.001–100 nM). Because of limited material available in the athymic mouse experiments, 2–3 concentrations of 125I-EGF (.3–3 nM) were used in those studies. Nonspecific binding was estimated by adding a 100-fold excess of unlabeled EGF.

Tyrosine kinase activity
Tyrosine kinase activity of isolated membranes was determined as described previously (24), with minor modifications described below. Solubilized cell membranes (50–100 µg protein in 100 µl buffer) were incubated with or without 10 nM EGF (20 µl of a 75-nM solution or other doses, as indicated in text) and with or without 5 mM [Val5]-angiotensin II (20 µl of a 37.5-mM solution). Reactions were started by the addition of 10 µl of 100 µM {gamma}32P-ATP (DuPont, specific activity of approximately 100 Ci/mmol) and continued for 5 min. Reactions were stopped by adding 150 µl cold 10% trichloroacetic acid, followed by addition of 10 µl of a 20-mg/ml BSA solution to aid precipitation. After 5 min on ice, samples were centrifuged (14,000 x g for 5 min) and supernatant spotted onto P81 phosphocellulose paper. Paper was washed with phosphoric acid, dried, and counted by liquid scintillation. In preliminary studies, assays were found to be linear over time and amount of enzyme used. ATP and substrate concentrations used were found to give maximum activity.

To verify that assays were dependent on added peptide, experiments were also conducted in which the substrate peptide was omitted or in which a peptide lacking tyrosine ([Phe4]-angiotensin II) was used. To verify that the observed responses were dependent on EGF receptor, EGF receptor was immunoprecipitated from solubilized membranes by adding 50 µg sheep anti-EGF receptor (UBI) or sheep IgG (Sigma Chemical Co., St. Louis, MO) followed by agarose-bound protein A and G (Oncogene Science, Inc., Manhasset, NY). After rocking at room temperature for 2 h, agarose beads were removed by centrifugation (2000 x g for 2 min), and the immunoprecipitation was repeated. Western blot analysis indicated that this procedure reduced EGF receptor content of preparations to undetectable levels. Supernatant was then used for kinase assays.

Statistical analysis
Data were analyzed, by ANOVA, as a randomized complete block design, with heifers considered random block effects and treatments considered fixed effects. Dunnett’s t test was used to compare various treatments with controls (25, 26). Unless otherwise stated, statements of difference indicate P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Studies on freshly removed tissue
In preliminary studies on tissue from four heifers, EGF or TGF-{alpha} increased DNA synthesis. After 1 day in culture, the increase was modest and was only observed with 10 nM of the growth factor. After 2 days in culture, as little as 0.1 nM increased DNA synthesis. Response to 1 or 10 nM EGF or TGF-{alpha} was an approximately 3-fold increase in DNA synthesis. After 3 days in culture, responses were similar to those observed after 2 days in culture (Fig. 1Go).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Effect of EGF and TGF-{alpha} on DNA synthesis of bovine mammary tissue from midpregnant heifers. Tissue from midpregnant heifers was cultured for 1, 2, or 3 days in the presence of the indicated concentration of EGF or TGF-{alpha}, and DNA synthesis was estimated as described in Materials and Methods (mean of four heifers, each in triplicate). SEM, not shown (for simplicity), averaged ± 11%. *, Significantly different from control (0 growth factor) within day of culture, P < 0.05.

 
Autoradiographic studies, performed on the same six heifers as used in athymic mouse studies, indicated that the majority of DNA synthesis (about 80%) occurred in epithelial structures. Autoradiographic analysis of percent S-phase epithelial cells followed a pattern similar to that of total DNA synthesis (Fig. 2Go). A significant increase in S-phase cells (approximately double basal level) was seen with 0.1 nM EGF or TGF-{alpha}. Maximum S-phase cells, occurring with 1 nM growth factor, was approximately 3 times basal level. The majority (80% average over all treatments) of S-phase cells were epithelial (not shown).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Autoradiographic analysis of EGF-induced DNA synthesis in bovine mammary tissue after 2 days in culture. Tissue was removed from midpregnant heifers and placed in culture with the indicated concentration of EGF or TGF-{alpha} for 2 days; and autoradiographic estimation of percent S-phase cells was performed as described in Materials and Methods (mean ± SEM of tissue from six heifers, each in triplicate). *, Significantly different from control (0 growth factor), P < 0.05.

