The Research On The Mechanism Of PRG4Modulation And TGFf-β1-induced Chondrogenic Differentiation Of Rat Temporomandibular Synovial Fibroblasts

Temporomandibular joint disorders (TMD) are common syndromes in stomatological clinic. Despite intensive research efforts, the pathophysiology of TMD has not been entirely elucidated because of their controversial etiology. As an important component of temporomandibular joint (TMJ), the synovium tissue appears to play an important role in the development of TMD. Many in vitro studies suggest that the synovial fibroblasts (SFs) isolated from synovium tissue have emerged as pivotal effector cells, which display unique biological characteristics that set their apart from other fibroblasts. This research uses rat temporomandibular synovial fibroblasts as a research topic and study the modulation of the lubrication function, chondrogenesis and lining layer construction, to explore the molecular mechanism of SFs participating in the process of adaptive change in TMJ.Part1The research on the mechanism of PRG4modulation of rat temporomandibular synovial fibroblastsObjectives:As a secreted mucinous glycoprotein, Proteoglycan4(PRG4) is as an essential lubricating molecule in synovial fluid, which plays a fundamental role in joint boundary lubrication. It is known that the alteration of PRG4expression is associated with changes of its chemical and physical environments. This study was designed to evaluate the combined effects of intermittent hydrostatic pressure (IHP) and TGF-β1or TNF-a on PRG4expression in rat temporomandibular SFs. The molecular mechanism of mechanical stress on PRG4expression was also investigated.Methods:Rat SFs were isolated and expanded in monolayer cultures and identified by immunofluorescence staining. Cells were subjected to IHP in the presence of TGF-β1or TNF-a. Quantitative real-time polymerase chain reaction (PCR) was applied to analyze the PRG4expression levels. Enzyme-linked immunosorbent assay (Elisa) was also used for the quantification of PRG4accumulation in the culture medium while immunofluorescence staining was used to detect intracellular PRG4protein expression. Western blot and immunofluorescence staining was used to detect the activation of target proteins. SB431542was employed to investigate the effect of Smad inhibition on the PRG4expression induced by IHP.Results:Our study showed that all the primary cultured cells were negative for CD68and positive for vimentin with a fibroblastic character, confirmed to be synovial fibroblasts (SFs). The combination of IHP and TGF-β1induced greater PRG4expression than either stimulus alone. In contrast, TNF-a inhibited PRG4expression, and this was partially alleviated by IHP. IHP can strengthen the Smad2/3phosphorylation in a time-dependent manner and induce nuclear translocation of Smad2/3; however, the protein content of phosphorylated P38did not vary significantly. In addition, SB431542greatly inhibited IHP-induced PRG4expression.Conclusions:Our study demonstrates a beneficial role of IHP, which can be used successfully in combination with TGF-β1to enhance PRG4production, and can partially counteract TNF-a-induced PRG4inhibition in isolated rat SFs. Smad signal acts as an essential mechanism to regulate PRG4expression induced by IHP.Part2TGF-β1regulates rat temporomandibular synovial fibroblasts into chondrogenic differentiation by RhoA/ROCK pathwayObjectives:Synovial fibroblasts (SFs) are synovium-derived mesenchymal cells, which have the potential to differentiate into chondrocytes and may play an important role in the regenerative response during arthritic diseases. TGF-β1is the most extensively examined growth factor for inducing chondrogenesis and cartilage matrix synthesis associated with cartilage regeneration. The objective of the present study was to investigate the possible role and mechanisms of RhoA/ROCK pathway during the process of chondrogenic differentiation of rat temporomandibular SFs promoted by TGF-β1.Methods:Chondrogenic differentiation of SFs was initiated in primary culture, in the presence of TGF-β1, and their actin reorganization was examined by FITC-phalloidin staining. The activation of RhoA/ROCK pathway was investigated using RhoA activation assay, quantitative real-time RT-PCR. Specific biochemical inhibitors, hydroxyfasudil and Y27632were employed to evaluate the function of RhoA/ROCK The roles of hydroxyfasudil and Y27632on gene expressions of chondrocyte-specific markers induced by TGF-β1were evaluated by quantitative real-time RT-PCR. To examine the effect of Y27632on Smad2/3phosphorylation induced by TGF-β1, Western blot analysis was also performed.Results:Stimulation of TGF-β1in SFs resulted in activation of RhoA/ROCK pathway and concomitantly induced cytoskeletal reorganization, which was specifically blocked by the RhoA/ROCK inhibitors, hydroxyfasudil and Y27632. TGF-β-induced gene expressions of type I collagen, type II collagen, aggrecan and Sox9were also inhibited by both hydroxyfasudil and Y27632, to different levels. Y27632treatment reduced phosphorylation of Smad2/3in a concentration-dependent manner.Conclusions:These results demonstrate that the RhoA/ROCK pathway plays a role in regulating the TGF-β1-stimulated transcription of chondrocyte-specific genes, through interaction with the TGF-β/Smad signaling pathway.Part3The role and mechanism of hydrostatic pressure and TNF-a on cadherin-11expression of rat temporomandibular synovial fibroblastsObjectives:Cadherin-11is selectively expressed on synovial fibroblasts (SFs) and plays a significant role in the formation of the lining layer of the synovium, synovial inflammation and the cartilage degradation. The aim of this study was to evaluate the regulation of cadherin-11distribution and expression in response to the hydrostatic pressure (HP) and inflammatory factor, TNF-α, and the signal transduction pathways of rat temporomandibular SFs.Methods:Cadherin-11expression in rat temporomandibular SFs was evaluated using immunofluorescence staining. HP was applied on cultured SFs at different levels for12h. Quantitative real-time RT-PCR and western blot were applied to analyze the cadherin-11expression levels. Time-course dependent expression of cadherin-11was also performed by stimulating with10ng/ml TNF-α. Extraction of total protein was for Western blot detection of a variety of signal-line protein expression. LY294002was employed to evaluate the function of PI3K/Akt.Results:Immunofluorescence analysis revealed that the cadherin-11is abundantly expressed at the edge of cell-to-cell junction, connecting tightly with F-actin cytoskeleton. Our study showed that the mRNA and protein expression of cadherin-11were significantly increased after HP stress and TNF-α treatment in a magnitude and time-course dependent manners. Both90kPa HP stress and10ng/ml TNF-α can significantly enhance PI3K and Akt phosphorylation, and Akt phosphorylation inhibitor LY294002can inhibit the phosphorylation level of Akt. We also found that HP stress and TNF-α significantly enhanced the expression of cadherin-11gene, and promoted cadherin-11protein expression. The enhancement could be prohibited by LY294002.Conclusions:Our study demonstrated that the exposure of SFs to HP and TNF-α could induce the expression of cadherin-11by the PI3K/Akt pathway, which was inhibited by the inhibitor of Akt pathway, LY294002.