- Calcium influx, required for keratinocyte differentiation, is enhanced by treatment with permeant cGMP. Moreover, the investigators have found a keratinocyte channel that is activated by cGMP. Finally, they have cloned complete and alternatively spliced isoforms of a cGMP channel that is similar to the calcium-permeable channel found in photoreceptor rod cells. The isoforms differ in their cGMP sensitivity, and their expression is unique in that it is developmentally regulated. The investigators hypothesize that this channel regulates keratinocyte differentiation through its role as cGMP-gated calcium influx pathway. The broad, long-term objective of these studies is to develop methods that modify keratinocyte differentiation by manipulating calcium influx into these cells. The health-relatedness of the project stems from the fact that abnormal keratinocyte differentiation underlies common skin diseases, both benign; psoriasis, abnormal wound healing; and malignant: squamous cell carcinoma. Thus, new approaches to modifying cell differentiation would have wide clinical applications.
The specific aims of the project are: 1) To correlate the exact proportion of each isoform with the degree of keratinocyte differentiation; 2) to compare channel characteristics in differentiating and undifferentiated keratinocytes; 3) to examine the channel's role in keratinocyte differentiation by overexpressing the native, alternatively spliced, or mutant isoforms in undifferentiated and differentiating keratinocytes. To accomplish these aims, the research design will first quantify the proportion of each isoform using the RNAse protection assay. Next, the functional difference conferred by the different isoforms will be examined by comparing 1) ion channel and whole cell current characteristics, using the patch clamp method; 2) calcium influx, using calcium-45 studies; and 3) intracellular calcium concentration, using Fluo-3 measurement. Finally, alteration of calcium influx and keratinocyte differentiation by overexpression of channel isoforms in keratinocytes will be tested.

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 #
5R29AR044341-02
Application #
2732898
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1997-08-01
Project End
2002-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Dermatology
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
McCormick, James A; Feng, Yuxi; Dawson, Kevin et al. (2004) Targeted disruption of the protein kinase SGK3/CISK impairs postnatal hair follicle development. Mol Biol Cell 15:4278-88
McKenzie, Roddie C; Oda, Yuko; Szepietowski, Jacek C et al. (2003) Defective cyclic guanosine monophosphate-gated calcium channels and the pathogenesis of psoriasis. Acta Derm Venereol 83:414-8
Aronchik, Ida; Behne, Martin J; Leypoldt, Laura et al. (2003) Actin reorganization is abnormal and cellular ATP is decreased in Hailey-Hailey keratinocytes. J Invest Dermatol 121:681-7
Behne, Martin J; Tu, Chia-Ling; Aronchik, Ida et al. (2003) Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores. J Invest Dermatol 121:688-94
Behne, Martin J; Meyer, Jamie W; Hanson, Kerry M et al. (2002) NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging. J Biol Chem 277:47399-406
Hu, Z; Bonifas, J M; Beech, J et al. (2000) Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease. Nat Genet 24:61-5
Oda, Y; Imanzahrai, A; Kwong, A et al. (1999) Epithelial sodium channels are upregulated during epidermal differentiation. J Invest Dermatol 113:796-801
Mauro, T; Bench, G; Sidderas-Haddad, E et al. (1998) Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE. J Invest Dermatol 111:1198-201