Metalloproteases play a leading role in maintenance of the extracellular matrix (ECM). We intend to define the nature, function, and regulation of collagenase and other members of this metalloprotease family at the molecular level. We plan to determine the structural organization of the normal human skin collagenase gene, including regulatory elements at the 5' end. The 5' end of the gene will be examined to identify the structural elements of conventional eucaryotic promoters, and chimeric genes will be used to identify regulatory elements needed for constitutive and inducible expression. The genomic arrangement and regulation of human skin collagenase, and its evolutionary relationship to other human and vertebrate collagenases and related members of this gene family, are major areas for continuing investigation. A knowledge of the normal human skin collagenase gene will enable us to determine the potential mutation site(s) responsible for the putative structurally altered RDEB collagenase. Any mutation will be confirmed by sequencing through the region of mutation in several RDEB cell strains. In vitro directed mutagenesis of normal collagenase would confirm that the mutation confers the phenotype of RDEB. Hydrated collagen lattices with embedded RDEB fibroblasts will be used to attempt to reconstruct the RDEB phenotype on the cellular level. Normal human skin collagenase and RDEB collagenase will be tested for catalytic effect on the type Vll collagen of anchoring fibrils, and their kinetic parameters determined. Stromelysin, another member of the gene family, with a broad range of substrate specificities, whose activity parallels that of collagenase in several instances, will also be assayed for its activity in RDEB. In addition, we have isolated a procollagenase activator molecule from skin explant culture medium, which may be an N-chloroamine. Future studies will focus on its range and mechanism of action. We have purified a secreted neutral metalloprotease from cultured human skin explants and fibroblasts which degrades gelatin and have isolated a cDNA clone coding for the complete progelatinase. Areas of investigation of this gelatinase molecule will focus on its structure, what cells (normal and tumor) produce it, whether its synthesis is coordinated with that of collagenase and stromelysin, what agents regulate its production, and its substrate and cleavage site specificities. This ECM metalloprotease gene family is crucial to our understanding of diseases of the skin and other organ systems and on basic cellular processes; proliferation, differentiation and tumorigenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR012129-27
Application #
2078288
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-04-01
Project End
1998-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
27
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Karelina, T V; Bannikov, G A; Eisen, A Z (2000) Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity. J Invest Dermatol 114:371-5
Karelina, T V; Eisen, A Z (1998) Interstitial collagenase and the ED-B oncofetal domain of fibronectin are markers of angiogenesis in human skin tumors. Cancer Detect Prev 22:438-44
Li, L; Eisen, A Z; Sturman, E et al. (1998) Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate. Biochim Biophys Acta 1405:110-20
Li, L; Akers, K; Eisen, A Z et al. (1997) Activation of gelatinase A (72-kDa type IV collagenase) induced by monensin in normal human fibroblasts. Exp Cell Res 232:322-30
Lee, A Y; Akers, K T; Collier, M et al. (1997) Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts. Proc Natl Acad Sci U S A 94:4424-9
Xia, T; Akers, K; Eisen, A Z et al. (1996) Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. Biochim Biophys Acta 1293:259-66
Karelina, T V; Goldberg, G I; Eisen, A Z (1995) Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors. J Invest Dermatol 105:411-7
Takagi, M; Konttinen, Y T; Santavirta, S et al. (1994) Extracellular matrix metalloproteinases around loose total hip prostheses. Acta Orthop Scand 65:281-6
Karelina, T V; Goldberg, G I; Eisen, A Z (1994) Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia. J Invest Dermatol 103:482-7
Seltzer, J L; Lee, A Y; Akers, K T et al. (1994) Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction. Exp Cell Res 213:365-74

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