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Endocrinology Vol. 139, No. 5 2519-2526
Copyright © 1998 by The Endocrine Society

ARTICLES

Mammogenic Hormones Differentially Modulate Keratinocyte Growth Factor (KGF)-Induced Proliferation and KGF Receptor Expression in Cultured Mouse Mammary Gland Epithelium1

Vadim K. Pedchenko and Walter T. Imagawa

Kansas Cancer Institute, Department of Molecular and Integrative Physiology, and Division of Etiology and Prevention of Hormonal Cancers, University of Kansas Medical Center, Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: Walter Imagawa, Ph.D., 1042 Lied Biomedical Research Facility, University of Kansas Medical Center, Kansas City, Kansas 66160-7810.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stromally derived keratinocyte growth factor (KGF) can play an important role in mammary gland development as a mesenchymal/stromal mediator of epithelial growth and morphogenesis. However, the possible coordinate regulation of mammary gland development by mammogenic hormones and KGF is unexplored. In these studies, the direct effect of mammogenic hormones on KGF-mediated mammary epithelial mitogenesis and expression of the KGF receptor was examined using primary mouse mammary epithelium growing in serum-free, collagen gel cell culture. Addition of KGF produced an average 7-fold increase in cell number after 10 days of culture. This effect of KGF was further increased in the presence of PRL (9-fold) or progesterone (P; 15-fold), with the combination of P and PRL (22-fold) producing the strongest synergistic stimulation. Estrogen did not show any additional stimulation of growth either alone or in combination with PRL and/or P. Ribonuclease protection analysis showed that epithelial cells grown in medium supplemented with P, but not PRL or estrogen, exhibited a 10-fold higher steady state level of KGF receptor (KGFR) messenger RNA (mRNA). KGFR expression was not induced by short term P exposure, suggesting an effect on mRNA stability rather than transcriptional activation. Time-course studies showed that an early decrease in the level of KGFR mRNA in basal cultures was significantly reduced by P addition. Measurement of RNA turnover after actinomycin D treatment showed that P increased the t1/2 of KGFR mRNA compared with basal medium. Thus, P and PRL may differentially potentiate the direct mitogenic effect of KGF: P partly by elevation of the level of KGFR mRNA, and PRL principally by intracellular pathways not affecting KGFR expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMARY gland development is regulated by the coordinated action of ovarian and pituitary hormones and, most likely, peptide growth factors produced locally and/or present in the systemic circulation (1). Although many growth factors can potentially affect the growth and differentiation of the mammary epithelium, there is a gap in our understanding of how these growth factors cooperate with the primary physiological regulators of mammary development, the mammogenic hormones, to orchestrate a complex developmental program. The identification of locally synthesized mammary stromal growth factors has prompted speculation that these factors may in part mediate the recognized influence of the stroma on normal and abnormal mammary growth and development (2). Therefore, a thorough understanding of their influence on the mammary epithelium and their interplay with mammogenic hormones is necessary.

Keratinocyte growth factor (KGF or FGF-7) is a member of fibroblast growth factor family (3) that was discovered as a heparin-binding protein secreted by cultured stromal fibroblasts. In epithelial tissues, KGF is produced only by associated stromal cells and targets only the epithelium (4), which expresses the KGF receptor (KGFR), a transmembrane tyrosine kinase encoded by the IIIb splice variant of FGF-2 receptor (FGFR-2) or bek (5, 6, 7). KGF is strongly mitogenic in vitro for a broad range of epithelial cells from different organs, including skin (8), cornea (9), stomach (10), liver (11), prostate (12, 13), ovary (14), lung (15), and mammary gland (16). Analysis of KGF and KGFR expression during embryonic development, including the mammary gland, has provided evidence that KGF is an important mesenchymal mediator of epithelial growth and differentiation (17, 18).

