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

Abstract

Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl 2 and an associated decrease of cell volume. We have previously reported that a Cl 2 conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca 21 concentration ([Ca 21 ] i ) in human chondrocytes. The molecular identity and pathologic function of these Cl 2 channels, however, remained to be determined. Here, we show that the ClC-7 Cl 2 channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl 2 currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4ʹ-diisothiocyanatostilbene-2,2ʹ-disulfonic acid (DIDS; IC 50 5 13 mM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca 21 ] i . ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca 21 ] i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

A New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function

“…RNA Extraction and Real-time PCR-Total RNA extraction from OUMS-27 cells, human articular cartilage and selected mouse tissues, and Q-PCR were performed as reported previously (8,9). Human total RNA was purchased from BioChain (Newark, CA).…”

Physiological and Pathological Functions of Cl⁻ Channels in Chondrocytes

“…In addition to cation channel currents, Cl − currents have been recorded in articular chondrocytes. 1) In articular chondrocytes, the resting membrane potential is regulated by Cl − conductance 29,30) in addition to K + conductance. 9,10) The dependence of resting membrane conductance to Cl − is stronger than that to K + , although there is still a balance between them in chondrocytes.…”

“…Pharmacological blockade and genetic knockdown of Cl − channels cause membrane hyperpolarization of chondrocytes. 30,31) In non-excitable cells such as chondrocytes, membrane hyperpolarization facilitates Ca 2+ entry through non-selective cation channels. An increase in [Ca 2+ ] i promotes the syntheses and secretion of extracellular matrix of cartilage.…”

“…29,35) Also, voltage-dependent Cl − currents sensitive to classical Cl − channel blockers, niflumic acid, DIDS, and anthracene-9-carboxylic acid (9-AC) as well as SITS, have been identified in a cell line of human chondrocytes, OUMS-27. 30,31) DNA microarray analyses revealed that two human (ClC-3 and ClC-7) and three rodent (ClC-3, ClC-4, and ClC-6) ClC transcripts are detectable in chondrocytes 36) ( Table 2). In addition, mRNA expression of ClC-3 has been observed in rat articular chondrocytes, although it is potentially responsible for swelling-activated Cl − channels.…”

“…Pharmacological blockade and genetic knockdown of Cl − channels also cause membrane hyperpolarization followed by [Ca 2+ ] i increase in OUMS-27 cells. 30,31) Sustained [Ca 2+ ] i increase is likely to induce extracellular matrix synthesis, cytokine production, and cell proliferation in chondrocytes. 1) In contrast, acidification at inflammatory region activates ClC-7 channels, causing membrane depolarization in OUMS-27 cells.…”