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© The Rockefeller University Press, 0021-9525/1997//473 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 473-483

Article

A Three-dimensional Collagen Lattice Induces Protein Kinase C-{zeta} Activity: Role in {alpha}2 Integrin and Collagenase mRNA Expression



Jiahua Xu and Richard A.F. Clark

Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8165

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor {alpha}2 subunit mRNA expression in both unstimulated or platelet-derived growth factor–stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239–249). Here we report that the collagen gel can signal protein kinase C (PKC)-{zeta} activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-{zeta} immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-{zeta} protein levels or intracellular location was observed. DNA binding activity of nuclear factor {kappa}B (NF-{kappa}B), a downstream regulatory target of PKC-{zeta}, was also increased by fibroblasts grown in collagen gel. The composition of the NF-{kappa}B/Rel complexes that contained p50, was not changed. The potential role of PKC-{zeta} in collagen gel–induced mRNA expression of collagen receptor {alpha}2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of {alpha}2 and MMP-1 mRNA in collagen gel–stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)–inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5' end of PKC-{zeta} mRNA sequences significantly reduced the collagen lattice–stimulated {alpha}2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-{zeta}, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for {alpha}2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-{zeta}.

Abbreviations used in this paper: AA, arachidonic acid; BIM, bisindolylmaleimide GF 109203X; CalC, calphostin C; CAM, cell adhesion molecule; CHX, cycloheximide; COL, collagen gel; DAG, diacylglycerol; DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; ECM, extracellular matrix; MMP-1, type I metalloproteinase; NF-{kappa}B, nuclear factor {kappa}B; PC-PLC, phosphatidylcholine-hydrolyzing phospholipase C; PDGF, platelet-derived growth factor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TC, tissue culture plates; TNF, tumor necrosis factor.

Funding for this work was provided by National Institutes of Health grant AG10114309 to R.A.F. Clark. J. Xu is supported by funding from the Dermatology Foundation and the School of Medicine, SUNY at Stony Brook.

Address all correspondence to Richard A.F. Clark, Department of Dermatology, School of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794-8165. Tel.: (516) 444-3843. Fax: (516) 444-3844.


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