In hormone-responsive tissues, KGF expression can be modulated by systemic hormones. KGF may act as a stromal andromedin in prostate (13, 19, 20) and seminal vesicle (21) or as a progestomedin in uterus (22). KGF is expressed in the mammary stroma of adult mice (23) and humans (24, 25) and changes in KGF expression during postnatal growth of the mouse mammary gland suggest that it may be hormonally regulated (26). We envision that mammogenic hormones can potentially regulate KGF bioactivity by modifying its synthesis in the mammary stroma, altering the sensitivity of the epithelium to KGF by regulation of the level and affinity of the KGFR or by modulation of postreceptor signaling pathways. In this report, we employed a collagen gel culture system permissive for hormone-responsive, three-dimensional epithelial cell growth and morphogenesis under defined conditions (27) to examine the modulation of KGF mitogenesis by mammogenic hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Progesterone (P) and 17ß-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO) and were prepared as 1000-fold stocks in ethanol. Ovine PRL was provided by the National Hormone and Pituitary Program, and bovine insulin and serum albumin (fraction V) were obtained from Sigma. Human recombinant KGF was provided by J. Rubin (NIH) and purchased from Promega (Madison, WI). All cell culture media were obtained from Life Technologies (Grand Island, NY). Collagenase (CLS type 2) was purchased from Worthington Biochemical Co. (Freehold, NJ). Rat tail collagen, solubilized in acetic acid, was prepared as described previously (28).

Cell culture
Epithelial cells were obtained by collagenase digestion of mammary glands from 3-month-old BALB/c virgin mice (Charles River, Wilmington, MA) followed by Percoll gradient centrifugation as described previously (27, 28). Animals were maintained and killed according to NIH guidelines and protocols approved by the institutional animal care and use committee of the University of Kansas Medical Center. Neutralized, isosmotic collagen solution was prepared by mixing collagen with a neutralizing mix composed of 0.34 N NaOH/10 x bicarbonate-free, HBSS (0.9:1, vol/vol) in a ratio of 85:15, respectively. For proliferation experiments, epithelial cells were mixed with neutralized collagen (2–3 x 105 cells/ml), and 0.5 ml collagen was pipetted into each well of 12-well culture plates. Triplicate wells were prepared for each medium combination to be tested. After gelation at room temperature, the collagen gels were overlaid with 0.5 ml culture medium. The basal medium used for cell growth was composed of a 1:1 mixture of Ham’s F-12 and DMEM buffered with 20 mM HEPES and 0.67 g/liter sodium bicarbonate and supplemented with 1 mg/ml BSA V, 10 µg/ml insulin, 100 U/ml soybean trypsin inhibitor, 1 µg/ml {alpha}-tocopherol succinate, and other additives as indicated in the figures. Cells were grown at 37 C in a 2% CO2-98% air atmosphere. Medium was changed every other day.

For determination of cell number, collagen gels containing cells were transferred to 12 x 75-mm glass tubes, acidified with 0.1 vol 25% acetic acid, and incubated at 37 C until the collagen had dissolved. Cells were pelleted by centrifugation at 1500 rpm for 5 min and extracted with 2 ml 70% ethanol overnight, and the cell pellets were dried for fluorometric DNA assay using diaminobenzoic acid and diploid mouse mammary tumor cells as standards (29).

RNA extraction, Northern blotting, and ribonuclease (RNase) protection assay
For RNA extraction, cells were grown in 100-mm culture dishes at 1–2 x 107 cells/dish, and total RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO) after partial dehydration of collagen gels by blotting on Whatman 3MM filter paper (Whatman, Clifton, NJ). RNA integrity was checked by formaldehyde denaturing agarose gel electrophoresis and staining with ethidium bromide. For RNase protection assay, a 148-bp PCR fragment corresponding to exon K (bp 1270–1417) of the mouse bek gene (6) was cloned into pGEM3Zf(-) transcription vector (Promega). After linearization with HindIII, antisense transcripts were synthesized with T7 RNA polymerase using the MAXIscript in vitro transcription kit from Ambion (Austin, TX) and 50 µCi [{alpha}-32P]ribo-UTP (800 Ci/mmol; DuPont New England Nuclear, Wilmington, DE). Full-length probe was recovered after electrophoresis of transcription products on a 5% polyacrylamide-7 M urea-Tris/borate/EDTA gel. A mouse ß-actin probe with a protected fragment size of 245 nucleotides (Ambion) was labeled to 200 times lower specific activity then the KGFR probe by the addition of 100 µM UTP to the transcription reaction and gel purified as described above. It was used as an internal control to monitor the amount and gel loading of RNA. The RNase protection assay was performed with 20 µg total RNA and 80,000 cpm labeled antisense probes for both KGFR and ß-actin using the RPA II kit (Ambion). After RNase digestion, protected fragments were separated on a 5% polyacrylamide-7 M urea-Tris/borate/EDTA gel and exposed to autoradiography film at -70 C with an intensifying screen. Signals from protected fragments were quantified on a Molecular Dynamics Personal Densitometer SI using ImageQuant 4.2 software (Molecular Dynamics, Sunnyvale, CA).

