Protein kinase C-δ transactivates platelet-derived growth factor receptor-α in mechanical strain-induced collagenase 3 (matrix metalloproteinase-13) expression by osteoblast-like cells

Abstract

Matrix metalloproteinase-13 (MMP-13, or collagenase 3) has been shown to degrade intact collagen and to participate in situations where rapid and effective remodeling of collagenous ECM is required. Mechanical strain induction of MMP-13 is an example of how osteoblasts respond to high mechanical forces and participate in the bone-remodeling mechanism. Using MC3T3-E1 osteoblast-like cells, we dissected the signaling molecules involved in MMP-13 induction by mechanical strain. Reverse transcription-PCR and zymogram analysis showed that platelet-derived growth factor receptor (PDGFR) inhibitor, AG1296, inhibited the mechanical strain-induced MMP-13 gene and activity. However, the induction was not affected by anti-PDGF-AA serum. Immunoblot analysis revealed time-dependent phosphorylation of PDGFR-α up to 2.7-fold increases within 3 min under strain. Transfection with shPDGFR-α (at 4 and 8 μg/ml) abolished PDGFR-α and reduced MMP-13 expression. Moreover, time-dependent recruitments of phosphoinositide 3-kinase (PI3K) by PDGFR-α were detected by immunoprecipitation with anti-PDGFR-α serum followed by immunoblot with anti-PI3K serum. AG1296 inhibited PDGFR-α/PI3K aggregation and Akt phosphorylation. Interestingly, protein kinase C-δ (PKC-δ) inhibitor, rottlerin, inhibited not only PDGFR-α/PI3K aggregation but PDGFR-α phosphorylation. The sequential activations were further confirmed by mutants ΔPKC-δ, ΔAkt, and ΔERK1. Consistently, the primary mouse osteoblast cells used the same identified signaling molecules to express MMP-13 under mechanical strain. These results demonstrate that, in osteoblast-like cells, the MMP-13 induction by mechanical strain requires the transactivation of PDGFR-α by PKC-δ and the cross-talk between PDGFR-α/PI3K/Akt and MEK/ERK pathways. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.