Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis

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Published: 2nd Feb 2022

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The transient receptor potential channel, vanilloid 5, induces chondrocyte apoptosis via Ca²⁺-CaMKII–dependent MAPK and Akt/mTOR pathwaysin a rat osteoarthritis model

Abstract

Chondrocyte apoptosis is a central pathological feature in cartilage in osteoarthritis (OA). Accumulating evidence suggests that calcium ion (Ca²⁺) is an important regulator of apoptosis. Here, we report that the transient receptor potential channel vanilloid (TRPV5) is upregulated in monoiodoacetic acid (MIA)-induced OA articular cartilage. Ruthenium red (a TRPV5 inhibitor) or (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) (an inhibitor of Ca2+/Calmodulin-Dependent Kinase II (CaMKII) phosphorylation) can relieve or even reverse the OA invivo. 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 which led us to demonstrate that overloaded calcium influx is important in activating CaMKII phosphorylation. 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 regulator of mammalian target of rapamycin (mTOR): 4E-binding protein, and S61 kinase. Taken together, our results demonstrate that the TRPV5 cation channel is functionally upregulated in OA articular cartilage. 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. Our results underscore an intriguing role for TRPV5 and p-CaMKII as mediators and potential drug targets in OA.

Introduction

Osteoarthritis (OA) is a common, debilitating disease with a large societal and economic burden that leads to physical and psychological sequelae in the elderly population. Knee involvement, in the form of pain and stiffness, occurs more frequently than for other joints in OA, causing the greatest burden to the population, and often leads to significant disability requiring surgical intervention[1]. Pathologically, OA is characterized by the progressive degeneration of articular cartilage[2]. The cartilage becomes hypocellular in OA and is often accompanied by lacunar emptying, suggesting that chondrocyte apoptosis is a central feature in OA progression[3]. Thus, finding a cause for chondrocyte apoptosis during OA may become a potential and urgent strategy against this disease.

It is widely accepted that calcium ion (Ca²⁺) is a major intracellular second messenger and plays an important role in apoptosis. The disruption of Ca²⁺ homeostasis, due to its sustained elevation in the cytoplasm, can trigger apoptosis[4]. The increase in intracellular calcium levels can result in the activation of the calcium sensor protein, calmodulin (CaM), and the combination of target proteins to form a Ca²⁺/calmodulin complex[5]. Our study highlighted how an increase in Ca²⁺ influx through Ca²⁺-selective channels, such as the transient receptor potential channel vanilloid 5 (TRPV5), can accelerate OA progression by activating calcium/calmodulin-dependent protein kinase II (CAMKⅡ)[6]. Our finding also indicates that TRPV5 has a specific effect on the cytosolic Ca²⁺ concentration and cascade events of OA pathophysiology.

CaMKII, a Ser/Thr specific protein kinase, is a general integrator of Ca²⁺ signaling. CaMKII is activated in the presence of Ca²⁺ and CaM, which leads to autophosphorylation, generating a Ca²⁺/CaM-independent form of the enzyme[7].

Increasing evidence indicates an elevation in cytoplasmic Ca²⁺ levels activates the mitogen-activated protein kinase (MAPK) cascade. Three distinct groups of MAPKs, including extracellular signal-regulated protein kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK, are also involved in chondrocyte apoptosis and cartilage degeneration in a rat osteoarthritis model[8-10].

Numerous studies have demonstrated that the development of Ca²⁺ can also be critical for the amino acid–mediated activation of mammalian target of rapamycin (mTOR)[11]. A serine/threonine (Ser/Thr) protein kinase, mTOR regulates differentiation, development and survival in various cells. Specifically, protein synthesis is regulated by mTOR via the phosphorylation and inactivation of translational repressor 4E-binding protein (4E-BP1), and through the phosphorylation and activation of S6 kinase (S6K1)[12]. The overexpression of mTOR is observed in human OA cartilage as well as in mouse experimental OA. Upregulated mTOR expression corresponds to increased chondrocyte apoptosis during OA[13].

However, evidence is lacking for a specific mechanism of mTOR and MAPK signaling in OA pathophysiology. This prompted us to study whether p-CaMKII activates MAPK and mTOR pathways to trigger chondrocyte apoptosis by elevated [Ca²⁺] influx through TRPV5 in OA.

