Keratins are the major structural proteins of epithelial cells in higher vertebrates, where they form 10 nm intermediate filaments (IFs) that significantly contribute to their trademark mechanical resilience. The greater than 40 keratin proteins are partitioned into two sequence types, I and II, both of which are required during filament polymerization in vivo and in vitro. In human, as in all mammalian species, type I and type II keratin genes are coordinately regulated in a pairwise, tissue- and differentiation-specific fashion, creating expression patterns that are tightly associated with the function of epithelia. Our studies are centered on K6, a type II keratin expressed in a variety of stratified epithelia under basal conditions but better known for its enhanced expression during wound repair and in chronic hyperproliferative diseases (psoriasis, carcinoma). We have shown the existence of multiple K6 isoforms in both human and mouse that are highly analogous in their primary sequence but differentially regulated at the transcriptional level. In doing so we have produced several tools including transgenic mouse strains that will enable us to examine three issues of broad and significant interest to skin biology. First, we will address the significance of having many K6 isoforms in the broader context of multiple type II keratin genes showing partially overlapping patterns of expression. We will inactivate the K6 alpha and K6 beta genes simultaneously by gene targeting and homologous recombination in 129 SvJ mice. The K6 null mice will be assessed in a variety of ways, including their ability to repair skin wounds and their susceptibility to two-step chemical carcinogenesis. We will attempt to complement the expected skin and oral mucosa phenotypes by re- introducing type II sequences in K6 null mice, such as K5 or K1, the two major type II keratins of skin. Second, we will pursue our characterization of the regulation of the K6 genes during wound repair in skin. We showed that the proximal 1.0 kilobase of 5'upstream sequence from the human K6a gene is both necessary and sufficient to confer highly-specific wound-inducible expression to a reporter gene in transgenic mice. The transgene is induced as early as 2-3 hours following injury to mouse skin, coinciding with the endogenous K6 genes. We will carry out a molecular analysis of the cis-acting DNA regulatory sequences that underlie this expression pattern, with the goal of identifying the transcription factors and signalling pathways that are responsible for keratinocyte activation early after skin injury. Third, we will investigate the molecular pathogenesis of inherited skin blistering diseases involving mutations in keratin proteins. We created a transgenic mouse model in which the expression of a dominant negative K6 mutant protein, and hence the skin blistering phenotype, are inducible. We will examine the micromechanical properties of keratin filaments and of skin keratincytes isolated from transgenic mice expressing the mutant protein. Relating these data to the cytoarchitecture and mechanical resilience of epidermal tissue in transgenic mice in vivo should lead us to pinpoint the cellular basis for the heightened sensitivity to mechanical trauma.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042047-05
Application #
6029969
Study Section
Special Emphasis Panel (ZRG4-GMA-1 (03))
Program Officer
Moshell, Alan N
Project Start
1995-06-15
Project End
2003-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Coulombe, Pierre A (2017) The Molecular Revolution in Cutaneous Biology: Keratin Genes and their Associated Disease: Diversity, Opportunities, and Challenges. J Invest Dermatol 137:e67-e71
Coulombe, Pierre A (2016) Discovery of keratin function and role in genetic diseases: the year that 1991 was. Mol Biol Cell 27:2807-10
Wang, Fengrong; Zieman, Abigail; Coulombe, Pierre A (2016) Skin Keratins. Methods Enzymol 568:303-50
Feng, Xia; Coulombe, Pierre A (2015) Complementary roles of specific cysteines in keratin 14 toward the assembly, organization, and dynamics of intermediate filaments in skin keratinocytes. J Biol Chem 290:22507-19
Feng, Xia; Coulombe, Pierre A (2015) A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes. J Cell Biol 209:59-72
van Steensel, Maurice A M; Coulombe, Pierre A; Kaspar, Roger L et al. (2014) Report of the 10th Annual International Pachyonychia Congenita Consortium Meeting. J Invest Dermatol 134:588-591
Alvarado, David M; Coulombe, Pierre A (2014) Directed expression of a chimeric type II keratin partially rescues keratin 5-null mice. J Biol Chem 289:19435-47
Chung, Byung-Min; Rotty, Jeremy D; Coulombe, Pierre A (2013) Networking galore: intermediate filaments and cell migration. Curr Opin Cell Biol 25:600-12
Pan, Xiaoou; Hobbs, Ryan P; Coulombe, Pierre A (2013) The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr Opin Cell Biol 25:47-56
Coulombe, Pierre A; Lee, Chang-Hun (2012) Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath. J Invest Dermatol 132:763-75

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