Epithelial-stromal cell interactions are important for normal development and function of the mouse mammary gland. The steroid hormone estrogen is required for epithelial cell proliferation and ductal development in vivo. Recent studies of estrogen receptor alpha knockout mice indicate that estrogen-induced proliferation is dependent upon the presence of estrogen receptor in mammary stromal cells, but not in epithelial cells. The purpose of the present study was to identify the underlying mechanism of estrogen-dependent stroma-derived effects on mammary epithelium. We have developed a minimally supplemented serum-free medium, collagen gel primary mammary coculture system to address the issue of stroma-derived, estrogen-dependent effects on epithelial cell proliferation. Conditioned medium from mammary fibroblasts or coculture with mammary fibroblasts caused increased epithelial cell proliferation and produced tubular/ductal morphology. Hepatocyte growth factor (HGF) was identified as the mediator of this effect, as the proliferative activity in fibroblast-conditioned medium was completely abolished by neutralizing antibody to HGF, whereas neutralizing antibodies to either epidermal growth factor or IGF-I had no effect. Treatment of mammary fibroblasts with estrogen increased the production of HGF. From these results we conclude that estrogen may indirectly mediate mammary epithelial cell proliferation via the regulation of HGF in mammary stromal cells and that HGF plays a crucial role in estrogen-induced proliferation in vivo.

IntroductionPuberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined.MethodsPubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL).ResultsP, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression.ConclusionsThese results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Diabetes mellitus accelerates cardiovascular microangiopathies and atherosclerosis, which are a consequence of hyperglycemia. The aldose reductase (AR) polyol pathway contributes to these microvascular complications, but how it mediates vascular damage in response to hyperglycemia is less understood. The RUNX2 transcription factor, which is repressed in diabetic animals, promotes vascular endothelial cell (EC) migration, proliferation, and angiogenesis. Here we show that physiological levels of glucose (euglycemia) increase RUNX2 DNA binding and transcriptional activity, whereas hyperglycemia does not. However, inhibition of AR reverses hyperglycemic suppression of RUNX2. IGF-1 secretion and IGF receptor phosphorylation by autocrine IGF-1 occur equally in euglycemic or hyperglycemic conditions, suggesting that reduced RUNX2 activity in response to hyperglycemia is not because of altered IGF-1/IGF receptor activation. AR also negatively regulates RUNX2-dependent vascular remodeling in an EC wounded monolayer assay, which is reversed by specific AR inhibition in hyperglycemia. Thus, euglycemia supports RUNX2 activity and promotes normal microvascular EC migration and wound healing, which are repressed under hyperglycemic conditions through the AR polyol pathway.

The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1a-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfb, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function. ß

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor κB ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland.