Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis

The hormonal stimulation of mammary gland morphogenesis is believed to occur through growth factor receptor signaling pathways. To determine the importance of the epidermal growth factor receptor (EGFR) pathway, we examined extracts of inguinal mammary glands from prepubertal and pubertal mice for tyrosine-phosphorylated EGFR and other erbB receptors. Tyrosine phosphorylation of both EGFR and erbB-2 was detected in normal female BALB/c mice at 5-6 weeks of age, but not during the prepubertal stage, e.g., 24 days of age. Treatment of mice with estradiol or epidermal growth factor also stimulated the formation of mammary EGFR/erbB-2 phosphotyrosine. Waved-2 mice, which have impaired EGFR kinase activity, exhibited less mammary development than did wild-type (wt) mice when both were evaluated at 36 days of age. Because EGFR knockout (KO) mice die shortly after birth, glands from the newborns were implanted under the renal capsules of female nude mice. Under these conditions, extensive ductal growth was observed in mammary glands from wt animals; in contrast, glands from EGFR KO mice failed to grow beyond rudimentary structures. Tissue recombinants revealed that the wt fat pad supported the morphogenesis of EGFR KO epithelium, whereas the EGFR KO fat pad did not. Taken together, these data suggest that EGFR is essential for morphogenesis of the mammary ducts and functions during this period of mammary development as a heterodimer with erbB-2 in the mammary stroma.     

A paracrine role for the epithelial progesterone receptor in mammary gland development

Recently generated progesterone receptor (PR)-negative (PR-/-) mice provide an excellent model for dissecting the role of progesterone in the development of the mammary gland during puberty and pregnancy. However, the full extent of the mammary gland defect in these mice caused by the absence of the PR cannot be assessed, because PR-/- mice do not exhibit estrous cycles and fail to become pregnant. To circumvent this difficulty, we have transplanted PR-/- breasts into wild-type mice, and we have demonstrated that the development of the mammary gland in the absence of the PR is arrested at the stage of the simple ductal system found in the young virgin mouse. Mammary transplants lacking the PR in the stromal compartment give rise to normal alveolar growth, whereas transplants containing PR-/- epithelium conserve the abnormal phenotype. Chimeric epithelia in which PR-/- cells are in close vicinity to PR wild-type cells go through complete alveolar development to which the PR-/- cells contribute. Together, these results indicate that progesterone acts by a paracrine mechanism on a subset of mammary epithelial cells to allow for alveolar growth and that expression of the PR is not required in all the cells of the mammary epithelium in order for alveolar development to proceed normally.     

Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development

Parathyroid hormone-related protein (PTHrP) was originally discovered as a tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in normal tissues and growing evidence suggests that it is an important developmental regulatory molecule. We had previously reported that overexpression of PTHrP in the mammary glands of transgenic mice impaired branching morphogenesis during sexual maturity and early pregnancy. We now demonstrate that PTHrP plays a critical role in the epithelial-mesenchymal communications that guide the initial round of branching morphogenesis that occurs during the embryonic development of the mammary gland. We have rescued the PTHrP-knockout mice from neonatal death by transgenic expression of PTHrP targeted to chondrocytes. These rescued mice are devoid of mammary epithelial ducts. We show that disruption of the PTHrP gene leads to a failure of the initial round of branching growth that is responsible for transforming the mammary bud into the rudimentary mammary duct system. In the absence of PTHrP, the mammary epithelial cells degenerate and disappear. The ability of PTHrP to support embryonic mammary development is a function of amino-terminal PTHrP, acting via the PTH/PTHrP receptor, for ablation of the PTH/PTHrP receptor gene recapitulates the phenotype of PTHrP gene ablation. We have localized PTHrP expression to the embryonic mammary epithelial cells and PTH/PTHrP receptor expression to the mammary mesenchyme using in situ hybridization histochemistry. Finally, we have rescued mammary gland development in PTHrP-null animals by transgenic expression of PTHrP in embryonic mammary epithelial cells. We conclude that PTHrP is a critical epithelial signal received by the mammary mesenchyme and involved in supporting the initiation of branching morphogenesis.     

