Murine Matrix Metalloproteinase 9 Gene. 5'-Upstream Region Contains Cis-Acting Elements for Expression in Osteoclasts and Migrating Keratinocytes in Transgenic Mice

in Journal of Biological Chemistry (1999), 274(9), 5588-96

Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown ... [more ▼]

Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between -2722 and -7745 allowed for expression in osteoclasts and migrating keratinocytes, i. e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the -2722 to -7745 base pair region. [less ▲]

Sera from patients with anti-GBM nephritis including goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen alpha chains.

in Nephrology Dialysis Transplantation (1996), 11(11), 2215-22

BACKGROUND: Goodpasture (GP) syndrome is defined by the clinical association of pulmonary haemorrhage with rapidly progressive glomerulonephritis. The disease is caused by pathogenic autoantibodies ... [more ▼]

BACKGROUND: Goodpasture (GP) syndrome is defined by the clinical association of pulmonary haemorrhage with rapidly progressive glomerulonephritis. The disease is caused by pathogenic autoantibodies directed against type IV collagen, which is a major structural component of glomerular basement membranes (GBM). METHODS: The non-collagenous domains (NC1) of all six human type IV collagen alpha chains was produced in E. coli as recombinant fusion proteins with glutathione-S transferase. Sera from 10 patients with different types of anti-GBM nephritis, including GP syndrome, were tested for reactivity with the six proteins using immunoblotting of denatured and reduced proteins and ELISA without reduction. RESULTS: All 10 sera reacted with the alpha 3 (IV) collagen chain by immunoblotting and ELISA. One serum also recognized the alpha 2(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains by immunoblotting. ELISA measurements revealed reactivity of several other sera with alpha 2(IV), alpha 4(IV) or alpha 6(IV) but not with alpha 5(IV) collagen chains. No reactivity was observed with the alpha 1(IV) chain. CONCLUSION: Autoantibodies in anti-GBM nephritis may not be directed only against the alpha 3(IV) collagen chain and they frequently recognize conformational epitopes. [less ▲]

Anti-GBM antibodies from patients with Goodpasture Syndrome react with the NC1 domain of recombinant alpha3 (IV) and alpha4 (IV) collagen chains

in Journal of the American Society of Nephrology [=JASN] (1993), 4

Basement membrane components and galactosylhydroxylysyl glucosyltransferase in suction blisters of human skin.

in British Journal of Dermatology (1982), 106(3), 257-66

Basement membrane components and collagen biosynthesis were studied in suction blisters in human skin. The basement membrane components were characterized by immunofluorescence using specific antibodies ... [more ▼]

Basement membrane components and collagen biosynthesis were studied in suction blisters in human skin. The basement membrane components were characterized by immunofluorescence using specific antibodies to type IV collagen, laminin and fibronectin, and collagen biosynthesis was studied by assaying galactosylhydroxylysyl glucosylatransferase. In suction blisters, the separation of epidermis and dermis occurred above the lamina lucida, indicating that the basement membrane, composed of lamina lucida and lamina densa, forms a mechanically strong entity. During the regeneration phase of blisters, type Iv collagen and laminin were not observed in the old epidermal blister roof. This indicates that keratinocytes when separated from the underlying basement membrane or connective tissue do not synthesize laminin or type IV collagen. Galactosylhydroxylysyl glucosyltransferase activity could be demonstrated in blister fluid and was about the same as in serum when expressed on the basis of protein in fresh blisters. It increased by 2-3 fold during the repair of blisters, indicating that there was local production of this enzyme. Further studies revealed that pure epidermis contained galactosylyhdroxylysyl glucosyltransferase and hydroxyprolineand this suggests that epidermis may synthesize some collagen type which, according to these studies, is not type IV (basement memebrane) collagen. [less ▲]

Assay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase.

in Analytical Biochemistry (1980), 107(1), 187-92