Materials and Methods

Animals and Development of MIA-Induced Rat OAModels

Male Sprague-Dawley Rats (2 months old, 220-230 g in weight) were used. All rats were housed in groups of five per cage under standard laboratory conditions with free access to food and water, and a constant room temperature (22°C) and humidity (45% to 50%). Rats were randomly divided into groups as described below (Fig. 1). Rats were given an intra-articular injection of MIA and ruthenium red (RR, Sigma USA) through the infra-patella ligament of left knee, at a dose of 1 mg in 50 μl sterile saline.

KN62 were dissolved in Dimethyl sulfoxide (DMSO) then diluted with sterile saline at a concentration of 5 mg/ml were administrated intra-articularly 50μl. Control (normal) animals were given an injection of equi-volume sterile saline.

Macroscopic Analysis

Joint space was monitored using the digital X-ray (MX-20, Faxitron X-Ray Corp., Wheeling, IL, US). X-rays were graded as follows: 0 = normal appearance; 1 = slight narrowing of the joint space; 2 = narrowing of the joint space but with no osteophytes;

3 = severe narrowing of the joint space with some osteophytes; 4 = severe narrowing of the joint space with many osteophytes. The articular appearance of macroscopic lesions was graded as follows: 0 = normal appearance; 1 = slight yellowish discoloration of the chondral surface; 2 = small cartilage erosions in load-bearing areas;

3 = large erosions extending down to the subchondral bone; 4 = large erosions with large areas of subchondral bone exposure. Each of the chondral compartments (the femoral condyles, the tibial plateaus, the patella, and the femoral groove). All samples were measured by three assessors who were blinded to the induction procedure.

Histological analysis and Immunohistochemistry (IHC)

Whole knee samples were fixed immediately in 4% paraformaldehyde, decalcified, embedded in paraffin, and cut into 4 μm tissue sections. Sections were stained with H&E. Immunohistochemistry staining was performed by procedure. antigen retrieval and blocking of the endogenous peroxidase activity, sections were then incubated with anti-CaMKII (phospho T286) antibody (ab32678) (Abcam, USA, 1:150 dilution) at 4°C overnight. Then the secondary antibody(zhongshanjinqiao, China) was applied for 30 min at room temperature. Staining was detected with DAB (3,3′-diaminobenzidine tetrahydrochloride).

Isolation, culture and identification of rat primary chondrocytes

Primary chondrocytes were isolated from rats as described [6]. Fresh medium was replaced every 2 days and chondrocytes reached approximately 80% confluence by days 4-5 as the P0 generation. Converged chondrocytes were then detatched with trypsin for subculture continually as generations P1, P2, and P3. Cells were used for experiments within the P3 generation. Immunocytochemistry was performed to identify chondrocyte phenotypes. Monolayer cells were incubated with anti-type II collagen antibody (Abcam, Cambridge, USA;1:400 dilution).

Immunofluorescence Staining

Chondrocytes were seeded at a density of 1×106 cells/well in completed growth medium in a 6-well plate for 24h respectively. Next day, three groups were pre-incubation with ruthenium red (10 μM), KN-62 (10 μM) and ruthenium red (10 μM) + KN-62 (10 μM) respectively for 30 minutes before 6 μM MIA incubation. The each well is at a final volume of 2ml completed growth medium. Cells were treated with MIA for 12 h. Treated cells were then fixed in neutral formalin-buffered solution for 30 minutes, washed 3 times with PBS following and incubated with primary anti-CaMKII (phospho T286) antibody (ab32678) (Abcam, USA, 1:100 dilution) overnight at 4°C. Samples were then incubated with secondary antibody (Abcam, USA, 1:100 dilution) for 1 h at 37°C. The chondrocyte nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) for 5 min. The stained chondrocytes were observed under a fluorescence microscope.