Role of growth hormone and insulin-like growth factor-I in mammary development

Mammary glands from 3- to 4-week-old mice were incubated in whole organ culture to determine the effects of GH, PRL, and insulin-like growth factor-I (IGF-I) on lobulo-alveolar development and milk protein expression. Virgin mice were implanted with pellets of estrogen and progesterone (1:1000). After 9 days, abdominal no. 4 glands were removed and place on siliconized lens paper in Waymouths' medium supplemented with insulin (Ins), aldosterone, hydrocortisone, and epidermal growth factor. Concentrations of bovine GH, ovine GH, rat GH, or ovine PRL added to the medium varied from 0-1 micrograms/ml. IGF-I was added to replace either Ins or PRL up to 1 microgram/ml. When glands were incubated with Ins, aldosterone, hydrocortisone, and 250 ng/ml PRL, they exhibited lobulo-alveolar development and expressed the milk protein beta-casein. When GH was substituted for PRL, little lobulo-alveolar development occurred, although beta-casein mRNA was expressed at low levels. Either PRL or GH at 1 microgram/ml induced lobulo-alveolar development and beta-casein mRNA. Addition of epidermal growth factor to whole organ culture with GH or PRL (1 microgram/ml) was equally effective in stimulating lobulo-alveolar development. IGF-I did not substitute for PRL, GH, or insulin in tissue maintenance. It is clear that GH at high concentrations can act directly on mouse mammary tissue to induce both lobulo-alveolar development and casein expression.     

Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development

Mesenchymal-epithelial tissue interactions are important for development of various organs, and in many cases, soluble signaling molecules may be involved in this interaction. Hepatocyte growth factor (HGF) is a mesenchyme-derived factor which has mitogenic, motogenic and morphogenic activities on various types of epithelial cells and is considered to be a possible mediator of epithelial-mesenchymal interaction during organogenesis and organ regeneration. In this study, we examined the role of HGF during lung development. In situ hybridization analysis showed HGF and the c-met/HGF receptor gene to be respectively expressed in mesenchyme and epithelium in the developing lung. In organ cultures, exogenously added HGF apparently stimulated branching morphogenesis of the fetal lung. In contrast, HGF translation arrest or neutralization assays resulted in clear inhibition of epithelial branching. These results suggest that HGF is a putative candidate for a mesenchyme-derived morphogen regulating lung organogenesis. We also found that HGF is involved in epithelial branching, in collaboration with fibroblast growth factor (FGF) family molecule(s). In mesenchyme-free culture, HGF alone did not induce epithelial morphogenesis, however, addition of both HGF and acidic FGF (aFGF) or keratinocyte growth factor (KGF), ligands for the KGF receptor, induced epithelial branching more extensively than that was observed in explants treated with aFGF or KGF alone. In addition, the simultaneous inhibition of HGF- and FGF-mediated signaling using neutralizing antibody and antisense oligo-DNA resulted in drastic impairment of epithelial growth and branching. Possible interactions between HGF and FGFs or other growth factors in lung development is given consideration.     

Importance of nidogen binding to laminin gamma1 for branching epithelial morphogenesis of the submandibular gland

Epithelial-mesenchymal interactions are major driving forces for the development of most solid organs. The importance of these interactions was first shown for the embryonic submandibular gland more than 40 years ago. We here present evidence that interactions between two basement membrane components, nidogen (entactin) and laminin gamma1 chain, could be important for epithelial-mesenchymal interactions in this gland. Nidogen mRNA was detected by in situ hybridization in the mesenchyme, and yet the protein was detected in epithelial and endothelial basement membranes. The role of nidogen-laminin interactions for epithelial morphogenesis was studied by applying antibodies to submandibular gland organ cultures. Antibodies reacting strongly with the nidogen-binding site of laminin gamma1 chain drastically perturbed branching epithelial morphogenesis. Electron microscopy of the epithelial-mesenchymal interface showed that blocking antibodies disrupted the formation of the basement membrane. Epidermal growth factor was shown to increase the expression of nidogen in mesenchyme, and could counteract the effect of the blocking antibodies. We suggest that nidogen could be an important mesenchymal factor for submandibular gland development.     

