Endothelin (Et-1) is a typical ligand capable of activating phospholipase C to generate diacylglycerol and inositol trisphosphate (IP 3) as proximate messengers. Stimulation of protein kinase C (pkc) by diacylglycerols and mobilization of intracellular Ca2+ by IP 3 activate pathways related to the modulation of gene transcription. Stimulation of transcription via activation of pkc occurs through enhancer sequences such as AP-1, however, much less is known about transcriptional activation that occurs via mobilization of intracellular Ca2 +. We have identified an enhancer region within the long terminal repeat of the VL30 endogenous retroviral element which confers transcriptional responsiveness to a reporter gene in the presence of elevated intracellular Ca2+. In Rat-1 fibroblasts, this enhancer is capable of responding to cellular signals such as activation of pkc or pka in the presence of elevated intracellular Ca2+ regulates the transcription of genes via Ca2+-responsive DNA sequences present in the VL30 long terminal repeat. We will investigate the dependence of Ca2+ activated transcription on the levels of intracellular Ca2+. Intracellular [Ca2+] will be experimentally manipulated using Et-1 to produce a transient elevation of intracellular [Ca2+] or thapsigargin and the ionophore A23187 to produce sustained increases in intracellular [Ca2+]. These experiments will relate the quantitative and temporal aspects of Ca2+ mobilization to Ca2+-activated transcription. A likely mechanism for Ca2+-activated transcription is via the phosphorylation of regulatory proteins. We will test whether the CAMP- responsive element binding protein (CREB), members of the fos/jun family of proteins, or other novel DNA-binding proteins are involved in VL30-mediated transcriptional regulation in response to elevated [Ca2+], and whether phosphorylation of these proteins occurs in response to increases in intracellular [Ca2+]. The specific DNA sequences responsible for the enhancement of transcription via the elevation of intracellular [Ca2+]. The specific DNA sequences responsible for the enhancement of transcription via the elevation of intracellular [Ca2+] have been identified by deletion mutagenesis and will be verified by competition with synthetic oligonucleotides. The functional significance of the responsive sequences will be tested using in vivo Dnase footprinting following manipulation of intracellular [Ca2+]. Putative trans-acting regulatory proteins interacting with the Ca2+-responsive element will be characterized using gel mobility shift assays and microinjection techniques, and will be purified using DNA-affinity chromatography. Identification of molecular clones encoding the protein(s) will be pursued if necessary. The results of these investigations should identify both DNA sequences and interactive proteins which are responsible for conferring transcriptional enhancement as a consequence of mobilization of intracellular Ca2+ by physiological agonists.
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