Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes

Abstract

In a variety of physiological and pathophysiological conditions, cells are exposed to acidic environments. Severe synovial fluid acidification also occurs in a progressive state of osteoarthritis (OA) affecting articular chondrocytes. In prior studies extracellular acidification has been shown to protect cells from apoptosis but the underlying mechanisms remain elusive. In the present study, we demonstrate that the inhibition of Cl− currents plays a significant role in the antiapoptotic effect of acidification in human articular chondrocytes. Drug-induced apoptosis was analyzed after exposure to staurosporine by caspase 3/7 activity and by annexin-V/7-actinomycin D (7-AAD) staining, followed by flow cytometry. Cell viability was assessed by resazurin, CellTiter-Glo and CellTiter-Fluor assays. Cl− currents and the mean cell volume were determined using the whole cell patch clamp technique and the Coulter method, respectively. The results reveal that in C28/I2 cells extracellular acidification decreases caspase 3/7 activity, enhances cell viability following staurosporine treatment and gradually deactivates the volume-sensitive outwardly rectifying (VSOR) Cl− current. Furthermore, the regulatory volume decrease (RVD) as well as the apoptotic volume decrease (ADV), which represents an early event during apoptosis, were absent under acidic conditions after hypotonicity-induced cell swelling and staurosporine-induced apoptosis, respectively. Like acidosis, the VSOR Cl− current inhibitor DIDS rescued chondrocytes from apoptotic cell death and suppressed AVD after induction of apoptosis with staurosporine. Similar to acidosis and DIDS, the VSOR channel blockers NPPB, niflumic acid (NFA) and DCPIB attenuated the staurosporine-induced AVD. NPPB and NFA also suppressed staurosporine-induced caspase 3/7 activation, while DCPIB and Tamoxifen showed cytotoxic effects per se. From these data, we conclude that the deactivation of VSOR Cl− currents impairs cell volume regulation under acidic conditions, which is likely to play an important role in the survivability of human articular chondrocytes.

Paclitaxel induces pyroptosis by inhibiting the volume‑sensitive chloride channel leucine‑rich repeat‑containing 8a in ovarian cancer cells

“…Following the activation of caspase proteins, gasdermin family proteins have been revealed to cleave to separate their N-terminal pore-forming domain from the C-terminal repressor domain, and finally the N-terminal is inserted into cell membranes and forms large oligomeric pores, disrupting ion homeostasis and subsequently inducing osmotic swelling (27,36). Several studies, including the present one, have demonstrated that the blockage of chloride channels led to cell swelling in both isotonic and hypotonic conditions (8,23,29,40). It was also demonstrated that PTX could inhibit the background-, hypotonicity-and cisplatin-induced chloride currents in A2780 ovarian cancer cells (22).…”

Genome-wide CRISPR screening of chondrocyte maturation newly implicates genes in skeletal growth and height-associated GWAS loci

“…While other chondrogenic cell lines, such as C28/I2, ATDC5, and C3H10T1/2, have been used for in vitro studies, they are not suitable for genome-wide growth-plate chondrocyte screening due to limitations of culture conditions and/or recapitulation of an articular cartilage phenotype. 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 Although use of the GPLC cell line enabled genome-wide screening, our chondrocyte maturation assay favors detection of KOs that have a proliferative advantage (most abundant KOs within the top/bottom 10% of mature/immature cells were selected). To account for this potential shortcoming, we considered background depletion of maturing cells.…”

Bio-responsive and multi-modality imaging nanomedicine for osteoarthritis theranostics

“…3 The extracellular matrix (ECM) of the articular cartilage mainly comprises type II collagen (Col II) and aggrecan (ACAN), and chondrocytes are wrapped in the lacuna, which is surrounded by a dense network of Col II and ACAN. 4,5 However, as the disease develops and progresses, many endogenous characteristic alterations become evident in the ECM, such as weakly acidic pH (5.0-6.5) of the synovial fluid 6 and overexpression of matrix metalloproteinases (MMPs; e.g., MMP3, MMP9, and MMP13). 7 However, these inherent traits of the damaged cartilage and microenvironment features in OA provide valuable references for its treatment.…”