Single-unit activity of cerebellar nuclear cells in the awake genetically dystonic rat

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.     

Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme

Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium.     

Removal of the membrane-anchoring domain of epidermal growth factor leads to intracrine signaling and disruption of mammary epithelial cell organization

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti-EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti-EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.     

Prolactin decreases epidermal growth factor receptor kinase activity via a phosphorylation-dependent mechanism

Previously, we have shown that prolactin inhibits epidermal growth factor (EGF)-induced mitogenesis in mouse mammary epithelial cells without altering the response to other growth promoting agents. This effect has been associated with reduced EGF-induced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation. Our current hypothesis is that prolactin induces an alteration in EGFR kinase activity via a phosphorylation-dependent mechanism. To test this hypothesis, we treated normal murine mammary gland cells with or without 100 ng/ml prolactin. EGFR isolated by wheat germ agglutinin affinity chromatography from nontreated cells exhibited substantial ligand-induced phosphorylation, and EGFR isolated from prolactin-treated cells displayed minimal EGF-induced EGFR phosphorylation, as well as decreased kinase activity toward exogenous substrates. The observed decrease in ligand-induced EGFR phosphorylation could not be attributed to either differential amounts of EGFR, decreased EGF binding affinity, or the presence of a phosphotyrosine phosphatase or ATPase. EGFR isolated from prolactin-treated cells exhibited increased phosphorylation on threonine. Removal of this phosphorylation with alkaline phosphatase restored EGFR kinase activity to levels observed in nontreated cells. Therefore, these results suggest that prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation and decreasing EGF-induced EGFR tyrosine phosphorylation.     

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.     

Pharmacokinetics and metabolism of [14C]dichloroacetate in male Sprague-Dawley rats. Identification of glycine conjugates, including hippurate, as urinary metabolites of dichloroacetate

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three-dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth-stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth-stimulating activity present in the mammary fibroblast-conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast-derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.     

Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12

Androgen has an important role in development of the prostate, and the actions of androgen are mediated, in part, by locally produced growth factors. These growth factors are postulated to mediate stromal-epithelial interaction in the prostate to maintain normal tissue physiology. Transforming growth factor-alpha (TGF-alpha) is one of the growth factors that can stimulate prostatic growth. The expression of TGF-alpha is thought to be regulated by androgen. The expression of epidermal growth factor receptor (EGFR), which is the receptor of TGF-alpha and EGF, also may be regulated by androgen. The hormonal and developmental regulation of TGF-alpha and EGFR messenger RNA (mRNA) levels in isolated epithelial and stromal cells from rat ventral prostate was investigated. The expression of mRNA for TGF-alpha and EGFR was analyzed by a quantitative RT-PCR (QRT-PCR) procedure developed. Observations from this assay demonstrated that both epithelial and stromal cells expressed the mRNA for TGF-alpha and EGFR. TGF-alpha mRNA expression was constant during postnatal, pubertal, and adult development of the prostate. EGFR mRNA expression was elevated at the midpubertal period and decreased with age. After castration of 60-day-old adult rats, both TGF-alpha and EGFR mRNA were significantly enhanced. TGF-alpha mRNA expression was stimulated by EGF in stromal cells (4.5-fold increase) but was not changed by any treatment in epithelial cells. EGFR mRNA levels were stimulated by EGF and keratinocyte growth factor treatment and inhibited by testosterone treatment in epithelial cells. Stromal cell EGFR mRNA levels were not affected by any treatment. Both testosterone and EGF stimulated incorporation of 3H-thymidine into prostatic stromal and epithelial cells. Anti-TGF-alpha antibody significantly inhibited testosterone-stimulated 3H-thymidine incorporation into stromal cells and epithelial cells. Immunocytochemical localization of TGF-alpha and EGFR demonstrated expression on the luminal surface of epithelial cells within prostatic ducts, and minimal expression was observed in stromal cells. Results indicate that testosterone does not directly regulate TGF-alpha mRNA levels but does inhibit EGFR mRNA levels. Interestingly, anti TGF-alpha antibody suppressed the effect of testosterone on 3H-thymidine incorporation into prostatic stromal and epithelial cells. This finding suggests that testosterone may act indirectly on prostatic cells to influence TGF-alpha actions. TGF-alpha mRNA levels were influenced by EGF in stromal cells only, and EGFR mRNA levels were influenced by testosterone, EGF, and keratinocyte growth factor in epithelial cells. These observations suggest that regulation of TGF-alpha and EGFR is distinct between the cell types. In conclusion, a network of hormonally controlled growth factor-mediated stromal-epithelial interactions is needed to maintain prostate development and function.     