Determination of intracellular Ca²⁺

The concentration of intracellular Ca²⁺ was determined using a green fluorescent dye, Fluo-4AM (Dojindo, Kumamoto, Japan). The chondrocytes were grouped like in Immunofluorescence Staining method above. Cells were treated with MIA for 12 h and washed 3 times with D-Hanks balanced salt solution without Ca²⁺. Subsequently, cell were loaded with 2 μmol/l Fluo-4AM (Dojindo, Japan) for 30 min at 37℃ in the dark, then washed twice with D-Hanks balanced salt solution without Ca²⁺ to remove the extracellular Fluo-4/AM. Imaging was performed using an OLYMPUS IX71 inverted microscope and analyzed with Image-Pro Plus 6.0. The measured average fluorescence intensity of each cell in the field (F) normalized with the non-specific background fluorescence (F₀) to obtain the fluorescence intensity (F/F0) [14].

Statistical data are provided as percentage variation of treatments group release vs control (0 μM MIA).

Detection of Apoptosis by Flow Cytometry

MIA-induced apoptosis of chondrocytes was detected using an annexin V-FITC apoptosis detection kit (KeyGEN, China). The chondrocytes were grouped like in Immunofluorescence Staining method above. Cells were treated with MIA for 12 h and resuspended in 500 μl of binding buffer (KeyGEN, China), followed by incubation with 5 μl annexin V-FITC and 5 μl of propidium iodide (PI) at room temperature for 15 min in the dark. Flow cytometry with cell Quest software (BD Biosciences, San Jose, CA) was carried out.

Western blot

Western blotting was performed as described[6] Total proteins were extracted from treated and tissues, and concentrations were determined using a bicinchoninic acid reagent assay (Beyotime Biotechnology, Shanghai, China). Proteins were separated by electrophoresis on SDS–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Blots were incubated with primary antibodies including Anti-TRPV5 antibody (ab77351) (Abcam, USA, 1:2000 dilution), anti-CaMKII antibody (ab52476) (Abcam, USA, 1:1000 dilution), anti-phospho -CaMKII antibody (ab32678) (Abcam, USA, 1:1000 dilution), anti–β-actin (ab8226) (Abcam, USA, 1:10000 dilution). Following antibodies were purchased from Cell Signaling Technology (Inc., Beverly, MA, USA) anti-phospho-JNK (#9255) (dilution 1:2000), 14 anti-JNK (#9252) (dilution 1:1500), anti-phospho-p38 (#9215) (dilution 1:1000), 15 anti-p38 (#9212) (dilution 1:1000), anti–phospho-Erk (#9106) (dilution 1:2000), anti-Erk(#9102) (dilution 1:2000), anti-phospho-Akt (#4051) (dilution 1:1000), and anti-Akt (#9272) (dilution 1:2000) anti-phospho-S6k1 (#9205) (dilution 1:2000), 18 anti-S6k1 (#9202) (dilution 1:1500), anti-4E-BP1 (#9452) (dilution 1:2000), anti-Phospho-4E-BP1(#2855) (dilution 1:1000).

Statistical Analysis

All experiments in this study were repeated three times. Quantitative analysis of the bands was performed with the Image J analysis software (Version 1.30v; Wayne Rasband, NIH, USA). All values are expressed as mean ± standard error of the mean (SEM). Statistical analysis of the results was carried out by paired t-test analysis. All statistical analyses were performed using SPSS 17.0 (IBM, Armonk, NY, USA).

Significance was set at P = 0.05 for all statistical analyses.

Results

Effects of ruthenium red or KN62 treatment can delay OA progression in the MIA-induced rat OA model

To explore whether ruthenium red (RR) and KN62 have protective roles for cartilage in MIA-induced OA, OA changes of the knee at 21 days reperfusion with MIA were examined by radiography and macroscopic examination. Radiography (Fig. 2A) revealed that the normal (control) group of rats had knee joints with a smooth surface.

