The long term objectives of this proposal are to understand the mechanisms that regulate growth and differentiation of adult human epidermis and thereby provide a rationale for the design of effective therapeutic modalities. Psoriasis is a common dermatological disease that affects 1- 2% of the American population. Psoriasis is characterized by inflammation, hyperplasia and altered differentiation of the epidermis. The cause of psoriasis is unknown, and it is a matter of some controversy whether the primary defect(s) resides in the epidermis, the dermis or the immune system. There is general agreement, however, that the primary site of expression of psoriasis is the epidermis, and specifically its major cell type the keratinocyte. Understanding the mechanisms that regulate keratinocyte growth and differentiation is therefore a major goal of psoriasis research. It is now well recognized that extracellular mediators, such as hormones, growth factors, and cytokines play a vital role in the regulation of cellular functions. Many of these mediators exert their effects by activation of the phospholipase C/protein kinase C signal transduction system. Data suggests that the phospholipase C/protein kinase C signal transduction system is activated in psoriatic lesions. It is hypothesized that misregulation of this system plays a key role in the pathophysiology of psoriasis. The focus of this proposal is to test this hypothesis.
The specific aims are 1) to investigate the mechanism of regulation of phospholipase C (PLC) by GTP-binding proteins, 2) to identify the precursors and pathways of metabolism of 1,2-diacylglycerol, which is the physiological activator of protein kinase C (PK-C), 3) to determine the levels of inositol phosphates, which elevate cytosolic calcium, in normal and psoriatic epidermis, 4) to determine the PK-C isozyme composition of normal and psoriatic epidermis, 5) to determine the mechanism of regulation of cross-linked envelope formation by PK-C, and 6) to characterize agonist- induced phosphoinositide hydrolysis in normal and psoriatic human keratinocytes. These studies will be conducted on samples of normal and psoriatic human epidermis obtained by keratome biopsy from volunteers, and on cultures of keratinocytes established from these biopsies. The methods to be employed include in vitro enzymatic assays using radiolabelled substrates, purification of proteins using column chromatography, analysis of lipids by thin layer and high pressure liquid chromatography, and measurement of PLC and PK-C steady state mRNA levels by hybridization to specific cDNA probes.
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