Hdud IM, Mobasheri A, Loughna PT. Effect of osmotic stress on the expression of TRPV4 and BK Ca channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes. Am J Physiol Cell Physiol 306: C1050 -C1057, 2014. First published March 26, 2014 doi:10.1152/ajpcell.00287.2013.-The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and largeconductance Ca 2ϩ -activated K ϩ (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BK Ca channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BK Ca channel is involved in responding to elevated intracellular Ca 2ϩ and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P Ͻ 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P Ͻ 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P Ͻ 0.001).cartilage; chondrocyte; mitogen-activated protein kinase; osmotic; transient receptor potential vanilloid 4 ARTICULAR CARTILAGE covers the ends of bones in diarthrodial joints to provide protection from shearing and compressive forces generated secondary to joint articulation. Cartilage consists of extracellular matrix (ECM) and chondrocytes (3,30). ECM is composed mainly of collagen type II and proteoglycan (PG), as well as other small protein and glycoprotein components. Chondrocytes are the only resident cells found in articular cartilage. Their metabolic activity is strongly influenced by environmental factors, including soluble mediators, ECM composition, and dynamic changes induced by mechanical loading (13,46). Mechanical loading of articular cartilage induces fluid flow, mechanical membrane deformation, hydrostatic pressure, and osmotic stress (45).The osmolarity of the tissue fluid that bathes chondrocytes in the cartilage ECM is different from that of most other tissues and typically exceeds 380 mosM (47). The presence of polyanionic PG molecules in the ECM attracts cations, such as Na ϩ
Ion channels play important roles in chondrocyte mechanotransduction. The transient receptor potential vanilloid (TRPV) subfamily of ion channels consists of six members. TRPV1-4 are temperature sensitive calcium-permeable, relatively non-selective cation channels whereas TRPV5 and TRPV6 show high selectivity for calcium over other cations. In this study we investigated the effect of time in culture and passage number on the expression of TRPV4, TRPV5 and TRPV6 in articular chondrocytes isolated from equine metacarpophalangeal joints. Polyclonal antibodies raised against TRPV4, TRPV5 and TRPV6 were used to compare the expression of these channels in lysates from first expansion chondrocytes (P0) and cells from passages 1–3 (P1, P2 and P3) by western blotting. TRPV4, TRPV5 and TRPV6 were expressed in all passages examined. Immunohistochemistry and immunofluorescence confirmed the presence of these channels in sections of formalin fixed articular cartilage and monolayer cultures of methanol fixed P2 chondrocytes. TRPV5 and TRPV6 were upregulated with time and passage in culture suggesting that a shift in the phenotype of the cells in monolayer culture alters the expression of these channels. In conclusion, several TRPV channels are likely to be involved in calcium signaling and homeostasis in chondrocytes.
Background: Chondrocytes are regularly exposed to load-induced stimuli and have the capability to sense and respond to applied mechanical stress. However, the mechanisms involved in chondrocyte mechanotransduction are not clearly understood. The purpose of this study was to explore the effects of cyclic equibiaxial mechanical stretch on the expression of α-BK and TRPV4 channels.
BackgroundSox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined.ResultsThe active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level.ConclusionsThe increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.
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