Periodic Mechanical Stress Stimulates the FAK Mitogenic Signal in Rat Chondrocytes Through ERK1/2 Activity

Abstract

Background/Aims: The biological effects of periodic mechanical stress on chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying chondrocyte mechanosensing and signaling in response to periodic mechanical stress remain to be determined. In the current study, we examined the effects of focal adhesion kinase (FAK) signaling on periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Methods and Results: Periodic mechanical stress significantly induced sustained phosphorylation of FAK at Tyr³⁹⁷ and Tyr^576/577. Reduction of FAK with targeted shRNA via transfection of NH₂-terminal tyrosine phosphorylation-deficient FAK mutant Y397F or Y576F-Y577F abolished periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis, accompanied by attenuated ERK1/2 phosphorylation. However, activation of Src, PLCγ1 and Rac1 was not prevented upon FAK suppression. Furthermore, pretreatment with the Src-selective inhibitor, PP2, and shRNA targeted to Src or suppression of Rac1 with its selective inhibitor, NSC23766, blocked FAK phosphorylation at Tyr,^576/577 but not Tyr,³⁹⁷ under periodic mechanical stress. Interestingly, FAK phosphorylation neither at Tyr³⁹⁷ nor at Tyr^576/577 was affected by PLCγ1 depletion when periodic mechanical stress was applied. In addition, Tyr³⁹⁷ and Tyr^576/577 phosphorylation levels were reduced upon pretreatment with a blocking antibody against integrin β1 under conditions of periodic mechanical stress. Conclusion: Our findings collectively suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through at least two pathways, integrin β1-Src-Rac1-FAK(Tyr^576/577)-ERK1/2 and integrin β1-FAK (Tyr³⁹⁷)-ERK1/2.

Focal Adhesion Kinase as a Cellular Mechano-Sensor

“…We here fill this gap and for the first time, to our knowledge, propose a mechano-biochemical combined signaling pathway, in which the first step is triggered by force. Introducing our proposed activation mechanism of FAK into the downstream signaling network allows to predict ERK activation kinetics as a function of external force, a dependency already observed previously in multiple experiments [123,134].…”

“…Phosphorylation, being if cis or trans, is re-quired to open FAK. Also, studies have showed that the auto-phosphorylation of Tyr397 is regulated by external force [33,123,124]. Thus, the opening motion of FAK is considered as the first reaction in our kinetic model.…”

Proteomic Analysis Reveals Grb2 as a Key Regulator of Periodic Mechanical Stress Transduction in Chondrocytes

“…The adhesive interactions of cells with their environment through the integrin family of transmembrane receptors have key roles in regulating multiple aspects of cellular physiology, including cell proliferation, viability, differentiation and migration16 [18]. Given that a clear role for integrins in mechnosensing has been established, and our previously studies revealed that mechanical induced proliferation and matrix synthesis were partly initiated by integrin β1 [7-9], we supposed the overexpression of Grb2 was associated with integrin. As expected, we observed the stress-induced enhancement of Grb2 expression was attenuated following integrin β1 down-regulation.…”

Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes

“…The periodic stress field with adjustable stress intensity and frequency was constructed by connecting a reciprocating intensifier pump to an air-tight cell culture device (Taixing Experimental Instrument Factory, Jiangsu, China) through a barrier-type pressure transducer (Tianjin Plastics Research Institute, Tianjin, China) as described previously [8, 9]. After passage to the second generation, chondrocytes were seeded at a density of 10 5 cells/ml on glass slides (diameter: 24 mm) or culture bottles (area: 75 cm 2 ).…”

“…Some studies have revealed that periodic mechanical stress can promote chondrocyte proliferation, migration and matrix synthesis of type II collagen and aggrecan [4-7]. In our previous studies, we found that a periodic stress field with a pressure range of 0–200 kPa at 0.1 Hz had the greatest effect on chondrocytes and promoted their proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway [8, 9]. There are likely other effects of periodic mechanical stress that have not been fully identifid in chondrocytes.…”