In contrast, obvious osteophytes as well as incomplete and thickened articular surfaces were observed in the knees of rats of the MIA 21 days group. However, treatment with ruthenium red, KN62, or ruthenium red combined with KN62 led to markedly less osteophytes and reduced pathological processes as observed in radiographic images of in rat knees. Macroscopic assessment revealed (Fig. 1B) MIA treatment resulted in marked cartilage erosion with large gray and losing its gloss areas even cartilage exfoliation. Changes in the subchondral bone was also explored 21 days after MIA injection; treatment with ruthenium red or KN-62 dramatically decreased cartilage degeneration induced by MIA injection. At the same time, treatment with ruthenium red combined with KN-62 showed the same protective effect as ruthenium red or KN-62 alone. Evaluation scores were consistent with the pathologic level of OA in radiographic images and macroscopic examination (Fig. 2C, 2D). These results suggest that in articular chondrocytes, the inhibition of TRPV5 using ruthenium red or the inhibition of CaMKII phosphorylation by KN-62 may be protective against the development of OA; the protective mechanisms involved may be related to TRPV5 and CaMKII phosphorylation.

Activation of CAMKⅡ phosphorylation correlated positively with TRPV5 function in parallel with the degree of osteoarthritis lesions

HE staining detected the severity of OA in different groups on histological analysis. In a comparison with the control group, the cartilage layer showed thinning, with a major loss of chondrocytes and lacunar emptying in the MIA 21 days group; cartilage block exfoliation even appeared (Fig. 3A). However, such cartilage degeneration was relieved and even reversed by treatment with RR and KN-62. These results suggest that treatment with both RR and KN-62 can change the process of osteoarthritis.

Interestingly, p-CAMKⅡ protein, immunolocalized by immunohistochemistry in articular cartilage, was positively associated with the corresponding OA lesion pathology. Light staining of p-CAMKⅡ was observed in normal articular cartilage (Fig. 3B), while staining intensity increased greatly after MIA 21days. However, at the same time, staining of p-CAMKⅡ was markedly weaker after MIA 21days, after treatment with RR and KN-62. TRPV5, p-CAMKⅡ, and CAMKⅡ proteins were quantified by western blotting; the expression of TRPV5 and p-CAMKⅡ increased by MIA 21 day (Fig. 4). RR and KN-62 treatment reduced the phosphorylation level of CAMKⅡ protein; however, TRPV5 and total CAMKⅡ protein remained unchanged.

These results indicate that upregulated p-CAMKⅡ in the OA lesion and upregulated CAMKⅡ phosphorylation was inhibited by KN62 and ruthenium red. These results also suggest channel function mediated by TRPV5 is related to the activation of CAMKⅡ phosphorylation, leading to the initiation of osteoarthritis.

Increased calcium in chondrocytes mediated by TRPV5 activated CAMKⅡ phosphorylation

Primary chondrocytes isolated from rats were used invitro. The calcium increase mediated by TRPV5 in chondrocytes was studied by monitoring intracellular cytosolic Ca²⁺ fluorescence intensity using a Fluo-4AM stain. The fluorescence intensity for the 6 μM MIA alone group was markedly higher than that of the 0 μM MIA group, while the fluorescence intensity was significantly reduced by treating with 10 μM ruthenium red or 10 μM ruthenium red + 10 μM KN-62 (Fig. 4A).

However, for the KN-62 treatment alone group, the fluorescence intensity did not decrease. A graph of the fluorescence intensity of calcium ions in each group is shown in Fig. 4B. This indicates that the increased calcium influx in response to treatment with MIA may be inhibited by the inhibition of TRPV5, but not by the inhibition of CaMKII phosphorylation. These results indicate that TRPV5 has a specific role in mediating extracellular Ca²⁺ influx in OA.

Primary chondrocytes were stained with anti-collagen II as an indication of functional chondrocytes. We observed p-CAMKII expression in chondrocytes in vitroby immunofluorescence staining. As shown in Fig. 6, p-CAMKII protein was aggregated into large clumps in the perinuclear areas of chondrocytes. The fluorescence intensity for cells of the 6 μM MIA alone group was obviously stronger than for cells of the 0 μM MIA group. However, the fluorescence intensity was reduced markedly by treating with 10 μM ruthenium red and 10 μM KN-62. Treatment with 10 μM ruthenium red combined with 10 μM KN-62 showed the same inhibition as treatment with 10 μM ruthenium red or 10 μM KN-62 alone. The two experiments above indicated that p-CAMKⅡ activation required calcium influx that was mediated by TRPV5, which was suppressed by ruthenium red. Also, CAMKII phosphorylation activated by calcium influx was abolished by KN62.