 
Scatchard analysis of EGF binding in tissue from 6 midpregnant heifers indicated the presence of a single affinity class of receptors (Fig. 3Go). Average dissociation constant (Kd) of the receptors was 0.81 ± 0.09 nM. Average maximum binding was 55 ± 6 fmol/mg membrane protein. Receptor binding was linear over amount of membrane protein added. Optimum binding was obtained after 2 h incubation at 37 C and was stable for at least 12 h (not shown). Because the Scatchard analysis was linear under the conditions used, subsequent studies with the limited membrane material from tissue grafted to athymic mice were performed using 2–3 EGF concentrations (0.3–3 nM) centered around the affinity of the receptor.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Scatchard analysis of EGF binding to bovine mammary tissue membranes. Tissue from midpregnant heifers was homogenized, membranes prepared, and 125I-EGF binding assessed as described in Materials and Methods. The graph shows one representative heifer. Mean ± SEM of six heifers was 55 ± 6 fmol binding/mg protein, and Kd was 0.81 ± 0.09 nM.

 
EGF was capable of inducing phosphorylation of [Val5]-angiotensin-II in solubilized bovine mammary membranes. A measurable background activity was observed, probably caused by other kinases present in the membrane preparations, but this was low, compared with EGF-stimulated phosphorylation, and the EGF effect was readily measured (Fig. 4Go). Angiotensin phosphorylation was substantially increased, in response to EGF, presumably caused by EGF-induced activation of the EGF receptor kinase. Dose responses to EGF indicated that significant kinase activity was induced by 0.1 nM EGF. The response to 1 nM EGF was approximately half-maximum, whereas a maximal response was observed at approximately 10 nM EGF, with relatively little increase beyond that concentration (Fig. 4Go). EGF- induced phosphorylation was dependent on added peptide substrate (Fig. 5Go, top) and was not observed if peptide lacking tyrosine was used. Furthermore, the EGF-induced phosphorylation was eliminated by immunoprecipitation of EGF receptor (Fig. 5Go, bottom), suggesting that the responses were EGF receptor dependent.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 4. EGF-induced tyrosine kinase activity in bovine mammary tissue. Membranes from midpregnant bovine mammary tissue were prepared, and EGF-induced [Val5]-angiotensin-II phosphorylation determined as described in Materials and Methods (mean ± SEM of six heifers). *, Significantly different from control (0 EGF), P < 0.05.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Specificity of EGF-induced kinase activity. Top, Basal and tyrosine kinase activity, in the absence or presence of 10 nM EGF, was assessed in soluble membrane fractions as described in Materials and Methods using either no peptide substrate (None), [Val5]-angiotensin II (Val-5, 5 mM), or [Phe4]-Angiotensin II (Phe-4, 5 mM) (mean ± SEM of tissue from six heifers). *, Significantly different from control (0 EGF), P < 0.05. Bottom, EGF receptor was immunoprecipitated from soluble membrane fractions, or sham immunoprecipitation was performed with sheep IgG and kinase activity (in the absence or presence of 10 nM EGF), assessed as described in Materials and Methods (mean ± SEM of tissue from six heifers). *, Significantly different from control (0 EGF), P < 0.05.

