Collagenase is a major gene product of induced rabbit synovial fibroblasts.

Abstract

The effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) were investigated on rabbit synovial fibroblasts. This agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or .apprx. 0.2-1 .mu.g secreted procollagenase protein/105 cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct Ag staining or by autoradiography after SDS PAGE [sodium dodecyl sulfate polyacrylamide gel electrophoresis] of [35S]Met-labeled proteins. The 2 procollagenase bands of MW 53,000 and 57,000, which migrated as a family of spots on 2-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (MW 55,000) as a major product (5% of total) that migrated as a single spot on 2-dimensional gels. Secreted procollagenase, preprocollagenase, and active collagenase (purified to homogeneity; specific activity 1.2 .times. 104 U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of MW 51,000 and a polypeptide of MW 47,000, were also induced by TPA. In contrast to the induction of these 4 polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.