Periodic Mechanical Stress Induces Extracellular Matrix Expression and Migration of Rat Nucleus Pulposus Cells Through Src-GIT1-ERK1/2 Signaling Pathway.

Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.

Gao G ，Li H ，Huang Y ，Yin J ，Jiang Y ，Xu N ，Zhou D ，Nong L ，Ren K ... -  《-》

Periodic Mechanical Stress Stimulates GIT1-Dependent Mitogenic Signals in Rat Chondrocytes Through ERK1/2 Activity.

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

Ren K ，Tang J ，Jiang X ，Sun H ，Nong L ，Shen N ，Gu Y ... -  《-》

Periodic mechanical stress induces the extracellular matrix expression and migration of rat nucleus pulposus cells by upregulating the expression of intergrin α1 and phosphorylation of downstream phospholipase Cγ1.

Gao G ，He J ，Nong L ，Xie H ，Huang Y ，Xu N ，Zhou D ... -  《-》

GIT1Y321 phosphorylation is required for ERK1/2- and PDGF-dependent VEGF secretion from osteoblasts to promote angiogenesis and bone healing.

Rui Z ，Li X ，Fan J ，Ren Y ，Yuan Y ，Hua Z ，Zhang N ，Yin G ... -  《-》

Periodic mechanical stress enhances rat chondrocyte area expansion and migration through Src-PLCgamma1-ERK1/2 signaling.

The signal transduction pathways of chondrocyte area expansion and migration under periodic mechanical stress remain a matter of debate. We explore this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, we find that both rat chondrocyte area and migration are significantly amplified. These changes are associated with increases in the phosphorylation of Src, PLCgamma1 and ERK1/2. Area expansion, migration and phosphorylation of PLCgamma1-Tyr(783) and ERK1/2-Thr(202)/Tyr(204) are inhibited (p<0.05) after pretreatment with Src inhibitor (PP2). We further demonstrate that area expansion, migration and phosphorylation of ERK1/2-Thr(202)/Tyr(204) are significantly inhibited (p<0.05) after pretreatment with PLCgamma1 inhibitor (U73122); the phosphorylation site of Src-Tyr(418) is not affected. After pretreatment with an ERK1/2 inhibitor (PD98059), area expansion and migration are inhibited (p<0.05), while the phosphorylation sites of Src-Tyr(418) and PLCgamma1-Tyr(783) are not affected. These findings suggest that periodic mechanical stress promotes chondrocyte area expansion and migration in part through the Src-PLCgamma1-ERK1/2 signaling pathway.

Nong L ，Yin G ，Ren K ，Tang J ，Fan W ... -  《-》