 
Mammogenesis studies
After transplantation to ovariectomized athymic mice, mammary tissue maintained the ability to initiate DNA synthesis in response to EGF. EGF-induced DNA synthesis in saline-treated mice (Fig. 1Go) was about 1.9 times control level (1 nM EGF vs. 0 EGF), which was less than the approximate 3-fold increase observed in freshly excised tissue (see Fig. 2Go). Furthermore, 0.1 nM EGF had no effect on DNA synthesis in tissue maintained in saline-treated mice, whereas this concentration of EGF approximately doubled DNA synthesis in freshly excised mammary tissue (Fig. 6Go). Progesterone alone resulted in DNA synthesis and EGF-induced DNA synthesis that were not significantly different from those in the saline-treated group. Treatment with estradiol alone resulted in 1 nM EGF inducing an approximately 2-fold increase in DNA synthesis and a 1.7-fold increase with 0.1 nM EGF. Estradiol and progesterone treatment for 10 days resulted in a 2.7-fold increase in S-phase cells, in response to 1 nM EGF, and a 2-fold increase in response to 0.1 nM EGF. These results suggest that a combination of estradiol and progesterone treatment of athymic mice is required to maintain EGF responses similar to those observed in tissue freshly excised from midpregnant heifers.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. Effect of estrogen and progesterone on EGF-induced DNA synthesis of bovine mammary tissue maintained in athymic nude mice. Mice were treated with either saline (C), estradiol (E), progesterone (P), or E + P for 10 days. Xenografted bovine tissue was removed and cultured for 2 days with indicated concentration of EGF, then DNA synthesis was estimated as described in Materials and Methods (mean of six heifers, each in triplicate). SEM, not shown (for simplicity), averaged ± 9%. *, Significantly different from 0 EGF within mouse treatment, P < 0.05.

 
EGF receptor content of bovine mammary tissue grafted to saline-treated athymic mice was approximately half the levels of freshly-excised mammary tissue (Fig. 7Go). EGF binding was increased about 50% by estradiol but was unaffected by progesterone. The combination of estradiol + progesterone resulted in an approximate doubling of EGF binding, to levels similar to that observed in freshly-excised tissue.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. Effect of estrogen and progesterone on EGF binding by bovine mammary tissue maintained in athymic nude mice. Mice were treated with either saline (control), estradiol (E), progesterone (P) or E + P for 10 days. Bovine mammary tissue was removed and EGF binding estimated as described in Materials and Methods (mean ± SEM of six heifers). *, Significantly different from control (C), P < 0.05.

 
EGF-induced kinase activity of bovine mammary tissue was detectable in all treatment groups. Kinase activity was parallel to EGF binding in tissue treated with saline, estradiol, progesterone, or estradiol + progesterone. When expressed per unit EGF binding, activity per receptor did not vary significantly among treatments (Fig. 8Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 8. Effect of estrogen and progesterone on EGF-induced tyrosine kinase activity of bovine mammary tissue maintained in athymic nude mice. Mice were treated with either saline (control), estradiol (E), progesterone (P), or E + P for 10 days. Bovine mammary tissue was removed and tyrosine kinase activity estimated as described in Materials and Methods (mean ± SEM of six heifers). *, Significantly different from control (C), P < 0.05.

 
Lactogenesis studies
In mice primed with ovarian steroids, 2 days treatment with saline resulted in basal and EGF-induced DNA synthesis similar to that observed after 10 days treatment with estradiol and progesterone or in freshly excised tissue. Treatment with hydrocortisone or with PRL alone decreased, but did not eliminate, EGF-induced DNA synthesis. Treatment with hydrocortisone + PRL essentially abolished EGF- induced DNA synthesis in bovine mammary tissue (Fig. 9Go).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 9. Effect of hydrocortisone and PRL on EGF-induced DNA synthesis of bovine mammary tissue maintained in athymic nude mice. Mice were primed for 10 days with E + P, then treated for 2 days with saline (control), hydrocortisone (HC, 100 µg/injection, 2 injections per day), PRL (Prl, 500 µg/injection, 2 injections per day), or HC + Prl. Xenografted bovine tissue was removed and cultured for 2 days with indicated concentration of EGF, then DNA synthesis was estimated as described in Materials and Methods. SEM, not shown (for simplicity), averaged ± 11% of indicated values. *, Significantly different from 0 EGF within mouse treatment, P < 0.05.

 
In tissue primed with estradiol + progesterone, hydrocortisone alone had no effect on EGF receptor content of bovine mammary tissue, relative to saline-treated control. In each case, receptor content was similar to that observed in freshly-excised tissue. PRL treatment decreased EGF binding to approximately 60% of that observed in controls, whereas hydrocortisone + PRL decreased EGF receptor content of bovine mammary tissue to less than half that observed in controls or in freshly-excised tissue from midpregnant heifers (Fig. 10Go). In all of these treatments, there was no evidence of a change in receptor affinity (not shown).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 10. Effect of hydrocortisone and PRL on EGF binding by bovine mammary tissue maintained in athymic nude mice. Mice were primed with estradiol + progesterone for 10 days and then were treated with either saline (control), hydrocortisone (HC), PRL (Prl), or HC + Prl for 2 days. Bovine mammary tissue was removed and EGF binding estimated as described in Materials and Methods (mean ± SEM of six heifers). *, Significantly different from control (C), P < 0.05.

