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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Osteoarthritis Cartilage. 2014 Jul 4;22(8):1197–1205. doi: 10.1016/j.joca.2014.06.031

Loss of Vhl in cartilage accelerated the progression of age-associated and surgically induced murine osteoarthritis

Tujun Weng 1,#, Yangli Xie 1,#, Lingxian Yi 1, Junlan Huang 1, Fengtao Luo 1, Xiaolan Du 1, Liang Chen 2, Chongyang Liu 3, Di Chen 4, Lin Chen 1,$
PMCID: PMC4150692  NIHMSID: NIHMS613541  PMID: 24999110

Abstract

Objective

To investigate the role of Vhl in maintaining the integrity of articular cartilage and in the development of experimental osteoarthritis (OA).

Method

Histology of articular cartilage and subchondral bone in both Vhl cKO and WT mice were analyzed by histopathology and micro-CT. Articular cartilage destruction and proteoglycan loss were scored in aged (12-month-old) mice as well as in mice with surgically induced OA. Apoptosis of cartilage in age-related and surgically induced OA was detected with TUNNEL assay. Expressions of VHL, Fas, LC-3, HIF-1α, HIF-2α, p-mTOR and MMP-13 in the knee joints were analyzed by immunostaining.

Results

No gross differences in cartilage were observed between Vhl cKO and WT mice at age 4 months. However, Vhl cKO mice displayed accelerated age-associated spontaneous OA and surgically induced OA. Cartilage destruction and proteoglycan loss were observed in the absence of Vhl. In addition, inactivation of Vhl resulted in up-regulation of HIF-2α and increased chondrocyte apoptosis and decreased expression of autophagy during OA development. Immunohistochemical staining also showed that Vhl deficiency led to increased expression of Fas, p-mTOR and MMP-13, and those genes were associated with cell apoptosis, autophagy and cartilage matrix breakdown, respectively.

Conclusion

Loss of Vhl in adult articular cartilage is associated with earlier dysregulation of cartilage homeostasis, characterized by an increased chondrocyte apoptosis, compromised chondrocyte autophagy, and an accelerated age-related and surgery-induced OA development. These results highlight the novel role of Vhl in maintaining joint homeostasis and OA development.

Keywords: von Hippel–Lindau gene (Vhl), hypoxia-inducible factor α (HIFα), Osteoarthritis, Apoptosis, Autophagy

Introduction

Osteoarthritis (OA) is a chronic degenerative disorder of synovial joints, and the cost of OA care is enormous and expensive. There is currently no available disease-modifying drug and major problem is poor understanding for the pathogenic mechanism of OA. Cartilage is an avascular connective tissue that physiologically lacks blood vessels or lymphatics, in which sole cell type chondrocyte lives by the nutrition diffusing through the matrix. To adapt this hypoxic environment, chondrocytes embedded in the cartilage matrix possess a capacity to maintain cartilage homeostasis throughout life. Hypoxia-inducible factors (HIFs) is a heterodimer which consists of an oxygen-sensitive α-subunit HIF-α (either HIF-1α or HIF-2α) and a ubiquitously expressed β-subunit HIF-β. Under a hypoxic environment, HIF-α will be accumulated and translocated to the nucleus, in which it dimerizes with HIF-1β to form a transcriptional complex and initiate expression of HIF-responsive genes. Under normoxic conditions, two prolyl residues within HIF-α subunit is hydroxylated and rapidly degraded by von Hippel-Lindau (VHL) tumor suppressor protein-mediated ubiquitination and proteosome degradation [1].