CAMKII phosphorylation is activated by an increase of calcium mediated by TRPV5 in chondrocytes, which then initiates chondrocyte apoptosis

We used flow cytometry to study apoptosis in chondrocytes. As shown in Fig. 7, flow cytometric analysis revealed that the percentage of apoptotic cells significantly increased for chondrocytes from the 6 μM MIA group of rats compared with chondrocytes from control group (0 μM MIA) rats. This suggests that MIA stimulation can lead to chondrocyte apoptosis which simulates the degeneration of articular cartilage in OA invitro. However, the percentage of apoptotic cells was dramatically attenuated in the presence of ruthenium red. Furthermore, the inhibition of CaMKII phosphorylation with KN-62 also markedly attenuated the percentage of apoptotic cells. Consistently, an attenuation of the percentage of apoptotic cells effect could also be detected for treatment with 10 μM ruthenium red combined with 10 μM KN-62. A graph of the percentage of apoptotic cells in each group is shown in Fig. 4B and is consistent with results shown in Fig. 4A. These results indicate that the activation of p-CAMKII requires calcium influx mediated by TRPV5, which is essential for chondrocyte apoptosis. Apoptosis in chondrocytes can be diminished and even reversed by ruthenium red and KN62, indicating apoptosis was mediated by TRPV5 and CaMKII phosphorylation.

Ca²⁺ influx mediated by TRPV5 elicited CaMKII phosphorylation and led to chondrocyte apoptosis by activating MAPK and mTOR pathways

We sought to determine whether Ca²⁺, mediated by the TRPV5-mediated induction of CaMKII phosphorylation, correlated with its activation of MAPK and mTOR signaling pathways. The expression of core proteins was determined by western blotting. As shown in Fig. 8A, chondrocytes were exposed to MIA (6 μM) and pretreated with ruthenium red, KN-93 or both ruthenium red plus KN-93. We found that TRPV5 was markedly upregulated after the stimulation of cells with 6 μM MIA.

The phosphorylation of CaMKII was also markedly upregulated with 6 μM MIA stimulation, but was attenuated by pretreatment with ruthenium red, KN-62 or ruthenium red plus KN-62. The phosphorylation of Erk1/2, JNK, and p38 MAPK in chondrocytes showed similar changes as for p-CaMKII protein. But Erk1/2, JNK, and p38 MAPK total proteins did not show a change. The phosphorylation of CaMKII activates MAPK signaling pathways by phosphorylating cascade proteins in the MAPK pathway; in chondrocytes, these showed a similar change as for p-CaMKII protein. We also found that the phosphorylation of Akt, S6K1, and 4E-BP1 in chondrocytes showed a similar change as p-CaMKII protein, simultaneously. But AKT, S6K1, and 4E-BP1 total proteins did not show any change. The activation of Akt, S6K1, and 4E-BP1 was inhibited markedly by ruthenium red and KN-62. These results indicate that phosphorylation of CaMKII activates Akt/mTOR signaling pathways by phosphorylating cascade proteins in the Akt/mTOR pathway. A graph of relative protein levels in repeated experiments is shown in Fig. 4B. The results indicate that TRPV5-mediated Ca²⁺ influx elicited chondrocyte apoptosis by inducing CaMKII phosphorylation that then activated the MAPK and Akt/mTOR pathways.

Discussion

Based on current knowledge, chondrocyte apoptosis may be the underlying factor for the initiation of OA[3]. Therefore, understanding the mechanism of chondrocyte apoptosis is essential for developing appropriate targeted therapies for OA treatment.

Considering the ethical problems of human experimentation and individual variability, in order to study the mechanism of chondrocyte apoptosis, we sought to establish a stable animal model of osteoarthritis. Therefore, an MIA-induced experimental OA rat model was developed to imitate the degeneration of articular cartilage observed in human disease. The MIA-induced experimental OA rat model has been widely used to study OA pathogenesis [15, 16]. The advantages of such a model are that it involves a quick and easy procedure, produces OA-like lesions, and displays functional impairment similar to that observed in human disease[17].

