Expression of Transient Receptor Potential Vanilloid (TRPV) Channels in Different Passages of Articular Chondrocytes

Abstract

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.

TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading

“…Previous studies have observed [Ca 2+ ] i signaling in response to mechanical loading (38,39); interestingly, these studies detected [Ca 2+ ] i signaling in chondrocyte-laden constructs without substantial preculture (<72 h), although chondrocytes synthesize small amounts of pericellular proteoglycans even within 2 d of culture in agarose (29) that could contribute to mechanically induced [Ca 2+ ] i signaling. Thus, the effects of loading with and without preculture, observed in this study as well as in previous studies, may be due to differences in the characteristics of the [Ca 2+ ] i signal with preculture, or perhaps other environmental and cellular factors that change over time (24,26,28,40). Although chondrocyte [Ca 2+ ] i signaling exhibits a high sensitivity to variations in osmolarity or volume (41)(42)(43)(44), there is little or no sensitivity to physiologic levels of other physical stimuli, such as direct membrane stretch, suggesting that membrane connections to the pericellular matrix and/or intracellular cytoskeleton are important for the generation and transduction of [Ca 2+ ] i transients.…”

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes

“…In nonexcitable cells such as chondrocytes, membrane hyperpolarization facilitates Ca 21 entry through nonselective cation channels (Gavenis et al, 2009;Funabashi et al, 2010b;Hdud et al, 2012). Blockage of Cl 2 channels by DIDS caused a significant hyperpolarization followed by an increase in [Ca 21 ] i .…”

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

“…The expression level of the BK Ca channel in EACs following exposure to hypo-osmotic, mild hypo-osmotic, and hyperosmotic stresses was monitored at different time points. Western blotting using a BK Ca channel-specific antibody was used to examine the effect of osmotic stress on BK Ca channel expression, as previously described (16). There were no significant changes in BK Ca channel expression following hypo-osmotic and mild hypo-osmotic stress after 90 min and 3 h, whereas 6 h of incubation under hypo-osmotic conditions induced a significant (1.5-fold) increase in BK Ca channel expression (P Ͻ 0.01; Fig.…”

“…A medium osmolarity of 380 mosM was used as the control condition. Western blotting using a TRPV4 channel-specific antibody, as described previously (16), was used to explore TRPV4 channel expression following hypoosmotic, mild hypo-osmotic, and hyperosmotic stress. Exposure of chondrocytes to hypo-osmotic stress for 6 h (P Ͻ 0.01), 3 h (P Ͻ 0.001), and 90 min (P Ͻ 0.001) increased TRPV4 channel expression by Ͼ1.5-fold (Fig.…”

“…Investigations of several other cell types have shown that entry of extracellular Ca 2ϩ and consequent activation of large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels are essential for initiation of RVD. Expression of TRPV4 and BK Ca channels varies be-tween tissues and cell types and was recently demonstrated in equine chondrocytes in vivo and in vitro (16). Expression of these channels at the mRNA and protein levels closely correlates with their activity (20,49).…”