The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions

The goals of this study were to determine the cellular sites of insulin-like growth factor (IGF) and IGF type-I receptor (IGF-IR) expression and to begin to elucidate functional roles for the IGFs during postnatal development of the murine mammary gland. Using in situ hybridization analyses, we determined that IGF-I, IGF-II, and IGF-IR messenger RNAs were expressed in the highly proliferative terminal end buds during pubertal ductal growth. Consistent with these data, IGF-I (in combination with mammogenic hormones) promoted ductal growth in pubertal stage mammary glands cultured in vitro. During postpubertal and pregnancy stages, IGF-II and IGF-IR continued to be expressed in ductal epithelium. Expression of IGF-II in ductal and alveolar epithelium correlated with the pattern of rapidly proliferating cells, as determined by incorporation of 5-Bromo-2'-deoxyuridine, suggesting a potential autocrine or paracrine role for IGF-II as a mitogen for ductal epithelial cells. IGF-I expression was reinitiated in mammary epithelium in the differentiated alveoli at the end of pregnancy, suggesting an additional role for this factor in maintenance of the alveoli during lactation. Taken together, these data support an in vivo role for locally-produced IGFs in promoting ductal growth during puberty and suggest that IGF-I and IGF-II may have distinct functions during pregnancy-induced alveolar development.     

3-(5-Alkylamino-4-isoxazolyl)-1,2,5,6-tetrahydropyridines: a novel class of central nicotinic receptor ligands

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated for its potential to predispose to breast cancer. Analysis of mammary gland differentiation and cell proliferation were used as biomarkers. Timed pregnant Sprague-Dawley CD rats were gavaged with 1 microg TCDD/kg on day 15 post-conception. Control animals were treated with the same volume of vehicle (sesame oil) on the same schedule. Mammary gland differentiation studies revealed that prenatal TCDD treatment, as compared with sesame oil treatment, resulted in significantly more terminal end buds and fewer lobules II in 50-day-old offspring, but no significant alterations to mammary gland differentiation in 21-day-old offspring. Terminal end buds are the most susceptible terminal ductal structures and lobules the least susceptible to carcinogenesis. Prenatal TCDD treatment did not alter labeling index in the mammary terminal ductal structures of 21- and 50-day-old rats, but the total proliferative compartment in terminal end buds of 50-day-old rats was larger. Prenatal TCDD treatment resulted in an increased number of chemically induced mammary adenocarcinomas in rats. TCDD delayed time of vaginal opening and caused disruption to the estrous cycle. Alteration to mammary gland differentiation (increased number of terminal end buds) is correlated with increased susceptibility to mammary cancer from prenatal exposure to TCDD.