It hs been recently reported that transgenic mice with chondrocyte-specific expression of Epas1, the gene encoding for HIF-2α, or overexpression of HIF-2α by intra-articular injection of Ad-Epas1, developed spontaneous cartilage destruction in knee joints [2, 3]. Conversely, mice with heterozygous genetic deletion of Epas1 are resistant to surgically induced OA development in knee joints [3]. Disruption of HIF-1α in chondrocytes led to dwarfism and marked shortening of the limbs in mice with massive cell death in the joint during development [4]. Although there is no direct genetic evidence for the role of HIF-1α in the OA development, induction of OA by HIF-1α inhibitor (2-methoxyestradiol) demonstrates the importance of HIF-1α in maintaining the homeostasis of articular cartilage [5]. HIF-1α and HIF-2α have nearly 50% homologous at the amino acid sequence, yet their function is remarkable different in cartilage. HIF-1α is essential for chondrocyte survival and differentiation in the joint development [4], while HIF-2α acts as a catabolic factor in cartilage mainly through promoting hypertrophic differentiation and increasing apoptosis [2, 6]. The overall effects of HIF-1α and HIF-2α in maintaining the articular cartilage homeostasis in adult have not been reported.

The Vhl tumor suppressor gene is best known as a component of E3 ubiquitin ligase of which major activity is to promote proteasomal degradation of HIF-1α and HIF-2α. The physiological role of Vhl in the skeleton development has been well established. Vhl null mice were embryonic lethal due to abnormalities of placental vasculogenesis [7]. Osteoblast-specific deletion of Vhl in mice led to increased expression of HIF-1α and VEGF The mutant mice developed extremely dense, heavily vascularized long bones [8]. Deletion of Vhl in chondrocytes increased matrix deposition and reduced cell proliferation during growth plate development [9]. However, little is known about the physiological role of VHL and total HIFs in maintaining the integrity of permanent joint cartilage.

The aims of this study were to explore the specific function of Vhl in the homeostasis of adult joint cartilage and experimental OA development using chondrocyte-specific and tamoxifen-inducible approach. We found that inactivation of Vhl in postnatal cartilage leads to accelerated age-associated and surgically induced OA development.

Methods

Animals

Vhlfl/fl mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and Col2a1-CreERT2 mice were generated in Dr. Di Chen’s lab [10–12]. Both of these two lines were maintained in C57BL/6J background. Vhlfl/fl mice were bred with Col2a1-CreERT2 transgenic mice to obtain Col2a1-CreERT2;Vhlfl/fl (hereafter referred to as Vhl cKO) mice. Only male Vhl cKO and Cre-negative control mice were used in this study. Vhl and Cre transgene were genotyped by PCR analysis as described previously [8]. All animal studies were approved by the Institutional Animals Care and Use Committee of Daping Hospital (Chongqing, China).

Surgical induced and aging related OA model

To specifically delete Vhl in adult articular cartilage, 8-week-old Vhl cKO mice and their Cre-negative control littermates were intraperitoneally injected with tamoxifen (1mg/10 g body weight, daily for 5 days, n = 8~10 each genotype). The destabilization of the medial meniscus (DMM) induced OA model was made in right knee joints of 10-week-old male Cre-negative and Vhl cKO mice as described previously [13]. Sham surgery was performed on left knee joints per animal with medial capsulotomy only. Mice were kept for an additional 12 weeks after surgery and then two side knee joints (DMM and Sham) per animal were harvested for histological analysis.

For the age-related OA model, Cre-negative and Vhl cKO male mice were treated with tamoxifen at age 8 weeks and were kept up to the age of 12 months (n = 3~5 each genotype). At that time mice were sacrificed and knee joints were harvested for assessment of spontaneous OA development and right knee joints per animal were used to histological assessment.

Histological assessment

Knee joints from Cre-negative and Vhl cKO mice were used to determine spontaneous or surgically induced cartilage degeneration as described previously [13]. Briefly, after dissected free of skin and muscle, knee joint samples were fixed in 4% paraformadehyde, decalcified in 20% formic acid, and embedded with paraffin. Serial sagittal sections (5-μm thick) were cut through the medial knee joints and three 5-μm sections were placed on each slide at nearly 50-μm intervals. Safranin O/Fast green stained sections were scored according to the OARSI recommended 0–6 subjective scoring system[14]. In addition, proteoglycan depletion in cartilage was scored (on a scale of 0–3) for the complement of cartilage degeneration [13]. The severity of cartilage destruction was expressed as a summed structure score (sum of the 5 highest scores in all slides) and as a maximal structure score for the medial femora and medial tibiae separately within each joint.

