Chondrocyte mechanotransduction is not well understood, but recently, it has been proposed that mechanically activated ion channels such as transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 are of functional importance in chondrocyte mechanotransduction. The aim of this study was to distinguish the potential contributions of TRPV4, Piezo1, and Piezo2 in transducing different intensities of repetitive mechanical stimulus in chondrocytes. To study this, TRPV4-, Piezo1-, or Piezo2-specific siRNAs were transfected into cultured primary chondrocytes to knock down (KD) TRPV4, Piezo1, or Piezo2 expression, designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells. Then we used Flexcell® Tension System to apply cyclic tensile strains (CTS) of 3% to 18% at 0.5 Hz for 8 h to the knockdown and control siRNA-treated cells. Finally, using a Ca2+ imaging system, stretch-evoked intracellular Ca2+ ([Ca2+] i) influx in chondrocytes was examined to investigate the roles of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different intensities of repetitive mechanical stretch stimulation. The characteristics of [Ca2+] i in chondrocytes evoked by stretch stimulation were stretch intensity dependent when comparing unstretched cells. In addition, stretch-evoked [Ca2+] i changes were significantly suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, indicating that any channel essential for Ca2+ signaling induced by stretch stimulation in chondrocytes. Of note, they played different roles in calcium oscillation induced by different intensities of stretch stimulation. More specifically, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to physiologic levels of strain (3% and 8% of strain), while Piezo2-mediated Ca2+ signaling played a central role in the response of chondrocytes to injurious levels of strain (18% of strain). These results provide a basis for further examination of mechanotransduction in cartilage and raise a possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of cartilage disease induced by repetitive mechanical forces. Impact statement Chondrocytes in cartilage are constantly subjected to load-induced stimuli and regulate their metabolic activities in order to maintain cartilage homeostasis. Therefore, mechanotransduction is important in chondrocytes and is vital for their role in cartilage function. Our results indicate that chondrocytes might sense and distinguish the different intensities of repetitive mechanical stimulus by using different mechanosensitive ion channels. Specifically, TRPV4 is mainly responsible for sensing physiologic levels of repetitive CTS stimulus, while Piezo2 mainly contributes to chondrocyte sensing noxious levels of repetitive CTS loading. These results provide a basis for further examination of mechanotransduction in cartilage and raise the possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of OA which is induced by injurious and repetitive mechanical stimulation.

Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.

BackgroundCorneal tensile strain increases if the cornea becomes thin or if intraocular pressure increases. However, the effects of mechanical stress on extracellular matrix (ECM) remodelling in the corneal repair process and the corneal anomalies are unknown.MethodsIn this study, the combined effects of interleukin-1β (IL-1β) on matrix metalloproteinases (MMPs) in corneal fibroblasts under cyclic stretching were investigated in vitro. Cultured rabbit corneal fibroblasts were subjected to 5, 10 or 15 % cyclic equibiaxial stretching at 0.1 Hz for 36 h in the presence of IL-1β. Conditioned medium was harvested for the analysis of MMP2 and MMP9 protein production using the gelatin zymography and western blot techniques.Results and conclusionsCyclic equibiaxial stretching changed the cell morphology by increasing the contractility of F-actin fibres. IL-1β alone induced the expression of MMP9 and increased the production of MMP2, and 5 % stretching alone decreased the production of MMP2, which indicates that a low stretching magnitude can reduce ECM degradation. In the presence of IL-1β, 5 and 10 % stretching increased the production of MMP2, whereas 15 % stretching increased the production of MMP9. These results indicate that MMP expression is enhanced by cyclic mechanical stimulation in the presence of IL-1β, which is expected to contribute to corneal ECM degradation, leading to the development of post-refractive surgery keratectasia.

Abstract. In order to understand the effect of mechanical stretch on corneal extracellular matrix remodeling, human keratoconus fibroblasts (HKCFBs) were subjected to cyclic stretch in vitro and the expression of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and inflammatory cytokines were evaluated. HKCFBs were seeded into a flexible membrane base and subjected to a cyclic stretch regimen of 10% equibiaxial stretch at a stretching frequency of 1 Hz for 6 h using a Flexcell tension unit. An antibody directed against interleukin-6 (IL-6 Ab) was used to investigate the roles of IL-6 on mechanical stretch mediated regulation of MMP in HKCFBs. Culture supernatants were assayed using an enzyme-linked immunosorbent assay for MMP-1 and -3, TIMP-1 and -2, and IL-6. Total RNA from the cells was extracted, and quantitative polymerase chain reaction was used to determine mRNA for MMP-1 and -3, TIMP-1 and -2, and IL-6. In stretched cells, levels of MMP-1 and -3 demonstrated an increase compared with unstretched cells, but levels of TIMP-1, and -2 revealed a decrease. Mechanical stretch significantly increased the mRNA expression and protein synthesis of IL-6 compared with unstretched cells. IL-6 induced MMP-1 and -3 expression, whereas no significant effects were observed in levels of TIMP-1 and -2 compared with the untreated control groups. Additionally, the IL-6 Ab markedly inhibited the stretch-induced increase in MMP-1 and -3 in culture supernatants in a dose-dependent manner. No significant differences in TIMP-1 and -2 protein levels were detected between stretched cells treated with IL-6 Ab and stretched cells without IL-6 Ab treatment. These results indicate that cyclical mechanical stretch augments IL-6 production and MMP expression, and reduces levels of TIMP in HKCFBs. Thus, it is suggested that IL-6 mediates the stretch-induced MMP expression.

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