 
Unlike the mammogenesis study, in which EGF receptor kinase activity per receptor was similar among treatments, treatment with lactogenic hormones did alter receptor kinase activity per receptor. In saline-treated or hydrocortisone-treated groups, receptor kinase activity, expressed per unit receptor binding, was similar to that observed in freshly excised tissue and in the mammogenesis studies. Treatment with PRL decreased EGF-induced tyrosine kinase to levels that were less than half that of saline-treated controls. The combination of hydrocortisone + PRL resulted in EGF receptor kinase activity that was less than 20% of control values (Fig. 11Go).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 11. Effect of hydrocortisone and PRL on EGF-induced tyrosine kinase activity of bovine mammary tissue maintained in athymic nude mice. Mice were primed with estradiol + progesterone for 10 days and then treated with either saline (control), hydrocortisone (HC), PRL (Prl), or HC + Prl for 2 days. Bovine mammary tissue was removed and tyrosine kinase activity estimated as described in Materials and Methods (mean ± SEM of six heifers. *, Significantly different from control (C), P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous results indicate that the bovine mammary gland has receptors for EGF (15) and that EGF, or its homolog TGF-{alpha}, is a potential mammogen for bovine mammary tissue (16, 17). Results of the present study indicate that bovine mammary tissue is capable of responding to EGF and TGF-{alpha}. In this respect, bovine mammary tissue seems similar to murine mammary tissue, which is known to respond to EGF under a variety of conditions (2, 3, 4, 5).

EGF binding to bovine mammary membranes indicated a single affinity class similar to that previously observed in bovine mammary tissue (15). This is in contrast to studies on mouse mammary tissue, which generally show the presence of two affinity classes (12). Although EGF binding was somewhat higher than previously reported for mouse mammary tissue (12), we observed lower levels of EGF binding than was observed by Spitzer and Grosse (15).

In addition to measuring EGF binding, we were also able to detect EGF-induced tyrosine kinase activity in bovine mammary tissue. Basal tyrosine kinase activity was detectable in all samples, which was probably because of the presence of other tyrosine kinases in samples. However, the EGF-induced kinase activity was sufficient to allow ready detection above this background. The EGF induction of tyrosine kinase followed a dose response pattern similar to that of EGF binding, with half-maximum EGF-stimulated tyrosine phosphorylation occurring near the Kd of the receptor. However, the tyrosine kinase dose response was substantially different from the response of DNA synthesis, with maximum DNA synthesis occurring at approximately 1 nM EGF. At this concentration, EGF receptor would seem to be only about half activated (based on Kd of slightly less than 1 nM and on EGF-induced tyrosine kinase activity). This suggests that maximum DNA synthesis in bovine mammary tissue does not require maximum activation of the EGF receptor.

The kinase assay used in these studies seemed to be measuring tyrosine phosphorylation of substrate peptide, because omission of the peptide or use of a peptide lacking tyrosine resulted in minimal phosphorylation. Furthermore, removal of EGF receptor from the soluble membrane preparations, by immunoprecipitation, eliminated EGF-induced tyrosine kinase activity. These results suggest that the assay measured EGF receptor-mediated tyrosine kinase activity.

The present study used athymic nude mice as a model to assess the effects of various hormone combinations on EGF receptor signaling in bovine mammary tissue. Previously, bovine mammary tissue has been shown to be readily maintained as sc xenografts in athymic mice (27, 28, 29, 30, 31). Bovine mammary tissue, maintained in athymic mice, can undergo hormone-induced growth in response to a variety of hormones, including estradiol, progesterone, PRL, GH, placental lactogen, and cholera toxin. Furthermore, hydrocortisone and PRL induce milk protein production in xenografted bovine mammary tissue, particularly after estrogen + progesterone priming.

