Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin α1 and discoidin domain receptor 2: a protein kinase C-dependent pathway

Methods. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITG alpha 1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), focal adhesion kinase (FAK) (FAK inhibitor 14), mitogen-activated protein kinase 8 (JNK) (SP600125) and protein kinase C (PKC) (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time PCR was performed to examine gene expression levels of MMP-13, ITG alpha 1 and DDR2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium. Results. Maintaining the chondrocyte's native pericellular matrix prevented MMP-13 up-regulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of ITG alpha 1 and DDR2 reduced MMP-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced MMP-13 gene expression levels. Gene expression levels of MMP-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor. Conclusion. Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both ITG alpha 1 and DDR2 modulate MMP-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of MMP-13.