Gastrin stimulation of the enterochromaffin-like (ECL) cell is a key step in the physiological pathway that leads to gastric acid secretion. Little is known about the ECL cell, or about the intracellular mechanisms that are activated in response to gastrin. The gene histidine decarboxylase (HDC) encodes the enzyme which produces histamine (from histidine), and which is highly expressed in the ECL cell of the oxyntic mucosa. Hypergastrinemic states lead not only to proliferation of ECL cells, but also to increased HDC mRNA levels, demonstrated in our laboratory by Northern blot and in situ hybridization analysis. The overall objective of the proposed studies is to characterize the cis- regulatory mechanisms which target HDC gene expression to the ECL cell, and which control Its response to gastrin. This laboratory has cloned the 5' flanking regulatory regions of both the human and rat HDC genes, and joined them to a luciferase reporter gene. Gastrin treatment of a gastric cancer (AGS) cell line transfected with both HDC-luciferase constructs and the cloned human CCK-B/gastrin receptor leads to a four- fold increase in luciferase activity. A dose response curve, time course studies, and antagonism with the specific CCK-B receptor antagonist L365,260 (ICso=5nM) indicate a highly specific response. Deletion analysis indicated that the gastrin response element was located within 100 nucleotides of the start site. Transfection studies in AGS cells have also identified a putative stomach specific cis-regulatory element containing a homeodomain-like ATTTA motif. This proposal aims to further characterize the gastrin response element, as well as the stomach- specific enhancer, through additional deletion and mutagenesis studies in AGS-B cells and in primary ECL cell preparations. Nuclear proteins which interact with these cis-acting DNA elements will also be studied. These in vitro studies will be extended through transgenic studies, which will determine which sequences in the HDC gene are necessary for correct tissue-specific and regulated expression in ECL cells in vivo. The expression of a hybrid consisting of the human growth hormone gene coding region under the control of the HDC gene 5' flanking sequences will be examined. Overall, the proposed studies will examine at a molecular level the regulation of HDC gene expression, one of the main targets of gastrin in the ECL cell, as well as a key signaling mechanism in acid secretion.
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