The present study indicated that estrogen + progesterone, previously shown to increase growth of bovine mammary tissue in athymic mice and in vivo (28, 30, 32), also increases EGF binding and EGF-induced DNA synthesis in bovine mammary tissue. This result seems similar to previous studies in mice (13) in which EGF receptor and EGF-induced mammary growth is modulated by ovarian steroid hormones. This seems to mimic effects seen during pregnancy. In mice, mammary EGF receptor is higher during early pregnancy (12).

Although changes in EGF-induced DNA synthesis in response to ovarian steroid hormones estradiol and progesterone are associated with changes in EGF receptor number in this and other studies (13, 33), it is unclear whether changes in EGF receptor number can account for observed differences, or if receptor signaling pathways are also modified. Because the EGF receptor is a tyrosine kinase, and ligand-induced tyrosine kinase activity is thought to be critical for many receptor functions (10, 34), we examined hormonal regulation of EGF-induced tyrosine kinase activity in bovine mammary tissue membranes. Results of these studies indicated that EGF-induced tyrosine kinase activity increased essentially in parallel with changes in receptor number. These results would indicate that estrogen and progesterone do not alter receptor kinase activity but increase receptor number. However, whether these hormones alter signaling downstream of the receptor kinase is unknown at this time.

An interesting observation, in the present study, was that PRL treatment, particularly in combination with hydrocortisone, decreased EGF-induced DNA synthesis of bovine mammary tissue. Previously, PRL, particularly in combination with estradiol and progesterone, was shown to increase DNA synthesis in bovine mammary tissue maintained in athymic mice (35), although the effect was small in the absence of ovarian steroid hormones. In the present study, the effect of PRL on basal DNA synthesis (after estrogen and progesterone withdrawal in vivo and a 2-day in vitro culture period) was small and not significant. However, the in vivo PRL treatment reduced the mitogenic actions of EGF, particularly in the presence of hydrocortisone. Because the combination of hydrocortisone and PRL has previously been shown to increase milk protein production in bovine mammary tissue grafted to athymic mice and primed with estradiol and progesterone (29, 30), these results suggest that a decline in EGF-induced DNA synthesis may be associated with differentiation of mammary tissue.

Inhibition of EGF-induced DNA synthesis in tissue treated with PRL seems to be, at least partly, reflected by decreased EGF receptor number. This observation would be in accordance with previous reports (12) that EGF receptor numbers in mouse mammary tissue decline during late pregnancy and lactation and that EGF receptor is lower in lactating bovine mammary tissue than in nonlactating tissue (15). PRL has also been shown to decrease EGF receptor numbers and mRNA in murine mammary epithelial cells after approximately 18 h of treatment (36).

Unlike the situation with estradiol and progesterone, the effect of PRL on EGF signaling may not be caused entirely by altered receptor number. EGF-induced kinase activity of bovine mammary tissue treated with PRL was less, on a per-receptor basis, than in control tissues, suggesting that PRL may inhibit not only receptor number but also receptor kinase activity. The inhibition of EGF-induced kinase activity is similar to that previously observed, in our laboratory, in mouse mammary epithelial cell cultures (37). In cell-free systems, the decreased EGF receptor kinase activity is associated with increased threonine phosphorylation of the receptor, and enzymatic removal of threonine phosphorylation restores receptor kinase activity. In cell cultures, PRL-induced threonine phosphorylation of the EGF receptor is, at least partly, dependent on activation of protein kinase C by PRL (38). Whether these same mechanisms are also operative in bovine mammary tissue is uncertain, but this possibility provides a plausible model for our observation that lactogenic hormones decrease EGF-induced signals in bovine mammary tissue.


   Acknowledgments
 
Technical assistance of In-Suh Yuh for care of athymic mice, Linda Kotolski for assistance in receptor binding assays, Dr. Ralph Stauffacher for animal surgery, and James Armbruster and the staff of the Dairy Cattle Center for assistance in obtaining bovine mammary tissue is gratefully acknowledged.


