Compressive force regulates gene expression of collagen-modifying enzymes and MMPs

Objectives: It has been well accepted that mechanical force affects bone architecture by regulating extracellular matrix formation. However, little is known about the underlying mechanisms by which the biosynthesis of collagen, the major matrix component in bone, is regulated by mechanical force. In this study, the gene expression of collagen, collagen-modifying enzymes and matrix metalloproteinases (MMPs), and their potential signaling pathways, in response to compressive force were analyzed using osteoblastic cells.

Methods: MC3T3-E1 osteoblastic cells were cultured in the 3D collagen gel (rat tail collagen, 2 g/ml) and three magnitudes of a compressive force (0.5, 1 and 2 g/cm2) were applied. Total RNA was isolated at two time points: 12 and 24 hours, and the following mRNA expression were analyzed by real-time PCR: Cbfa1/Runx2, Osteopontin (OPN), type I collagen alpha 2 chain (Col1a2), lysyl hydroxylase (LH) 1-3, lysyl oxidase (LOX), matrix metalloproteinase (MMP) 2, 9, 13, 14, and GAPDH (internal control), and relative gene expression were calculated. Furthermore, after a compressive force was applied for 30 or 60 min, following 4 hours serum starvation, the phosphorylation of FAK and subsequent three MAPKs (ERK, p38 and JNK) was determined by Western blot analysis with specific antibodies.

Results: Gene expression of OPN, LH2, LOX and MMP-13 were significantly upregulated upon 12 and 24 hours of compression, while Col1a2 and Cbfa1/Runx2 expression did not change. For the signaling pathway, p-FAK was upregulated at 60 min of compression. Among the three MAPK pathways, upregulation of p-ERK and p-p38 were observed at 60 min of compressive force.

Conclusions: Compressive force may upregulate the genes that are associated with both collagen stability (i.e. cross-linking) and degradation potentially through MAPK pathway.