The mammalian regulatory gastrin-releasing peptide (GRP) binds with high affinity to its G protein-coupled GRP-receptor (GRP-R) and elicits important physiologic actions in the gastrointestinal tract, such as pancreatic enzyme and gastric acid secretion. The human GRP-R (hGRP-R) also mediates potent signals of cell proliferation in human intestinal and extraintestinal cells both in vivo and in vitro. Owing to its aberrant expression in a large proportion of gastrointestinal malignancies, the hGRP-R can therefore be activated via paracrine and autocrine ligand action and function as a growth factor in the carcinogenesis of gastrointestinal epithelia and promote tumor growth. However, molecular mechanisms of hGRP-R up-regulation, its aberrant expression and hGRP-R dependent intracellular signaling cascades related to cell proliferation have not been elucidated. We identified in gastrointestinal cell line that hGRP-R expression is transcriptionally up-regulated by increased levels of intracellular cAMP and by compounds stimulating the protein kinase C (PKC) pathway. Analysis of critical cis-regulatory elements within the hGRP-R promoter determined two cAMP responsive element (CRE) sites are binding transcription factors CREB, ATF-1, and ATF-2, and up-regulate hGRP-R responsiveness to cAMP dependent signals in a cooperative manner. Furthermore, ligand-specific hGRP-R activated intracellular signaling in duodenal cancer cells caused sustained CREB phosphorylation and transactivation in a PKC-dependent, but calcium and ERK1/2-independent manner. Accordingly, we propose to precisely characterize transcripiton factors capable of binding to the identified hGRP-R core promoter region and their mechanism of action to up-regulate hGRP-R expression. Likely candidates include the CREB/ATF-1, C/EBP, c-myb, and Oct-1 transcription factors. Meanwhile, mechanisms of Bn-induced cell proliferation will be examined, specifically the Bn-mediated EGF-receptor transactivation and subsequent activation of the MAP kinase pathway in gastrointestinal malignancies. Based on our preliminary data, we will further explore the regulation of potential downstream target genes via Bn-dependent CREB activation as they might directly relate to apoptotic cell death and cell proliferation. Taken together, our current results suggest a novel, likely cooperative mechanism of CREB transcription factor action in hGRP-R regulation relevant in gastrointestinal carcinogenesis. Moreover, data indicate several Bn-induced signaling pathways might be associated with cell proliferation in gastrointestinal malignancies. Therefore, the proposed studies are critical to further unravel the mechanisms of hGRP-R regulation and receptor-dependent signaling relevant to cell proliferation. Results will likely contribute to the general understanding of mechanisms of gene expression and signaling in cancer biology.
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