   Footnotes
 
1 This work was supported by the University of Wisconsin College of Agricultural and Life Sciences, United States Department of Agriculture Hatch project WIS 3769, and NIH Grant HD-24094. Back

Received April 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cohen S, Savage Jr CR 1974 Recent studies on the chemistry, and biology of epidermal growth factor. Recent Prog Horm Res 30:551–574
  2. Coleman S, Silberstein GB, Daniel CW 1988 Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor. Dev Biol 127:304–315[CrossRef][Medline]
  3. Imagawa W, Bandyopadhyay GK, Nandi S 1990 Regulation of mammary epithelial cell growth in mice and rats. Endocr Rev 11:494–523[CrossRef][Medline]
  4. Yang J, Guzman R, Richards J, Imagawa W, McCormick K, Nandi S 1980 Growth factor- and cyclic nucleotide-induced proliferation of normal and malignant mammary epithelial cells in primary culture. Endocrinology 107:35–41[Abstract]
  5. Taketani Y, Oka T 1983 Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture. Endocrinology 113:871–877[Abstract]
  6. Massague J 1983 Epidermal growth factor-like transforming growth factor. J Biol Chem 258:13606–13613[Abstract/Free Full Text]
  7. Pike LJ, Marquardt H, Todaro GJ, Gallis B, Casnellie JE, Bornstein, P Krebs EG 1982 Transforming growth factor and epidermal growth factor stimulate the phosphorylation of a synthetic, tyrosine-containing peptide in a similar manner. J Biol Chem 257:14628–14631[Abstract/Free Full Text]
  8. Vonderhaar BK 1987 Local effects of EGF, {alpha}-TGF and EGF-like growth factors on lobulealveolar development of the mouse mammary gland in vivo. J Cell Physiol 132:581–584[CrossRef][Medline]
  9. Snedeker SM, Brown CF, DiAugustine RP 1991 Expression and functional properties of transforming growth factor alpha and epidermal growth factor during mouse mammary gland ductal morphogenesis. Proc Natl Acad Sci USA 88:276–280[Abstract/Free Full Text]
  10. Gill GN 1990 Regulation of EGF receptor expression and function. Mol Reprod Dev 27:46–53[CrossRef][Medline]
  11. Taketani Y, Oka T 1983 Biological action of epidermal growth factor and its functional receptors in normal mammary epithelial cells. Proc Natl Acad Sci USA 80:2647–2650[Abstract/Free Full Text]
  12. Edery M, Pang K, Larson L, Colsi T, Nandi S 1985 Epidermal growth factor receptor levels in mouse mammary glands in various physiological states. Endocrinology 117:405–411[Abstract]
  13. Vonderhaar BK 1985 Hormones and growth factors in mammary gland development. In: Veneziale CM (ed) Control of Cell Growth and Proliferation. Van Norstand Reinhold Co., New York, pp 11–33
  14. Haslam SZ, Counterman LJ, Nummy KA 1992 EGF receptor regulation in normal mouse mammary gland. J Cell Physiol 152:553–557[CrossRef][Medline]
  15. Spitzer E, Grosse R 1987 EGF receptors on plasma membranes purified from bovine mammary gland of lactating and pregnant animals. Biochem Int 14:581–588[Medline]
  16. Peri I, Shamay A, McGrath MF, Collier RJ, Gertler A 1992 Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and des-3-IGF-I in bovine mammary gland in vitro. Cell Biol Int Rep 16:359–368[Medline]
  17. Shamay A, Pines M, Waksman M, Gertler A 1990 Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins. Mol Cell Endocrinol 69:217–226[CrossRef][Medline]
  18. Koff MD, Plaut K 1995 Expression of transforming growth factor-alpha-like messenger ribonucleic acid transcripts in the bovine mammary gland. J Dairy Sci 78:1903–1908[Abstract]
  19. Plath A, Einspanier R, Peters F, Sinowatz F, Schams D 1997 Expression of transforming growth factors alpha and beta-1 messenger RNA in the bovine mammary gland during different stages of development and lactation. J Endocrinol 155:501–511[Abstract]
  20. Sheffield LG 1997 Mastitis increases growth factor messenger ribonucleic acid in bovine mammary glands. J Dairy Sci 80:2020–2024[Abstract]