For Northern blot analysis, 15 µg total RNA from cultured mammary epithelial cells were electrophoresed on formaldehyde agarose gels and transferred overnight in 4 x SSC to a Hybond N+ membrane (Amersham, Aylesbury, UK). After methylene blue staining to ensure equal loading and integrity of RNA (30) and alkaline fixation (31), the membrane was prehybridized for 3 h at 60 C in a solution containing 0.2 M Na2HPO4-H3PO4 (pH 7.2), 1% BSA, 45% formamide, 10% dextran sulfate, 7% SDS, 1 mM EDTA, 1 x Denhardt’s solution, and 10 µg/ml denatured salmon sperm DNA. The membrane was hybridized for 16 h in the same solution with 32P-labeled antisense riboprobe for KGFR (107 cpm/ml); washed once for 30 min at room temperature and three times for 30 min at 65 C in 40 mM Na2HPO4-H3PO4 (pH 7.2), 1 mM EDTA, and 1% SDS; and subjected to autoradiography.

Measurement of KGFR RNA stability
Epithelial cells (1–2 x 107/60-mm culture dish) were cultured for 3 days in collagen gels in basal medium with or without 50 ng/ml P. Fresh medium containing 10 µg/ml actinomycin D was then added, and total RNA was isolated at varying time points. The level of KGFR transcripts was determined by RNase protection assay and quantified by densitometry.

Statistical analysis
All data were expressed as the mean ± SEM and were tested for statistical significance (P < 0.05) using paired Student’s t test and single factor ANOVA. When multiple comparisons were performed, the data were analyzed by one-way ANOVA followed by the Student-Newman-Keul or Dunn’s test. Differences were considered significant at P < 0.05 for all comparisons. The number of independent experiments is indicated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-course effect of KGF on growth of mammary epithelial cells
Time-course experiments were performed to examine the effect of KGF on the kinetics of normal mammary epithelial cell growth during 10 days in culture. As shown in Fig. 1AGo, growth stimulation by KGF was observed after a 3- to 4-day lag period. The growth rate was maximal from days 4–8. Under these experimental conditions, a slow decrease in epithelial cell number occurred in serum-free basal medium, possibly as a result of cell damage during tissue dissociation and the absence of growth/viability-promoting factors in the serum-free medium. After 10 days, the cell number was 9 times higher in the presence of KGF compared with basal medium. Besides stimulation of proliferation, KGF also strongly induced stellate outgrowths of epithelial colonies growing within the collagen matrix (Fig. 1BGo).



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  		Figure 1. A, Time course of the effect of KGF on mammary epithelial cell growth. Cells were cultured for various times in collagen gels in basal medium only (•) or in basal medium supplemented with 15 ng/ml KGF ({blacksquare}). Data shown are the mean ± SEM of six independent experiments. *, P < 0.05 compared with cell number in basal medium only (by paired Student’s t test). B, Morphology of mouse mammary epithelial cell colonies in serum-free collagen gel culture after 6 days of growth in basal medium or in the presence of KGF (15 ng/ml). Phase contrast microscopy, x40.

