Excessive mechanical stress induces chondrocyte apoptosis through TRPV4 in an anterior cruciate ligament-transected rat osteoarthritis model

Abstract

Aims

Chondrocyte apoptosis is the most common pathological feature of cartilage in osteoarthritis (OA). Excessive mechanical stress can induce chondrocyte apoptosis and destroy cartilage tissue. Transient receptor potential channel vanilloid 4 (TRPV4) is a mechanosensitive ion channel that mediates chondrocyte response to mechanical stress. Here, we investigated the potential role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress.

Main methods

Using a rat OA anterior cruciate-ligament transection (ALCT) model, we detected immunolocalization of calmodulin protein and mRNA and protein levels of TRPV4, calmodulin, and cleaved caspase-8 in articular cartilage. Primary chondrocytes were isolated and cultured in vitro, and Fluo-4AM staining was used to assess intracellular Ca²⁺ levels in order to evaluate TRPV4-mediated Ca²⁺ influx. Flow cytometry and western blot were performed to detect apoptosis and apoptosis-related protein levels in chondrocytes, respectively.

Key findings

TRPV4 was upregulated in ALCT-induced OA articular cartilage, and we found that administration of a TRPV4 inhibitor attenuated cartilage degeneration. Additionally, TRPV4 specifically mediated extracellular Ca²⁺ influx, leading to chondrocyte apoptosis in vitro, which was inhibited by transfection of TRPV4 small-interfering RNA or administration of a TRPV4 inhibitor. Moreover, increased Ca²⁺ influx triggered apoptosis by upregulating FAS-associated protein with death domain and cleaved caspase-3, -6, -7, and -8 levels, with these effects abolished by TRPV4 knockdown or TRPV4 inhibition.

Significance

These results indicated that TRPV4 was upregulated in OA articular cartilage, and that excessive mechanical stress might induce chondrocyte apoptosis via TRPV4-mediated Ca²⁺ influx, suggesting TRPV4 as a potential drug target in OA.

Introduction

Osteoarthritis (OA) is the most common joint disease and also referred to as “immortal cancer”. Although not fatal, it results in a disability rate of 53%, and its incidence continues to increase. OA can cause joint stiffness, swelling, pain, or disability, and as age and joint-wear increase, OA incidence continues to significantly increase [1]. The knee joint shows the highest incidence of OA, which is characterized by progressive articular-cartilage degeneration [2]. Articular cartilage depends solely on resident cells (chondrocytes) to maintain the extracellular matrix, with chondrocyte apoptosis a central feature of cartilage degeneration in OA [3].

Numerous studies show that OA progression is related to not only to biochemical factors but also mechanical stress [4]. Excessive mechanical-stress loading is associated with articular-cartilage degradation and plays an important role in the occurrence and development of excessive chondrocyte apoptosis [5]. A recent study reported that apoptosis of growth-plate chondrocytes is regulated by mechanical stress, and that appropriate stretch stress can effectively promote cell proliferation and differentiation, whereas excessive stress promotes their apoptosis [6]. Additionally, Li et al. [7] showed that chondrocytes tend to undergo late-stage apoptosis under compressive loading, during which caspase-12 is significantly upregulated. Chondrocytes can convert external mechanical signals into intracellular metabolic signals via mechanosensitive channels [8], among which transient receptor potential channel vanilloid 4 (TRPV4) is one of the most important mechanosensitive ion channels and widely distributed in the organs and tissues of various organisms [9]. Mechanical stimulation can regulate metabolic responses by activating TRPV4 and increasing intracellular Ca²⁺ concentrations [10]. Previous studies indicated that TRPV4 protein is highly expressed in articular chondrocytes, and that TRPV4-mediated Ca²⁺ signaling plays a central role in the transduction of mechanical signals to support cartilage extracellular-matrix maintenance and joint health [8,11]. However, the role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress remains unknown.

This study determined the potential regulatory mechanism of TRPV4 in mediating Ca²⁺ influx and initiating chondrocyte apoptosis in a rat model of OA induced by mechanical stress.