  21. Sheffield LG, Yuh IS 1988 Influence of epidermal growth factor on growth of bovine mammary tissue in athymic nude mice. Domest Anim Endocrinol 5:141–147[CrossRef][Medline]
  22. Burton KA 1956 A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323[Medline]
  23. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  24. Sheffield LG, Aylsworth CF, Welsch CW 1987 Cyclic nucleotides and protein phosphorylation in mouse mammary glands: effect of estrogen and progesterone administered in vivo. Proc Soc Exp Biol Med 185:283–290[Abstract]
  25. Gill JL 1978 Design and Analysis of Experiments in the Animal and Medical Sciences. Iowa State University Press, Ames, vol 1
  26. Gill JL 1978 Design and Analysis of Experiments in the Animal and Medical Sciences. Iowa State University Press, Ames, vol 2
  27. Welsch CW, McManus MJ, DeHoog JV, Goodman GT, Tucker HA 1979 Hormone-induced growth and lactogenesis of grafts of bovine mammary gland maintained in the athymic "nude" mouse. Cancer Res 39:2046–2050[Medline]
  28. Sheffield LG, Welsch CW 1986 Transplantation of bovine mammary tissue to athymic nude mice: growth subcutaneously and in mammary gland free fat pads. J Dairy Sci 69:1141–1147
  29. Sheffield LG, Eppler CM, Tucker HA, Welsch CW 1988 Influence of recombinant DNA derived bovine growth hormone on {alpha}-lactalbumin production by bovine mammary tissue maintained in athymic nude mice. J Dairy Sci 71:68–74
  30. Sheffield LG, Welsch CW 1988 Cyclic nucleotide concentration and protein kinase activities of bovine mammary tissue maintained in athymic nude mice: effects of mammogenic and lactogenic hormones. J Dairy Sci 71:75–83
  31. Sheffield LG 1989 Influence of cholera toxin (an adenylate cyclase activator) on DNA synthesis of bovine mammary tissue in vitro and in athymic nude mice. J Dairy Sci 72:41–48
  32. Tucker HA 1981 Physiological control of mammary growth, lactogenesis, and lactation. J Dairy Sci 64:1403–1421
  33. Vanderboom RJ, Sheffield LG 1993 Estrogen enhances epidermal growth factor induced DNA synthesis in mammary epithelial cells. J Cell Physiol 156:367–372[CrossRef][Medline]
  34. Gill GN, Chen WS, Lazar CS, Glenney Jr JR, Wiley HS, Ingrahm HA, Rosenfeld MG 1988 Role of intrinsic protein tyrosine kinase in function and metabolism of the epidermal growth factor receptor. Cold Spring Harb Symp Quant Biol 53:467–475
  35. Vega JR, Sheffield LG, Bremel RD 1989 Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice. Proc Soc Exp Biol Med 192:135–139[Abstract]
  36. Fenton SE, Sheffield LG 1993 Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function. Mol Biol Cell 4:773–780[Abstract]
  37. Quijano Jr VJ, Sheffield LG 1998 Prolactin decreases EGF-induced EGF receptor tyrosine phosphorylation via a phosphorylation-dependent mechanism. J Biol Chem 273:1200–1207[Abstract/Free Full Text]
  38. Fenton SE, Sheffield LG 1997 Prolactin inhibits EGF-induced DNA synthesis in mammary epithelium via early signaling mechanisms: possible involvement of protein kinase C. Exp Cell Res 236:285–293[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J DAIRY SCIHome page
R. M. Akers
Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows.
J Dairy Sci, April 1, 2006; 89(4): 1222 - 1234.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
V. C.-L. Lin, E. H. Ng, S. E. Aw, M. G.-K. Tan, E. H.-L. Ng, and B. H. Bay
Progesterone Induces Focal Adhesion in Breast Cancer Cells MDA-MB-231 Transfected with Progesterone Receptor Complementary DNA
Mol. Endocrinol., March 1, 2000; 14(3): 348 - 358.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sheffield, L. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sheffield, L. G.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*ESTRADIOL
*HYDROCORTISONE
Medline Plus Health Information
*Hormones

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals