BACKGROUND/AIMS: The present study aimed to analyze the mechanisms by which periodic mechanical stress is translated into biochemical signals, and to verify the important role of signaling molecules including phosphatidylinositol-3-kinase (PI3K)-Akt, protein kinase C (PKC), and epidermal growth factor receptor (EGFR) in chondrocyte proliferation. The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress need further investigation. METHODS: Two steps were undertaken in the experiment. In the first step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, GF109203X, Gö6976, rottlerin, and AG1478. They were maintained under static conditions or periodic mechanical stress for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. In the second step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, AG1478, and rottlerin. They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis. RESULTS: Proliferation was inhibited by pretreatment with PKC or PKCδ inhibitor GF109203X or rottlerin and by short hairpin RNA (shRNA) targeted to PKCδ, but not by PKCα inhibitor Gö6976 in chondrocytes in response to periodic mechanical stress. Meantime, rottlerin and shRNA targeted to PKCδ also attenuated EGFR, Akt, and ERK1/2 activation. Furthermore, inhibiting EGFR activity by AG1478 and shRNA targeted to EGFR abrogated chondrocyte proliferation and phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK)1/2 subjected to periodic mechanical stress, while the phosphorylation site of PKCδ was not affected. In addition, pretreatment with the PI3K-Akt-selective inhibitor LY294002 and shRNA targeted to Akt reduced periodic mechanical stress-induced chondrocyte proliferation and phosphorylation of ERK1/2, while the phosphorylation levels of EGFR and PKCδ were not inhibited. CONCLUSION: These findings suggested that periodic mechanical stress promoted chondrocyte proliferation through PKCδ-EGFR-PI3K-Akt-ERK1/2. They provide a stronger viewpoint for further investigations into chondrocyte mechanobiology under periodic mechanical stress and the ways to improve the quality of tissue-engineered cartilage.
BACKGROUND/AIMS: The present study aimed to analyze the mechanisms by which periodic mechanical stress is translated into biochemical signals, and to verify the important role of signaling molecules including phosphatidylinositol-3-kinase (PI3K)-Akt, protein kinase C (PKC), and epidermal growth factor receptor (EGFR) in chondrocyte proliferation. The effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively in recent years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to periodic mechanical stress need further investigation. METHODS: Two steps were undertaken in the experiment. In the first step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, GF109203X, Gö6976, rottlerin, and AG1478. They were maintained under static conditions or periodic mechanical stress for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. In the second step, the cells were pretreated with shRNA targeted to Akt or EGFR or PKCδ or control scrambled shRNA. Moreover, they were pretreated with LY294002, AG1478, and rottlerin. They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis. RESULTS: Proliferation was inhibited by pretreatment with PKC or PKCδ inhibitor GF109203X or rottlerin and by short hairpin RNA (shRNA) targeted to PKCδ, but not by PKCα inhibitor Gö6976 in chondrocytes in response to periodic mechanical stress. Meantime, rottlerin and shRNA targeted to PKCδ also attenuated EGFR, Akt, and ERK1/2 activation. Furthermore, inhibiting EGFR activity by AG1478 and shRNA targeted to EGFR abrogated chondrocyte proliferation and phosphorylation levels of Akt and extracellular signal-regulated kinase (ERK)1/2 subjected to periodic mechanical stress, while the phosphorylation site of PKCδ was not affected. In addition, pretreatment with the PI3K-Akt-selective inhibitor LY294002 and shRNA targeted to Akt reduced periodic mechanical stress-induced chondrocyte proliferation and phosphorylation of ERK1/2, while the phosphorylation levels of EGFR and PKCδ were not inhibited. CONCLUSION: These findings suggested that periodic mechanical stress promoted chondrocyte proliferation through PKCδ-EGFR-PI3K-Akt-ERK1/2. They provide a stronger viewpoint for further investigations into chondrocyte mechanobiology under periodic mechanical stress and the ways to improve the quality of tissue-engineered cartilage.