 
Effect of mammogenic hormones and KGF on growth
KGF stimulated epithelial cell proliferation in a dose-dependent manner, with a maximal effect at about 20 ng/ml (Fig. 2Go). Similar dose dependencies were reported for KGF growth stimulation of human keratinocytes (8). This dose dependency resembles closely saturable specific binding of [125I]KGF to the mouse keratinocytes described by Bottaro et al. (32), indicating that the observed effect is modulated by a high affinity KGF receptor. As shown previously, P and PRL are the mammogenic hormones that directly stimulate the growth of mouse mammary epithelial cells cultured within collagen gels in phospholipid-supplemented, serum-free medium (27). In these experiments, optimal concentrations of PRL and P alone stimulated growth by at most 2-fold (P < 0.05, by Student-Newman-Keuls test), but when these optimal concentrations of hormones were combined with KGF, they synergistically stimulated growth, resulting in nonparallel KGF dose-response curves (Fig. 2Go) and a more than additive stimulation (Fig. 3Go). For PRL, P, and P plus PRL, the sums of the mean increase in cell number over basal for hormone(s) alone and KGF alone compared with the observed increase in cell number over the basal value for the combination of hormone(s) and KGF (shown in parentheses) were, respectively, 6.6 x 105 (8.5 x 105), 7 x 105 (14 x 105), and 9.3 x 105 (21.7 x 105). 17ß-Estradiol did not stimulate proliferation or alter KGF mitogenesis alone or when combined with P and/or PRL.



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  		Figure 2. Dose-dependent stimulation of epithelial cell proliferation by KGF in the presence of mammogenic hormones. Mammary epithelial cells were cultured for 10 days in basal medium only (•), 1 µg/ml PRL ({blacksquare}), 50 ng/ml P ({blacktriangleup}), or 0.5 ng/ml E ({blacktriangledown}) with various concentrations of KGF. The results represent the mean ± SEM (n = 6). *, P < 0.05 (by Student-Newman-Keuls test) compared with cell number in basal medium at the same KGF concentration.

 


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  		Figure 3. Growth of mammary epithelial cells in response to different combinations of hormones and KGF. Cells were cultured for 10 days in the presence of different hormones/hormone combinations without or with KGF (15 ng/ml). Hormone concentrations are the same as indicated in Fig. 2Go. Data represent the mean ± SEM (n = 6), and significant differences among groups are shown by different letters (P < 0.05, by Student-Newman-Keuls test).

 
P showed a much stronger synergism than PRL (P < 0.05; data combined from 12 independent experiments). The proliferative effect of P was examined in further experiments in which the concentration of P was varied, whereas KGF was held constant at 20 ng/ml (Fig. 4Go). These data show that KGF plus P at P concentrations above 5 ng/ml clearly stimulated more than additive proliferation (the sum of the effect of KGF alone and that of P alone). Mammogenic hormones did not significantly change the sensitivity of the cells to KGF as would be indicated with a shift in the dose-response curves; the small difference between the P and PRL curves at the lowest concentration of KGF was not statistically significant (P > 0.05, by Student-Newman-Keuls test).



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  		Figure 4. Dose-dependent stimulation of mammary epithelial cell proliferation by P in the presence and absence of KGF. Cells were cultured for 10 days in basal medium, to which were added different concentrations of P with (dark bars) or without KGF (20 ng/ml). Data represent the mean ± SEM (n = 3). Significant differences between groups are indicated by different letters (P < 0.05, by Student-Newman-Keuls test).

 
Effects of mammogenic hormones on KGFR expression by mammary epithelial cells
To investigate the effects of mammogenic hormones on KGFR expression, epithelial cells were cultured for 10 days in basal medium to which mammogenic hormones were added singly or in combination. Northern hybridization analysis (Fig. 5Go) revealed a 4.2-kb transcript [also reported for mouse keratinocytes (5)] that is elevated in the presence of P. Further comparison of hormonal effects by RNase protection assay showed about a 10-fold increase in the steady state level of this transcript in cells grown in the presence of P (Fig. 6Go, A and B). The marked effect on proliferation of the combination of PRL and P was not mirrored in this combination’s effect on KGFR expression; the messenger RNA (mRNA) level was similar to that observed for P alone. Estrogen (E) alone did not affect the level of KGFR transcripts, but in two of six experiments there was a higher level of KGFR mRNA when E was combined with P than when P was administered alone. We suggest that this effect may be related to E’s ability to stimulate P receptors in mammary cells cultured in this system (33, 34) and is more evident when the endogenous P receptor level happens to be low in mammary epithelium isolated after enzymatic digestion of the mammary gland. KGFR expression was specific for epithelial cells, as KGFR mRNA was not detected in parenchyma-free fat pads or primary cultures of mouse mammary fibroblasts (data not shown).



