VITAMIN-D-3 REGULATION OF STROMELYSIN-1 (MMP-3) IN CHONDROCYTE CULTURES IS MEDIATED BY PROTEIN-KINASE-C

Matrix metalloproteinases (MMPs) are a group of enzymes with the poten tial to degrade extracellular matrix proteins. One of the MMPs, strome lysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enz yme in remodeling of the extracellular matrix during endochondral deve lopment, a process which is regulated by the vitamin D metabolites, 1, 25-(OH)(2)D-3 and 24,25-(OH)(2)D-3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derive d from growth zone (GC) and resting zone (RC) rat costochondral cartil age were treated with 1 alpha,25-(OH)(2)D-3 (1,25) and 24R,25-(OH)(2)D -3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activi ty was enriched in the matrix vesicle fraction, only the plasma membra ne enzyme was affected by the treatment; 1,25 and 24,25 caused a marke d decrease in plasma membrane stromelysin-1 activity in their target c ells. Since plasma-membrane protein kinase C (PKC) activity is stimula ted by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylat ion. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma(32)P]-ATP and anti-stromelysin-1 immunoprecipitates were analy zed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Select ive inhibitors of PKC activity demonstrated that phosphorylation was i nhibited by H7 and low concentrations of H8, but not by HA1004, indica ting that PKC, not PKA, was responsible. Protein phosphatase 2A(1) (PP 2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone c ell plasma membranes with 24,25, but not 1,25, resulted in phosphoryla tion of stromelysin-1, demonstrating that the nongenomic effect was me tabolite-specific. This suggests that this may be one mechanism by whi ch vitamin D metabolites regulate stromelysin-1 activity and that PKC- dependent phosphorylation inhibits the metalloproteinase. (C) 1996 Wil ey-Liss, Inc.