Since the TRPV family was first discovered in early 1997[18] and was systematically proposed in 2001 [19], TRPV proteins have been investigated in the etiologies of many diseases. TRPV5 is a member of the TRPV subfamily that functions as a facilitative Ca²⁺ transporter. Our study delineated that Ca²⁺ increases via intracellular influx through TRPV5 can inhibit chondrocyteautophagy in OA[6]. In this study, we also have comprehensively demonstrated TRPV5 expression in cartilage and that the upregulation of TRPV5 participated in the development of OA in the MIA-induced rat model. We also found p-CaMKII was significantly upregulated in OA cartilage in a positive linear relationship with TRPV5 protein (Fig. 3, Fig. 4). In this study, the inhibition of TRPV5 using ruthenium red had a protective effect against the development of OA equal to the inhibition of CaMKII phosphorylation (Fig. 2). We speculate that the activation of p-CaMKII linked with OA may be promoted by TRPV5-mediated Ca²⁺ influx.

It has been previously reported that abnormal TRPV5 can cause Ca²⁺ influx overload in HEK293[20] and mice ear hair cells[21]. Recently, our study delineated that a Ca²⁺ increase via intracellular influx through TRPV5 can inhibit chondrocyte autophagy in OA[6]. Consistently, in the study, we noted that upregulated TRPV5 was able to increase the elevation of Ca²⁺ influx in chondrocytes. Calcium ion, as a second messenger, mediates a variety of physiological responses of cells and has direct or indirect roles in mediating apoptosis[4]. Ca²⁺ was shown to be important in apoptosis that involved the production of calcium entry-dependent reactive oxygen species (ROS) [22], mitochondrial depolarization and DNA fragmentation [23]. Although most studies regarding the role of Ca²⁺ in apoptosis have mainly focused on its increased release from the endoplasmic reticulum, the role of Ca²⁺ influx through TRP channels has also been demonstrated recently [24]. Our results show that calcium influx though the TRPV5 channel can induce the apoptosis of chondrocytes which can be diminished and even reversed by ruthenium red and KN62 (Fig. 5) [25]. We speculate that the activation of p-CaMKII promoted by TRPV5-mediated Ca²⁺ influx may act as a core effect in chondrocyte apoptosis.

CaMKII, a multifunctional Ser/Thr kinase ubiquitously expressed in chondrocyte [26], is prominent among Ca²⁺-sensitive processes [27]. It is activated upon binding of CaM, which undergoes autophosphorylation. Based on the unique regulatory properties of CaMKII and our recent findings that TRPV5 induces Ca²⁺ influx contributing to chondrocyte apoptosis, we speculate that CaMKII is an “interpreter” of the TRPV5 induction of Ca²⁺ signaling, leading to apoptosis in chondrocytes. In the present study, we observed that exposure of chondrocytes to MIA resulted in p-CaMKII (Fig. 6), which was consistent with an increased apoptosis rate. If intracellular Ca²⁺ influx is inhibited with ruthenium red or the activation of CaMKII blocked, chondrocyte apoptosis can be attenuated and even reversed (Fig. 7). Increasing evidence indicates that p-CaMKII activates the MAPK cascade [28].

Consistently, in this study, we noted that phosphorylation of Erk1/2, JNK, and p38 MAPK in chondrocytes all increased under p-CaMKII activation. However, the phosphorylation of Erk1/2, JNK, and p38 MAPK was abolished when the phosphorylation of CAMKII was inhibited by KN-62 (Fig. 8). In this study, we noticed that the elevation of p-CAMKII did not alter the total cellular protein expression of JNK1/2, but induced both phosphorylation of JNK2 (the upper band) and JNK1 (the lower band). However, in contrast, p-CAMKII preferentially induced p-JNK1 in neurons [29]. We speculate both p-JNK2 and p-JNK1 are critical to the phosphorylation of c-Jun. Four isoforms of p38 (-α, -β, -γ, and -δ) have been identified in chondrocytes in OA [30]. In this study, an antibody to phospho-p38 (Thr180/Tyr182; Cat. #9215, Cell Signaling) was used that could not differentiate the -α, -β, -γ, and –δ isoforms of p38. Therefore, currently, we do not know what isoforms of p38 MAPK are activated by p-CaMKII. Since various isoforms of p38 have unique cellular functions, this suggests that the identification of the exact isoforms of p38 in OA is important.