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  		Figure 5. Northern blot analysis of KGFR expression in mouse mammary epithelial cells. A, Fifteen micrograms of total RNA from cells grown for 2 days within collagen gels in either basal (B) or P-supplemented medium were loaded per lane and hybridized with 32P-labeled antisense KGFR probe as described in Materials and Methods. B, Methylene blue staining of nylon membrane after RNA transfer showing the positions of 18S and 28S ribosomal RNA.

 


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  		Figure 6. Effects of hormones on KGFR expression in mammary epithelial cells. A, Cells were cultured for 10 days in serum-free basal medium (B) supplemented with mammogenic hormones as indicated. KGFR expression was analyzed by RNase protection assay as described in Materials and Methods. Actin transcripts were analyzed to control for RNA integrity and loading. The sizes of the protected actin and KGFR fragments are, respectively, 245 and 148 nucleotides. B, Quantitation of KGFR level based on densitometric analysis. The data (mean ± SEM; n = 6) were normalized to the KGFR level in cells grown in basal medium by dividing the value in hormone-treated groups by the basal value. Significant differences (P < 0.05, by Dunn’s test) between groups are shown by different letters.

 
Characterization of the effect of P on KGFR expression
Only P among the mammogenic hormones tested caused a significant change in the level KGFR mRNA. To determine the mechanism of the P effect on KGFR expression, we compared the level of KGFR transcripts in cells cultured for different times in the presence or absence of P (Fig. 7Go). The level of KGFR mRNA was highest in freshly isolated epithelial cells and declined rapidly during the first 2 days of cell culture, but stabilized thereafter. After 6 days in culture, KGFR mRNA levels in basal and P-containing medium were, respectively, 10% and 57% of their original values. Thus, P inclusion in the culture medium reduced the magnitude of the initial decline, resulting in about a 6-fold higher level of steady state mRNA by this time. This decline in KGFR mRNA levels appears to be unrelated to an effect of P on cell viability, as PRL, which is also growth promoting, had a relatively minor effect on the mRNA level. In addition, during this time interval the morphologies of the cultures in basal and P-containing medium were similar.



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  		Figure 7. Effect of P on the level of KGFR mRNA in mouse mammary epithelial cells: time course. Total RNA was extracted from cells grown for various times in basal medium only (•) or supplemented with 50 ng/ml P ({blacksquare}), and 10 µg were used for RNase protection assay. The data (mean ± SEM; n = 4) were normalized to KGFR level in freshly isolated epithelial cells. *, P < 0.05 compared with message level in cells grown in basal medium at the same time point (by paired Student’s t test).

 
As these experiments were performed over a long time course, it was possible that an inductive effect of P on KGFR transcription could be missed. Therefore, experiments spanning shorter time intervals and conducted at the plateau phase of KGFR mRNA were performed to look for such an effect of P. It has been shown that the in vitro proliferative response of mouse mammary epithelial cells to mammogenic hormones is diminished by prior culture in their absence (27), and that the E can induce P receptors in culture (33, 34). Therefore, to determine whether P could elevate KGFR mRNA levels, epithelial cells were cultured for 2 days in either basal or E (2 nM)-supplemented medium (to help maintain P receptors). Cultures were then exposed to P for 4, 12, 24, or 48 h, followed by RNA extraction. In neither medium was there a change in KGFR expression in response to P (data not shown). Additionally, nuclear run-on assays were performed with nuclei isolated from epithelial cells grown for 3 days in medium with or without P. No enhancement by P of the hybridization signal to KGFR riboprobe immobilized on nylon membranes was detected (data not shown). These results are indicative of an absence of transcriptional activation of KGFR by P.

