The Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis via Ca2+ CaMKII–Dependent MAPK and Akt/ mTOR Pathways in a Rat Osteoarthritis Model

Abstract

Background/Aims: Chondrocyte apoptosis is a central pathological feature of cartilage in osteoarthritis (OA). Accumulating evidence suggests that calcium ions (Ca²⁺) are an important regulator of apoptosis. Previously, we reported that the transient receptor potential channel vanilloid (TRPV5) is upregulated in monoiodoacetic acid (MIA)-induced OA articular cartilage. Methods: The protein levels of TRPV5, phosphorylated Ca²⁺/calmodulin-dependent kinase II (p-CaMKII), and total CaMKII were detected in vivo using western blotting techniques. Primary chondrocytes were isolated and cultured in vitro. Then, p-CAMKII was immunolocalized by immunofluorescence in chondrocytes. Fluo-4AM staining was used to assess intracellular Ca²⁺. Annexin V-fluorescein isothiocyanate / propidium iodide flow cytometric analysis was performed to determine chondrocyte apoptosis. Western blotting techniques were used to measure the expression of apoptosis-related proteins. Results: We found that ruthenium red (aTRPV5inhibitor)or(1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaze (KN-62) (an inhibitor of Ca^2+/calmodulin-dependent kinase II (CaMKII) phosphorylation) can relieve or even reverse OA in vivo. We found that TRPV5 has a specific role in mediating extracellular Ca²⁺ influx leading to chondrocyte apoptosis in vitro. The apoptotic effect in chondrocytes was inhibited by KN-62. We found that activated p-CaMKII could elicit the phosphorylation of extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal kinase, and p38, three important regulators of the mitogen-activated protein kinase (MAPK) cascade. Moreover, we also showed that activated p-CaMKII could elicit the phosphorylation of protein kinase B (Akt) and two important downstream regulators of mammalian target of rapamycin (mTOR): 4E-binding protein, and S61 kinase. Conclusion: Our results demonstrate that upregulated TRPV5 may be an important initiating factor that activates CaMKII phosphorylation via the mediation of Ca²⁺ influx. In turn, activated p-CaMKII plays a critical role in chondrocyte apoptosis via MAPK and Akt/mTOR pathways.

K+ and Ca2+ Channels Regulate Ca2+ Signaling in Chondrocytes: An Illustrated Review

“… The chondrocyte responds to the change in [Ca 2+ ] i , by sometimes generating [Ca 2+ ] i waves or oscillations [ 51 ]. These can activate cascades of Ca 2+ -dependent enzymes, (CaMK2 [ 6 , 7 ], NFAT, and calcineurin [ 8 , 11 ]); enhanced secretion of cytokines, catabolic factors and paracrine substances [ 52 , 53 ]; increased Ca 2+ -dependent secretion of essential extracellular matrix ([ 2 , 10 , 38 , 54 ]) and significant changes in cellular transcription activity [ 10 , 54 ] and/or altered proliferation as well as differentiation [ 11 , 51 , 55 ]. …”

“…In articular joints (elbows, shoulders, knees), at least two types of non-excitable cells need to be studied: synovial fibroblasts and chondrocytes. In healthy articular joints, both of these cells synthesize and secrete essential lubricants [ 2 ] (hyaluronan, lubricin) and both also exhibit altered function associated with progressive development of rheumatoid and/or osteoarthritis [ 3 , 4 , 5 , 6 , 7 , 8 ] and related pain [ 9 ].…”

“…It is well known that the precise regulation of intracellular Ca 2+ levels ([Ca 2+ ] i ) is required for normal matrix metabolic and secretory functions, as well as cartilage differentiation [ 10 , 11 ]. Dysregulation of [Ca 2+ ] i homeostasis is very likely to be an important factor in the initiation and rate of progression of chronic articular joint diseases such as arthritis [ 3 , 6 , 7 , 8 ] and related inflammation [ 12 ].…”

Progesterone induces cell apoptosis via the CACNA2D3/Ca2+/p38 MAPK pathway in endometrial cancer

“…An increase in cytoplasmic Ca 2+ levels activates the MAPK cascade (26,27). Three distinct groups of MAPKs, including ERK1/2, JNK, and p38 MAPK, are important for cancer cell apoptosis (28). To examine the signaling pathways behind the effects of CACNA2D3 and P4 on Ishikawa cell apoptosis, the activity of ERK, JNK and p38 MAPK pathways was measured following CACNA2D3 overexpression or P4 treatment.…”

Phosphorylated claudin-16 interacts with Trpv5 and regulates transcellular calcium transport in the kidney

“…Reagents, Antibodies, Cell Lines, and Animals. The following antibodies were used: anti-GFP (IP grade; Abcam), anti-Myc (Roche), anti-Flag (IP grade; Sigma), anti-claudin-16 (35), anti-NCC (Santa Cruz Biotech), anti-NCC (Millipore), anti-Aqp2 (Santa Cruz Biotech), anti-parvalbumin (Swant), anti-calbindin-D28K (Swant), anti-THP (R&D Systems), anti-tubulin (University of Iowa Hybridoma Bank), anti-Trpv5 (Abcam) (46), and anti-Trpv5 (Alpha Diagnostics) (47). Anti-CLDN16-P antibody was made against the phosphorylated peptide AKMYAVD(pT)RV.…”