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ERá is a critical regulator in breast cancer and mammary gland development. Deregulation of ER signaling correlates with abnormal mammary gland development and breast cancer. However, the role of epithelial ER remains to be clarified in vivo and the mechanism of ER signaling regulation is far from comprehensive. We hypothesize that 1) mammary epithelial ER plays critical roles in mammary gland development during pregnancy and lactation and that 2) novel, as yet identified factors in ER transcriptional regulation are involved in breast cancer development. The loxP-Cre system was used to generate epithelial ERKO mice. The well characterized MMTV-Cre and WAP-Cre transgenic mice were used to delete ER in mammary epithelial cells at different developmental stages. Early expression of MMTV-Cre arrested mammary gland development at the neonatal stage. Successive pregnancy and lactation activated epithelial ER ablation, which compromised side-branching, alveolar development, and epithelial proliferation. Further analysis revealed a massive loss of luminal epithelial cells presumably caused by apoptosis. The abnormal mammary gland development decreased milk production, thereby, caused growth retardation in the offspring. Similar phenotypes were also observed in MMTV-ERKO females in lactation. Thus, we concluded that epithelial ER is essential for mammary gland development during pregnancy and lactation stages. To further pursue the molecular mechanism of ER signaling regulation, a human mammary gland cDNA library was screened to identify novel factors that interact with ER. One novel ERá binding protein identified in the screen contains two conserved LXXLL motifs (NR-box) and a coiled-coil domain. The protein product, which we named NRCC, consists of 3 isoforms that vary in their N-terminal region. NRCC isconserved in vertebrates and its mRNA was detected in human breast cancer cells and mouse breast tumors. We found that NRCC-A interacts with ERá and enhances ERá transcriptional activity in human cancer cells. Moreover, NRCC-A co-localized with ERá in the cell nucleus and was recruited to ER target gene promoters. SiRNA analysis indicated that NRCC proteins are important for endogenous ERá-mediated transcriptional activity and estrogen dependent cell proliferation. Taken together, these data indicate that NRCC-A is a novel coactivator for ERá.

HoxA11 is one of the homeobox genes with similarity to the Drosophila homeotic gene, Abdominal-B. The Abd-B type Hox genes have been shown to be expressed in the developing limbs, axial regions and the most caudal regions of the body. From the study of HoxA11 knockout and HoxA11/D11 double knockout mice, HoxA11 has been shown to be involved in appendicular and axial skeleton development, kidney development and reproductive system development (Small et al. 1993, Davis et al. 1995, Li et al. 1995). In the HoxA11 knockout mice, there are axial skeleton phenotypes, limb phenotypes and urogenital malformation. The homozygous male and female KOs are sterile. There is no kidney phenotype in HoxA11 KOs. Hox11 paralogues are functional redundant. The HoxA11 and HoxD11 double knockout mouse have severe limb, reproductive system and kidney phenotypes. There are different kidney phenotypes in the double KO mouse: some mice have no kidney, some have one or two smaller kidneys. Though the nephrons look normal, there are fewer nephrons in the double KO kidneys. The rare adult kidneys have a thick wall of cortical tissue and poorly developed renal papillae. One wild-type allele of either HoxA11 or HoxD11 was sufficient for normal kidney development. It is not known what genes are regulated by HoxA11 in the kidney development. The goal of this project is to identify the HoxA11 downstream targets in the kidney. Gain of function and loss of function methods have been used to identify the HoxA11 downstream targets in kidney. Drosophila Engrailed repressor and HoxA11 Homeodomain fusion protein was stable expressed in mouse kidney K3 cells to inhibit expression of the HoxA11 targets. The gene expression profiles of En-A11HD positive and negative cells have been analyzed on genechips. Expression of some genes was changed in the En-A11HD positive cells. The expression of some cell proliferation factors and oncogene are decreased, while the expression of some cell differentiation factors is increased in the En-A11HD positive cells. Immunofluorescence experiments result shows that EnA11HD inhibit cell proliferation in mouse kidney k3 cells. HEK293 cells that stably express tetracycline controlled HoxA11 have been established. The RNA from HoxA11"on" and HoxA11 "off" 293 cells have been analyzed on genechips. Expression of some genes have been shown to be different in the A11 positive and A11 negative cells. In accordance with the chip result, integrin associated protein (IAP, CD-47), zinc-finger transcription factor LD5-1 and Bravo (Nr-CAM precursor) have been confirmed to be expressed at higher levels in the HoxA11 positive cells by Northern blot. A novel band that increased in intensity in mRNA from in the A11 positive cells was detected by Northern with IAP probe.