To explore the possibility that the observed difference in KGFR mRNA level between basal and P-treated cultures might be caused by an effect of P on mRNA stability, we determined the half-life of the KGFR mRNA in cell cultures treated with actinomycin D. As shown in Fig. 8Go, A and B, the rate of decay of the KGFR mRNA was slower for cells grown in the presence of P (t1/2, 161 ± 13 min; n = 3; P < 0.05) than for those grown in basal medium (t1/2, 95 ± 11 min; n = 3). Taken together, these results suggest that the P-dependent increase in the KGFR level in mammary epithelial cells is primarily due to the stabilization of KGFR mRNA.



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  		Figure 8. Effect of P on KGFR mRNA stability in mammary epithelial cells. A, Cells were maintained for 3 days, then treated with actinomycin D (10 µg/ml) as described in Materials and Methods, and KGFR expression was analyzed by RNase protection assay. B, Quantitation of KGFR level based on densitometric analysis. The KGFR level was normalized to that in cells before actinomycin D treatment (0 h). The results represents the mean ± SEM for three independent experiments. Curves were generated by single exponential fitting of experimental data (Sigma Plot software). Basal, t1/2 = 96 ± 13; P, t1/2 = 175 ± 15; the t1/2 was calculated from the coefficient of decay. *, P < 0.05 compared with cells maintained in basal medium (by paired Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We began these studies to analyze direct interactions among individual mammogenic hormones and KGF in exploration of the hypothesis that KGF is a component of a local hormonal regulatory network in the mammary gland. In vivo experiments in which KGF was administered to mice (23) or rats (35) suggested that KGF could stimulate mammary epithelial cell proliferation in the presence and absence of the ovary. An ovary-independent effect of KGF on mammary development was shown in ovariectomized mice, in which KGF administration caused ductal dilation and mild ductal hyperplasia, possibly indicating a proliferative effect of KGF independent of comodulation by ovarian steroids. The effect of KGF in vitro confirms that KGF alone can directly stimulate growth. If ovariectomized mice were treated with E and P followed by combined treatment with KGF, then hyperplasia of ductal cells increased, causing ductal thickening (35). Similarly, in ovary-intact female rats, KGF injection caused a massive intraductal hyperplasia (23). These data indicate that E and/or P may synergize with KGF in stimulating ductal proliferation. The contribution of pituitary hormones was not assessed in these in vivo experiments.

In vitro studies were initiated to address questions concerning the roles that individual mammogenic hormones and KGF might play in the regulation of mammary gland development. Our findings suggest that E, P, and PRL may differentially affect KGF-mediated proliferation in the mammary gland. P and PRL, but not E, could synergize with KGF in stimulating mammary epithelial cell proliferation. In addition, P and PRL appear to amplify the epithelial response to KGF via different mechanisms. For example, P was a significantly stronger synergist than PRL. The basis for the synergistic effect of P could be explained by its greater ability than PRL to raise the steady state level of KGFR mRNA by increasing the mRNA half-life and, if KGFR mRNA correlates with protein level, the level of KGFR. Although P can elevate the KGFR mRNA level, we cannot establish that this is the only mechanism for the synergistic effect of P on growth stimulation without being able to specifically block this effect with subsequent monitoring of the proliferative response. We also cannot eliminate the possibility that the effect of P is indirect, perhaps via the stimulation of matrix molecule (type IV collagen, laminin) synthesis. Our working assumption is that the greater decline in KGFR mRNA during culture in basal medium than in P-stimulated cultures reflects the loss of P stimulation present in vivo. A similar effect of P on the level of epidermal growth factor receptor estimated by radioligand binding has been reported in the ductal epithelium of mammary glands of mature mice (36). Unfortunately, the high homology between KGFR and FGFR-2 hampers direct measurement of KGFR protein by specific antibodies, and radioligand binding studies have been hampered due to the presence of cell surface heparin-containing proteoglycans that bind KGF with high affinity (32). A histochemical technique using KGF-Fc fusion proteins to localize and compare the receptor level is another approach (37). Clearly, PRL differs from P in synergizing with KGF without significantly modifying KGFR expression, suggesting that PRL and KGF may activate downstream postreceptor signaling pathways with common intermediates and/or modulate one another’s receptor activities via cross-talk. In addition, the roughly additive proliferative response when P and PRL are added together with KGF is compatible with the interpretation that these two hormones use different mechanisms.

E, alone or in combination with P or PRL, had no effect on proliferation when combined with KGF. These results are in agreement with previous findings that E is not directly mitogenic for mouse mammary epithelial cells in vitro (27). Similarly, E alone did not affect KGFR expression, but in some experiments it could enhance the effect of P, which we hypothesize may be due to P receptor induction. These data suggest that E does not affect KGF-mediated proliferation of normal mammary epithelial cells by directly affecting the responsiveness of the epithelium to KGF. In vivo experiments in mice have shown that E treatment can increase KGF expression in mammary stroma (our unpublished results), indicating the E may affect KGF synthesis in the stroma, whereas P and PRL affect the magnitude of the mitogenic response of the epithelium to KGF. Thus, local stimulation of ductal growth by E in vivo (38) may be due in part to an induction by E of growth factors such as KGF or epidermal growth factor/transforming growth factor-{alpha} in the surrounding stroma, as has been previously discussed (1, 39, 40) and offered as a possible explanation for at least some of the E-directed effects on mammary gland growth. Recently, a transgenic model has been developed in which KGF expression, under the control of the mouse mammary tumor virus promoter, caused a massive pregnancy-dependent ductal hyperplasia (41) as a prelude to the development of tumors. On the basis of our in vitro results, we would predict that this response is regulated by hormones, not only by their effect on KGF expression, but also by a direct synergistic effect of KGF with P and PRL on the proliferative response of the epithelium.

The physiological significance of these findings is intriguing because the predominant effect of KGF overexpression or injection in vivo appears to be a stimulation of the proliferation of ductal cells. KGF cannot induce lobular morphogenesis (23) when administered alone to virgin rats or to E/P-supplemented mice, but there is evidence to suggest that it may affect alveolar differentiation as well. KGF stimulates alveolar cell proliferation if it is administered to male rats that already show some alveolar development, and in pregnant rats stimulates lobular as well as ductal DNA synthesis (23). More recently, a transgenic mouse strain containing a mouse mammary tumor virus-driven dominant negative KGFR lacking the intracellular kinase domain has been generated (42). In this strain, lobuloalveolar development during pregnancy was inhibited, indicating a requirement for KGF. Ductal development was not inhibited, although expression of the transgene was low in virgin animals. As P, PRL, and E are required for lobular development, it is tempting to speculate that KGF, if overexpressed or injected systemically in virgin animals, is able to thwart this hormonally regulated developmental program, resulting in a massive intraductal hyperplasia. It is clear that KGF can target ductal cells, and this activity implies that KGF can potentially stimulate ductal morphogenesis [especially during postpubertal ductal growth (43)] and is consistent with the observation that KGF expression is high in mammary glands from virgin mice but declines by midpregnancy (26) with the concomitant appearance of a lower mol wt transcript (whose significance has yet to be determined) (26). KGF may be required for alveolar development insofar as it may synergize (at physiological levels) with P and/or PRL in the initiation of alveolar development. Further work examining the effect of KGF on the growth of alveolar epithelium and the hormonal induction of differentiation is needed to examine this issue.

In summary, we have demonstrated that P and PRL synergistically potentiate the mitogenic effect of KGF in primary cultures of mouse mammary epithelium. This effect is mediated by epithelial KGFR, whose level of expression is modulated by P. In other experiments we have found expression of KGF in primary culture of mammary stoma (unpublished). Taken together, these data strongly suggest a role for KGF as a paracrine mediator of stromal-epithelial interactions. Thus, the hormonal regulation of the KGF-KGFR axis could play a significant role in the growth and development of mammary gland.


   Acknowledgments
 
The authors thank Drs. Stuart A. Aaronson, Paul Finch, and Jeffrey Rubin for providing riboprobe vectors and KGF.


   Footnotes
 
1 This work was supported by Grant CA-68414–01 from the NIH/NCI. Back

Received October 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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