It is commonly accepted that mTOR is a master kinase, which positively regulates protein synthesis, cell growth, proliferation and survival[31]. Akt/mTOR signaling is crucial for chondrocyte survival[32]. We have demonstrated that p-CaMKII activates the Akt/mTOR signaling pathway, promoting chondrocyte apoptosis. We found that p-CaMKII activated the phosphorylation of Akt, S6K1, and 4E-BP1 in chondrocytes under 6 μM MIA stimulation. As expected, we found that pre-treatment with KN-62 or ruthenium red markedly attenuated Cd-induced phosphorylation of Akt, S6K1, and 4E-BP1, as well as that of chondrocyte apoptosis (Fig. 8). In contrast, several studies have shown that blocking the Akt/mTOR pathway suppresses proliferation and promotes apoptosis in many kinds of tumors [33, 34]. As mTOR controls cap-dependent translation [31], we hypothesize a possible mechanism whereby the activation of mTOR would increase protein synthesis, which may then consume a lot of energy (ATP) and generate high levels of ROS. If mTOR is activated continuously, ATP would become exhausted, leading to apoptosis.

In summary, we found that the TRPV5 cation channel is functionally up regulated in OA articular cartilage. Up regulated TRPV5 may be an initiating factor that activates CaMKII phosphorylation via the mediation of Ca2+ influx. Activated p- CaMKII plays a critical role in contributing to chondrocyte apoptosis via MAPK and Akt/mTOR pathways (Fig. 9). Our results underscore an intriguing role for TRPV5 and p- CaMKII as mediators and potential drug targets in OA.

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Figure legends

Figure 1. Experimental animals grouped flowchart

Figure 2. Macroscopic and radiographic analyses and effects of ruthenium red or

KN62 treatment can delay OA progression in the MIA-induced rat OA model.

(A) Macroscopic photographs of tibial plateaus of the rat knee joints (B) X-ray photographs of the

total knee joints (C) Radiographic scores measuring joint destruction (D) Macroscopic scores

measuring joint destruction. Data are presented as mean ± SEM (n = 3). *P < 0.05 vs. control;

**P < 0.05, ***P < 0.05, ****P < 0.05 vs. 6 μM MIA without therapy treatment. ^#P < 0.05vs.

control; ^##P< 0.05, ^###P< 0.05, ^####P< 0.05 vs. 6 μM MIA without therapy treatment. MIA,

monosodium iodoacetate; RR, ruthenium red; KN-62,

(1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine

Figure 3. Evaluation of the protective effect of the TRPV5 inhibitor (ruthenium red) and CAMKⅡ phosphorylation inhibitor (KN-62) by histological analysis and expression of p-CAMKⅡ correlated positively with the progression of osteoarthritis in an MIA-induced rat OA model. (A) Photomicrographs showing representative hematoxylin and eosin (H&E) staining of rat knee joints. (B) Photomicrographs showing representative immunohistochemical analyses of p-CAMKⅡ expression of the articular chondrocyte in each group. Brown staining indicates specific p-CAMKⅡ protein, and blue staining indicates the nucleus. The distribution of brown staining is positively correlated with p-CAMKⅡ protein expression. (×40 and ×400 [inset] magnification for 2A, B and C). TRPV5, transient receptor potential channel vanilloid 5; MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; p-CAMKⅡ, Ca2+/Calmodulin-Dependent Kinase II phosphorylation.

Figure 4. TRPV5 and p-CAMKⅡ protein expression in the articular cartilage of different group (A) TRPV5, p-CAMKⅡI protein expression from different stimulated groups at each time point as detected by Western blotting. (B) A bar graph showing the level of TRPV5, p-CAMKⅡ proteins in various groups. *P < 0.05 vs. untreated control, **P < 0.05, ^#P< 0.05, 8 ^##P< 0.05, ^###P< 0.05, ^#P< 0.05, ^##P< 0.05, ^###P< 0.05 vs. MIA 21 day. Each column represents the mean ± SEM (n = 3). MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; p-CAMKⅡ, Ca2+/Calmodulin-Dependent Kinase II phosphorylation.

