Estrogen plays a pivotal role in the proper development and physiological function of reproductive, cardiovascular, nervous and immune system of both male and female. Environmental chemicals that interfere with the estrogen functions could cause various cellular abnormalities, thus endanger our health. In order to address the question on how environmental factors interfere with estrogen functions and design strategy for intervention, we need to understand the intricate molecular event associated with gene expression under the influence of estrogen. With molecular approach, estrogen receptor alpha (ERa)-mediated transactivation of lactoferrin gene and estrogen receptor-related receptor alpha (ERRa) gene whose product are important in innate immune defense and energy balance, respectively were investigated. For the lactoferrin gene, we identified and characterized several DNA elements that play important roles in estrogen responsiveness of the gene. Other than the classical estrogen response element of the human lactoferrin gene, an additional DNA element which binds the ERRa gene was found involving in the estrogen response either positively or negatively. With the state-of-art technology such as chromatin-immunoprecipitation assay (ChIP), yeast-two hybrid system, co-immunoprecipitation (Co-IP) etc, we studied the mechanism on how ERRa modulates estrogen response and found that the ERRa can compete or enhance the interaction between ERa and the coactivators, corepressor or the DNA binding elements of their target genes. As demonstrated by both biochemical and crystal structure studies, ERRa is a constitutive activator and can modulate many ERa-regulated physiological pathways and influence ERa-targeted gene expression in the absence of known ligand. Interestingly, ERRa gene itself is an estrogen target as demonstrated by a series of studies from our laboratory for the past few years. Recent clinical studies have implicated ERRa in breast cancer progression and served as a negative prognostic marker. It was found that a high level of ERRa expression occurs in the aggressive breast cancer cell especially in the ERa-negative breast cancer cells. For the past year, our laboratory was investigating the estrogen regulation of ERRa gene expression in ERa-positive and ERa-negative breast cancer cells. Since the regulation of gene expression is so dependent on the dynamics of chromatin structure and accessibility, we examined the chromatin structural changes and coregulator loading at the multi-hormone-response elements of the ERRa gene promoter. We found increasing ERRa expression and changing chromatin architecture that includes increase in restriction enzyme hypersensitivity and histone acetylation around the response region after estrogen stimulation in both ERa-positive MCF-7 and ERa-negative SKBR3 breast cancer cells. In MCF-7 cells, coactivators were recruited and corepressors released while ERa tethered with Activating Protien 1 (AP1) and ERRa to the response element and mediated estrogen induced ERRa gene expression. In SKBR3 cells, estrogen stimulated ERRa expression could be blocked by MAPK inhibitor suggests an ERa-independent mechanism was involved in estrogen regulation. Further investigation demonstrated that ERRa regulation by estrogen in ER-negative cells is mediated through GPR30, a orphan GPCR. This finding has expand the molecular mechanisms of estrogen action and the potential pathologic effect of estrogen. To understand the in vivo function of ERRa, generating ERRa-null (ERRa KO) or tissue specific ERRa-null mouse will be useful especially since the receptor is active without ligand. We have designed and completed the construction of construct to produce the ERRa KO and ERRa floxed mouse. At the present time, several positive ES cells were identified and is in the expanding stage. Recently, we discovered that the lactoferrin gene is also regulated by bacterial lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) in normal mammary gland cells. Major portion of the LPS and dsRNA-induced lactoferrin was released into the culture medium where it blocked the TNF-a production in exposed cells. We found that the specific inhibitors to PKC, NF-kB, p38 and JNK, but not inhibitor to PKA attenuated the induction of lactoferrin expression by LPS and dsRNA. These data suggested that lactoferrin was induced by LPS and dsRNA through PKC, NF-kB and MAPK pathways which in turn plays a feedback inhibitory role in innate inflammation. Overall, the two models used in the molecular mechanism of estrogen action study demonstrated the complexity of gene regulation and the intricate relationship of estrogen signaling pathway with other cellular pathways in regulation of lactoferrin and ERRa gene expressions. Environmental pollutants such as the endocrine disruptors could alter the body's innate regulatory mechanisms by deregulation of the lactoferrin gene expression or enhancing the cancer progression by stimulation of ERRa expression.
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