Protein deprivation not only has immediate regulatory effects on gene expression, but can also have long-term effects through chromatin-associated mechanisms. However, there are significant gaps in our knowledge about the impact of dietary protein fluctuation on basic cell functions, including transcription. At the level of individual tissues/cells, dietary protein deficiency is reflected as amino acid (AA) limitation and therefore, AA deprivation of cultured cells is a useful model to investigate the genomic mechanisms of transcriptional control. Dietary protein limitation in vivo or AA deprivation in cell culture activates a multi-pathway AA response (AAR) that is one of several stress-activated signaling mechanisms that culminate in ATF4 synthesis. A microarray study identified members of the FOS/JUN family of transcription factors as AA-regulated genes in human HepG2 hepatocellular carcinoma (HCC) cells. Subsequent studies showed that cJUN regulates several downstream genes that are known AAR targets. Two additional novel observations were made. First, the induction of the cJUN gene is ATF4-independent, and therefore, must contain a novel AA response element (AARE), which may be linked to a novel signaling pathway. Second, the induction of cJUN occurs in human HCC cells, but not in primary human hepatocytes or in a non-transformed immortalized hepatocyte cell line. The proposed studies are innovative because: 1) they will provide the first investigation of a protein/AA responsive gene by an ATF4-independent mechanism, and 2) they will establish the mechanism for regulation of the cJUN gene in an animal tumor model in vivo, as well as in cultured human hepatoma cells and normal hepatocytes. Our global hypothesis is that the cJUN gene contains a novel AARE and is activated in HCC cells by an ATF4-independent signaling pathway, and that elevated cJUN expression supports continued HCC proliferation during dietary protein or AA limitation. The proposed experiments will test the following hypotheses: (Hypothesis I) The cJUN gene is the target of a novel ATF4-independent AA signaling pathway;(Hypothesis II) The cJUN gene exhibits novel AA-responsive transcriptional mechanisms;and (Hypothesis III) cJUN modulates AAR genes and cell proliferation during protein/AA limitation.
Sufficient dietary protein/amino acid availability is an important factor in maintenance of health, especially during pregnancy, fetal/neonatal development, and in the progression of a wide range of diseases, including diabetes and cancer. Despite these important relationships, the influence of dietary protein/amino acid fluctuation on fundamental cellular processes and pathological states, such as the development and maintenance of tumors, are topics about which we know relatively little. The proposed research will investigate the mechanisms that lead the FOS/JUN family of proteins, major regulators of gene expression and cell proliferation, to be induced by amino acid limitation of human tumor cells, but not their normal counterparts.
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