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

Running title: WEI et al: TRANSIENT RECEPTOR POTENTIAL CHANNEL, VANILLOID 5, INDUCES CHONDROCYTE APOPTOSIS.

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

EffectsofrutheniumredorKN62treatmentcandelayOAprogressioninthe

MIA-induced rat OAmodel

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 osteoarthritislesions

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 chondrocyteapoptosis

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 mTORpathways

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.

Figure9. 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.

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