In the past few years, we have demonstrated that GHRH can be produced by cells of the immune system and stimulate lymphocyte GH production and influence immune function. It is our hypothesis that lymphocyte-derived GHRH may play an autocrine or intracrine function in the regulation of lymphocyte GH synthesis which in turn may regulate the production of lymphocyte-derived IGF-1 molecules. Thus it appears that the role of GHRH in the immune system may be as important as its function in the neuroendocrine system. This proposal is concerned with the regulation of GH gene expression by GHRH and identifying the mechanism of lymphocyte-derived GHRH and GH induction of lymphocyte proliferation. Our preliminary data have identified positive (-299/-193) and negative (-193/-107) regulatory sites in the GH promoter involved in lymphocyte GH expression. The negative regulatory effect appears to be mediated by Spl/3 transcription factors. By DNase protection and gel shift analysis we plan to identify the transcription factor(s) binding to the -299/-193 region of the GH promoter that promote GH gene expression. We will investigate whether GHRH induces GH by relieving the inhibition mediated by the Spl/3 transcription factors in the -193/-107 region of the GH promoter and/or GHRH induces GH by acting to promote transcription via the positive response element in the -299/-193 region of the GH promoter. In addition to studying the mechanism of GHRH induction of GH, we will study the mechanism of GHRH and GH-induced lymphocyte proliferation. Specifically, we will determine if the proliferative effect is cell-cycle specific and involves synthesis of specific cyclin proteins. Further, we will identify signaling pathways utilized by endogenous GHRH and GH and whether they interact with GH receptor-mediated events in cells of the immune system. This will be accomplished in cells overexpressing GHRH and GH, compared to GH treatment, and the effect on activation of STAT proteins, MAP kinase activation and protein kinase C activation. Finally, we will investigate the possibility that the synthesis of lymphocyte GH in vivo occurs after treatment of animals with GHRH. It is anticipated that the results from our experiments will identify novel regulatory mechanisms that cells of the immune system utilize to produce these neuroendocrine peptides and establish the importance of lymphocyte GHRH as a cytokine-like factor involved both in the synthesis of lymphocyte GH and in cellular proliferation. It is further anticipated that the new understanding gained from the results will identify important sites for drug intervention against various immunodeficiencies including AIDS, autoimmunity, and aging.
