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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AR039691-05
Application #
3457157
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1989-03-01
Project End
1994-02-28
Budget Start
1993-03-13
Budget End
1994-02-28
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Reynolds, N J; Baldassare, J J; Henderson, P A et al. (1994) Translocation and downregulation of protein kinase C isoenzymes-alpha and -epsilon by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts. J Invest Dermatol 103:364-9
Baldassare, J J; Tarver, A P; Henderson, P A et al. (1993) Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq. Biochem J 291 ( Pt 1):235-40
Reynolds, N J; Talwar, H S; Baldassare, J J et al. (1993) Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-alpha in normal human skin fibroblasts and keratinocytes. Biochem J 294 ( Pt 2):535-44
Fisher, G J; Tavakkol, A; Leach, K et al. (1993) Differential expression of protein kinase C isoenzymes in normal and psoriatic adult human skin: reduced expression of protein kinase C-beta II in psoriasis. J Invest Dermatol 101:553-9
Elder, J T; Sartor, C I; Boman, D K et al. (1992) Interleukin-6 in psoriasis: expression and mitogenicity studies. Arch Dermatol Res 284:324-32
Kim, N I; Cooper, K D; Fisher, G J et al. (1992) Psoriatic skin reveals the in vivo presence of an epidermal IL-1 inhibitor. Arch Dermatol Res 284:71-6
Klein, S B; Fisher, G J; Jensen, T C et al. (1992) Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways. J Cell Physiol 151:326-36
Zhang, Q Y; Hammerberg, C; Baldassare, J J et al. (1992) Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts. J Immunol 149:1402-8
Baldassare, J J; Henderson, P A; Burns, D et al. (1992) Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels. J Biol Chem 267:15585-90
Tavakkol, A; Elder, J T; Griffiths, C E et al. (1992) Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin. J Invest Dermatol 99:343-9

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