The broad long-term objective of this application is to understand the molecular mechanisms through which protein kinase C (PKC) isoenzymes regulate growth and differentiated functions in human skin keratinocytes. The PKC family is comprised of ten phospholipid-dependent, serine/ threonine protein kinase isoenzymes. Accumulating evidence suggests that PKC isoenzymes possess functional specificities. PKC isoenzymes are activated within keratinocytes in response to extracellular agonists such as hormones, growth factors, and cytokines, which bind to specific cell surface receptors. Activation of PKC isoenzymes initiates intracellular protein phosphorylation cascades, which modulate keratinocyte growth, differentiation, gene expression, cytoskeletal organization, cell-cell and cell-matrix interactions, receptor expression, enzyme activities, and synthesis and release of pro-inflammatory cytokines and eicosanoids. PKC isoenzymes are also directly activated by phorbol esters, and are the major molecular mediators of biological responses to these agents. In skin phorbol esters cause hyperplasia, accelerated terminal differentiation, inflammation, and promote tumor formation. Thus PKC isoenzymes are key proximal regulators of fundamental cutaneous functions, and as such their study is directly pertinent to many common neoplastic. hyperproliferative, inflammatory skin diseases, including cancer, psoriasis, and atopic dermatitis. PKC isoenzymes, PKC-alpha, PKC- delta, and PKC-eta, are localized within keratinocytes in distinct cell layers in human epidermis. PKC-alpha is expressed in suprabasal layers, PKC-delta is expressed in the basal layer, and PKC-eta is expressed in the granular layer. It has been hypothesized that these three PKC isoenzymes possess unique functions consonant with their specific sites of expression, i.e. PKC-alpha, PKC-delta, and PKC-eta participate in regulation of inflammatory responses, growth, and terminal differentiation, respectively. The focus of this proposal is to test this hypothesis.
The specific aims are to determine the consequences of l) enrichment, 2) depletion, and 3) inhibition of PKC-alpha, PKC-delta, and PKC-eta on keratinocyte growth, differentiation, and synthesis and release of inflammatory cytokines and eicosanoids.
The aims will be accomplished through specific manipulation of PKC-alpha, PKC-delta, and PKC-eta levels and activities in keratinocytes, using molecular biology techniques. 1) Wild type PKC-alpha, PKC-delta, and PKC-eta genes will be inserted into keratinocytes to elevate the levels of their protein products, 2) antisense PKC-alpha, PKC-delta, and PKC-eta genes will be inserted into keratinocytes to block endogenous gene transcription, and thereby reduce their protein levels, and 3) dominant negative kinase deficient mutant PKC-alpha, PKC-delta, and PKC-eta genes will be inserted into keratinocytes to specifically inhibit their endogenous activities.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR042419-01A2
Application #
2081661
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1995-08-01
Project End
1998-07-31
Budget Start
1995-08-01
Budget End
1996-07-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Dermatology
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109