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

BACKGROUND/AIMS: 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. METHODS: 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 ß1 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. RESULTS: 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 ß1, 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. CONCLUSION: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin ß1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin ß1-FAK(Tyr397)-ERK1/2.

Periodic Mechanical Stress Stimulates Cav-1-Dependent IGF-1R Mitogenic Signals in Rat Chondrocytes Through ERK1/2.

BACKGROUND/AIMS: The biological effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to mechanical stimuli remain to be determined. In the current study, we analyzed the mechanisms by which periodic mechanical stress is translated into biochemical signals and verified the key role of non-integrin mechanosensors including Caveolin-1 (Cav-1), and insulin-like growth factor-1 receptor (IGF-1R) in chondrocyte proliferation. METHODS: Two steps were undertaken in the experiment. In the first step, the cells were maintained under static conditions or periodic mechanical stress for 0 h and 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to Cav-1 or IGF-1R or control scrambled shRNA. Moreover, they were pretreated with their selective inhibitors methyl ß-cyclodextrin (MCD) or Linsitinib (OSI-906). They were maintained under static 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. RESULTS: Periodic mechanical stress significantly induced sustained phosphorylation of Cav-1 at Tyr14 and IGF-1R at Tyr1135/1136. Proliferation was inhibited by pretreatment with Cav-1 inhibitor MCD and by shRNA targeted to Cav-1 in chondrocytes in response to periodic mechanical stress. Meantime, MCD and shRNA targeted to Cav-1 also attenuated IGF-1R, and extracellular signal-regulated kinase (ERK)1/2 activation. In addition, inhibiting IGF-1R activity by Linsitinib and shRNA targeted to IGF-1R abrogated chondrocyte proliferation and phosphorylation level of ERK1/2 subjected to periodic mechanical stress, while the phosphorylation site of Cav-1 was not affected. CONCLUSION: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through Cav-1-IGF-1R-ERK1/2.

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

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND/AIMS: Periodic mechanical stress could significantly promote chondrocyte proliferation and matrix synthesis. However, the mechanisms underlying the ability of chondrocyte detecting and responding to periodic mechanical stimuli have not been well delineated. METHODS: Quantitative proteomic analysis was performed to construct the differently expressed proteome profiles of chondrocyte under pressure. Then a combination of Western blot, quantitative real-time PCR, lentiviral vector and histological methods were used to confirm the proteomic results and investigate the mechanoseing mechanism. RESULTS: Growth factor receptor-bound protein 2 (Grb2), a component of integrin adhesome, was found a 1.49-fold increase in dynamic stress group. This process was mediated through integrin ß1, leading to increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) respectively and then produce the corresponding biological effects. CONCLUSION: This was the first time to demonstrate Grb2 has such an important role in periodic mechanotransduction, and the proteomic data could facilitate the further investigation of chondrocytes mechanosensing.

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling.

BACKGROUND/AIMS: Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin ß1-ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. METHODS: Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. RESULTS: Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr402 and CaMKII at Thr286. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr286 and Pyk2- Tyr402 were abolished after pretreatment with blocking antibody against integrinß1 exposed to periodic mechanical stress. CONCLUSION: Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinß1-CaMKII-Pyk2-ERK1/2 signaling cascade.

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

BACKGROUND/AIMS: In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress. In our previous studies, we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway, and we used proteomic analysis to detect quantitative changes in chondrocytes under periodic mechanical stress. Despite these results, the effects and mechanisms of periodic mechanical stress are still not fully understood, so in this study we extended our study using phosphoproteomic techniques. METHODS: We used phosphoproteomic techniques to detect phosphorylation changes in chondrocytes under periodic mechanical stress and combined the results with the quantitative proteomic data to further explore the underlying mechanisms. Data were obtained by phosphorylation inhibition, quantitative real-time PCR (qPCR) analysis, western blot analysis and immunofluorescence assay. RESULTS: From phosphoproteomic analysis, a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified. The number of significant differentially expressed proteins and phosphopeptides was 97 and 108, respectively (ratio >1.20 or <0.83 at p<0.05). Periodic mechanical stress increased glycogen synthase kinase 3-beta (GSK3-beta) phosphorylation at Y216, promoted the phosphorylation of beta-catenin, decreased beta-catenin levels and suppressed the expression of type I collagen. In contrast, inhibition of GSK3-beta by TWS119, which specifically inhibits the phosphorylation of Y216, suppressed the phosphorylation of beta-catenin, which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen. CONCLUSIONS: We successfully constructed differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress, and discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of GSK3-beta and beta-catenin.

Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2.

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes.

In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin ß1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin ß1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin ß1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.

Effects of periodic mechanical stress on cytoskeleton dependent lipid raft-induced integrin ɑ1 activation in rat nucleus pulposus cells.

Extracellular matrix (ECM) production and nucleus pulposus (NP) cell migration increase under periodic mechanical stress (PMS), but the underpinning regulatory mechanism remains unclear. This work aimed to examine the regulatory effects of cytoskeleton-lipid raft-integrin α1 signaling in NP cells exposed to PMS. Briefly, In NP cells, cytoskeleton rearrangement, lipid raft aggregation and integrin α1 expression in the stress and control groups were assessed by immunofluorescent staining and immunoblot. In addition, cell migration and ECM gene expression were detected by a scratch test and quantitative reverse transcription polymerase chain reaction (qRT­PCR), respectively. As a result, PMS up-regulated ECM gene expression and enhanced NP cell migration (both P < 0.05), accompanied by increased integrin α1, lipid raft, caveolin-3, F-actin and ß-tubulin amounts. Pretreatment with the lipid raft inhibitor methyl-ß-cyclodextrin (MßCD) or small interfering RNA (siRNA) targeting caveolin-3 resulted in decreased ECM mRNA synthesis and cell migration induced by PMS (both P < 0.05); meanwhile, integrin α1 expression was also reduced. F-actin and ß-tubulin inhibition by cytochalasin D and colchicine, respectively, not only reduced ECM mRNA synthesis and cell migration (both P < 0.05), but also disrupted lipid raft and caveolin-3 amount increases induced by PMS in NP cells. In conclusion, PMS promotes ECM mRNA up-regulation and cell migration through the cytoskeleton-lipid raft-integrin α1 signaling pathway, inhibiting cytoskeleton and lipid rafts could block the cellular effects.

Periodic Mechanical Stress Inhibits the Development of Osteoarthritis via Regulating ATF3-Akt Axis.

Purpose: The development of osteoarthritis (OA) has been linked to mechanical factors. Studies suggest that periodic mechanical stress (PMS) may be a factor contributing to cartilage repair and the onset of OA. Therefore, this study was designed to explore the effects and underlying mechanisms of PMS on OA development. Patients and Methods: Firstly, surgery and interleukin (IL)-1ß were used for the establishment of rat/cell models of OA, respectively. Subsequently, activating transcription factor (ATF) 3 expression was knocked down in OA rats, and OA chondrocytes were treated with different heights (0, 1, 2, 4, 8 cm) of PMS or si-ATF. Safranin O staining was used to observe the histological changes in the rat knee joint, and enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of tumor necrosis factor (TNF)-α, IL-6, and IL-8 in vivo and in vitro. Further, the expression of extracellular matrix (ECM) proteins in the rat knee joint was assessed immunohistochemistry. Flow cytometry was used to evaluate chondrocyte apoptosis. Lastly, Western blot was performed to detect the expression of related proteins of the protein kinase B (Akt) signaling pathway and ECM. Results: The OA rat model was successfully constructed. Further experiments indicated that the knockdown of ATF3 not only alleviated joint swelling, pain, inflammatory response and pathological damage, but also promoted ECM synthesis and the phosphorylation of Akt in OA rats. In vitro experiments showed that PMS (4 cm) effectively inhibited cell apoptosis, decreased the levels of TNF-α, IL-6 and IL-8, promoted ECM synthesis, and activated the Akt signaling pathway in osteoarthritic chondrocytes. However, ATF3 overexpression reversed the positive effects of PMS on osteoarthritic chondrocytes. Conclusion: PMS can effectively inhibit the development of OA, and its protective effects may be attributed to the down-regulation of ATF3 expression and activation of the Akt signaling pathway.

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.