Several examples of steroid hormone mediated regulation of mRNA stability are known. However, the biochemical basis of this regulation is not known. Estrogen-mediated accumulation of the avian apolipoprotein (apo)II mRNA, in the liver, is in part due to its stabilization. ApoII mRNA has a t 1/2 of 13 hours in the presence of estrogen. The withdrawal of estrogen reduces the t l/2 to 1.5 hours. Preliminary studies using a novel in vitro assay has identified the expression of a cytosolic element in the rooster liver, in response to estrogen., specifically stabilized apoII and certain other mRNAs. This apoII mRNA stabilizing activity was inactivated by heating at 60 degrees C, and by treatment with proteinase K, but not micrococcal nuclease, indicating that the activity was in fact due to a protein that did not require a nucleic acid component for its function. Interestingly, the expression of this estrogen-regulated mRNA stabilization factor was induced by certain xenobiotic agents such as tamoxifen (Nolvadex), Araclor, chlordecone (Kepone) and hexchlorophene but not by clomiphere (Clomid), DDT (chlorophenethane), methoxychlor, toxaphene, bisphenol, dieldrin and Lindane (hexachlorocyclohexane). Clomiphene, toxaphene and bisphenol acted as estrogen antagonists in the presence of estrogen, and prevented stimulation of the gene. Studies also showed an induction by estrogen of a subset of apoII mRNA-binding proteins (mRNPs). The studies proposed in this application will elucidate the biochemical mechanisms underlying the estrogen-dependent stabilization of apoII mRNA, by initially purifying the mRNA stabilizing protein to homogeneity and cloning the cDNA encoding it. This cDNA will be used to predict the amino acid sequence of the protein and to overproduce the protein and its mutated versions, and characterize its functions and mechanism of action at the biochemical level using the in vitro apoII mRNA stability assay. The genomic clone containing the promoter will be obtained, and the transcription regulatory elements will be characterized by mutational analyses in transfection studies using estrogen-receptor containing chicken hepatoma cell line LMH/2A. The transcription elements and factors involved in the regulation of the gene by estrogen as well as the xenobiotics will be identified. Despite the critical need for a thorough understanding of the effects of environmental xenobiotics on human health, a knowledge of cellular xenobiotic-toxicity and their effects at the cellular level, particularly on gene expression, is lacking. This is essential for early detection of biological effects of xenobiotics prior to any clinical manifestations. The cognate protein in the human will be identified by sequence homology, and its functions characterized. This study will help us understand the role of xenobiotics in gender-specific disorders including certain types of cancers (breast, prostate and testicular), gender-bending effects and infertility. The findings from the proposed work may also help us look for biochemical effects that lead to the higher incidence of osteoporosis, arteriosclerosis and coronary artery disease in post-menopausal women. In addition to the studies on transcriptional and post-transcriptional effects of xenobiotics, this study proposes to develop a rapid and efficient molecular procedure using RT-PCR, for screening estrogenic compounds prior to human, animal or environmental exposure. In addition, estrogen induction of a subset of apoII mRNPs is suggestive of their role in the regulation of the mRNA at the cytoplasmic level. Purification, cloning and characterization of these proteins are essential to our understanding of the molecular mechanisms underlying estrogen-regulation of apoII mRNA stability.
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