Chondrocyte gene expression and intracellular signaling pathways in cartilage mechanotransduction

Abstract

Chondrocytes respond to in vivo mechanical loads by regulating the composition of the cartilage extracellular matrix. This study utilized three loading protocols that span the range of forces and flows induced by in vivo loading. Constant (static) compression of cartilage explants induces a transient hydrostatic pressure buildup and fluid exudation from the compacted matrix until relaxation leads to a new equilibrium compressed state. Dynamic compression induces cyclic matrix deformation, hydrostatic pressures, fluid flows, and streaming currents. Dynamic tissue shear causes cyclic matrix deformation only. After applying these loading protocols to intact cartilage explants for 1 to 24 hours, we used real-time PCR to measure the temporal expression profiles of selected genes associated with cartilage homeostasis. In concurrent experiments, we assessed the involvement of intracellular signaling pathways using molecular inhibitors. In order to interpret the results we developed two techniques that reliably clustered intermediate-sized datasets using principal component analysis and k-means clustering. Mechanical loading regulated a variety of genes including matrix proteins, proteases, protease inhibitors, transcription factors, cytokines, and growth factors. Static compression transiently upregulated matrix proteins, however, mRNA levels were suppressed by 24 hours.

(cont.) Dynamic compression and dynamic shear increased matrix protein transcription particularly after 24 hours. In contrast, matrix proteases were upregulated by all 24 hour loading regimes, particularly static compression. Taken together these results demonstrate the functionally-coordinated regulation of chondrocyte gene transcription in response to mechanical forces, and support the hypothesis that dynamic loading is anabolic for cartilage and static loading is anti-anabolic. Intracellular calcium release, cAMP activation of protein-kinase-A, and the phosphorylation of MAP kinases (ERK1/2, p38), were all identified as signaling events necessary for mechanically-induced transcription. In addition, we measured the immediate, transient increase in mRNA levels of transcription factors downstream of the MAP kinase pathway (c-Fos and c-Jun), in response to all three loading types. The prevention of protein synthesis during static compression suppressed mechanically-induced transcription suggesting that signaling molecules are synthesized in response to mechanical forces. Comparison of this well characterized model of normal cartilage mechanotransduction to what occurs within diseased cartilage will hopefully provide insight into the mechanisms driving the progression of osteoarthritis.