Figure 5. Fluorescent images of rat primary chondrocytes labelled with the Ca2+ indicator dye Fluo-4AM (A) Ca²⁺ fluorescence relative intensity in different chondrocyte treatment groups (all photomicrographs are shown at ×200 magnification). (B) Bar graph showing the level of relative fluorescent intensity in each group. *P < 0.05; **P < 0.05;; ****P < 0.05; #P >0.05; Each column represents mean ± SEM (n = 3). MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; p-CAMK, Ca2+/Calmodulin-Dependent Kinase II phosphorylation.

Figure 6. P-CAMKⅡ expression in different chondrocyte treatment groups. Expression of (A) p-CAMKⅡ (red) was determined by immunofluorescence staining and blue staining indicates the nucleus (original magnification ×200). MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; p-CAMK, Ca2+/Calmodulin-Dependent Kinase II phosphorylation.

Figure 7. Each group chondrocyte apoptosis detected by flow cytometry with Annexin V-FITC/PI staining of chondrocytes (A) Each group apoptotic chondrocytess distribution in flow cytometry machine. (B) Bar graph showing the apoptosis rate of each group. *P < 0.05 difference vs. untreated group (control); ^#P < 0.05; **P < 0.05, ***P < 0.05, ****P < 0.05 difference vs. 6 μM MIA without therapy treatment. Each column represents mean ± SEM (n = 3). MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine.

Figure 8. Ca²⁺ mediated through TRPV5 elicited CaMKII phosphorylation activation leading to chondrocyte apoptosis by activating the MAPK and Akt/mTOR pathways (A) TRPV5, p-CAMKⅡ, p-JNK, p-Erk, p-38, p-Akt, p-S6k1, p-4E-BP1 protein expression from groups were detected by western blotting. (B) A bar graph showing relative levels of TRPV5, 12 p-CAMKⅡ, p-JNK, p-Erk, p-38, p-Akt, p-S6k1, p-4E-BP1 proteins. *P < 0.05; **P < 0.05; **^#P< 13 0.05; ***^#P< 0.05; ****^#P<0.05; ^#*P< 0.05; ^##*P< 0.05; ^###*P< 0.05 ^####*P< 0.05 vs. 14 untreated control. **P< 0.05; ***P< 0.05; ****P< 0.05; ^#P<0.05; ^##P< 0.05; ^###P< 0.05; ^####P < 0.05 vs. 6 μM MIA without therapy treatment. MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; TRPV5, transient receptor potential channel vanilloid 5; p-CAMKⅡ, Ca2+/calmodulin-dependent kinase II phosphorylation; MAPK, mitogen-activated protein kinase; p-JNK, c-Jun N-terminal kinase phosphorylation; p-Erk, extracellular signal-regulated protein kinase phosphorylation; p-Akt, protein kinase B phosphorylation; 4E-BP1, 4E-binding protein; p-S6k1, S6 kinase 1 phosphorylation.

Figure 9. Diagram of the signaling cascade The up-regulated TRPV5 could be an initiating factor that activate CaMKII phosphorylation via the mediation of Ca2+ influx. Activated p-CaMKII play a critical role in contributing to chondeocyte apoptosis via MAPK and Akt/mTOR pathways. MIA, monosodium iodoacetate; RR, ruthenium red; KN-62, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; TRPV5, transient receptor potential channel vanilloid 5; p-CAMK Ⅱ , Ca2+/calmodulin-dependent kinase II phosphorylation; MAPK, mitogen-activated protein kinase; p-JNK, c-Jun N-terminal kinase phosphorylation; p-Erk, extracellular signal-regulated protein kinase phosphorylation; p-Akt, protein kinase B phosphorylation; mTOR, mammalian target of rapamycin; 4E-BP1, 4E-binding protein; p-S6k1, S6 kinase 1 phosphorylation. 3 75 male Sprague-Dawley rats {about 2 months old, 220- 230g in weight, diifferences < 5g)

thro

malle SD rats were inji ugh Joint cavity, acqu

21 days

ectedl MIA ired OA for