Assessment of subchondral bone

Tibiae from Cre-negative and Vhl cKO mice at 4 months of age were scanned with a Scanco viva CT 40 instrument and analyzed for subchondral bone density. Briefly, serial 12.5-μm tomographic images were acquired at the condition of 70 kV and 113 mA. Constant thresholds (200) were performed in binary images to segment bone from bone marrow. 2D and 3D images were obtained and region of interest (ROI) in subchondral bone was defined as a sclerotic area contouring from the bone surface above the growth plate. The bone density of the ROI in the tibial condyle was defined as Mean/Density (mg HA/ccm), which was calculated with the Scanco software. Histomorphometric analyses were performed on subchondral bone in Safranin O/Fast green stained sections according to the methods described previously[15].

Immunohistochemistry

Knee joint sections were deparaffinized by xylene and deprived endogenous peroxidase activity with 3% H2O2, then blocked with normal goat serum for 30 minutes. Sections were incubated overnight with rabbit anti-VHL polyclonal antibody (1:50 dilution, Anbo, USA), rabbit anti-Fas polyclonal antibody (1:50 dilution, Boster, China), rabbit anti-MMP-13 polyclonal antibody (1:50 dilution, ProteinTech, Chicago, USA), rabbit anti-HIF-2α polyclonal antibody (1:100 dilution, Abcam, USA), rabbit anti-HIF-1α polyclonal antibody (1:100 dilution, Boster, China), rabbit anti-LC-3 polyclonal antibody (1:150 dilution, Cell Signaling Technology, USA), rabbit anti-p-mTOR polyclonal antibody (1:100 dilution, Abcam, USA). After rinsed with PBS, horseradish peroxidase (HRP)-conjugated the secondary antibody was applied and stained with DAB kit.

Isolation and culture of chondrocytes

The knee joint isolated from 5-day-old Col2a1-CreERT2; Vhlfl/fl mice and digested with 0.1% collagenase (Gibco) to obtain primary chondrocytes. Chondrocytes were maintained in 6-well plates at 5×105cells per well, and on culture day 3 the cells were treated with 4OH-TM (1μM) or vehicle for 48 hours.

Western blot

Proteins were lysed from chondrocytes with RIPA buffer contained protease inhibitors (Roche). 20μg proteins each sample were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% milk resolved in TBST buffer and probed with diluted antibodies. Primary antibodies used include: LC-3 (Abcam), Beclin1(Cell Signaling Technology), β-actin (Sigma), HIF-1α (Abcam), HIF-2α (Abcam), VHL (Abcam).

Apoptosis assay

Articular cartilage chondrocyte apoptotic cell were determined using in situ cell death detection kit (POD) of Roche according to the protocols described.

Statistical analysis

Results were reported as the mean with 95% confidence intervals. Unpaired Student’s t-test was used to determine statistical difference between groups using the IBM SPSS Statics 20 program. P values less than 0.05 was considered significant.

Results

Mice lacking Vhl in postnatal cartilage show normal articular cartilage at age 4 months but accelerated cartilage degeneration at age 12 months

Previous study showed that mice lacking Vhl in chondrocytes developed a severe dwarfism [9]. To determine the direct effects of Vhl in joint cartilage maintenance, we generated chondrocyte-specific Vhl knockout mice at adult stage in a tamoxifen-inducible fashion using Col2a1-CreERT2 transgenic mice. The efficiency of Cre mediated recombination in postnatal articular chondrocytes was confirmed in previous studies [11, 13].

Both Vhl cKO and their Cre-negative littlemates mice received tamoxifen administration at 8 weeks of age, and the gross morphological features of the joints were analyzed at age 4 months. No gross abnormalities in the knee joints and no significant structural changes or proteoglycans loss in the articular cartilage were observed in either the Vhl cKO or Cre-negative mice (Fig 1A). Since subchondral bone plays an important role in the pathogenesis of OA [16], the total subchondral bone mineral density and bone volume per tissue volume (BV/TV) in subchondral bone were determined by micro-CT analysis and histomorphometric analyses, repectively. Although increased trabecular bone mass was observed in the Vhl cKO mice as reported previously [17], no significant difference in subchondral bone mineral density was found between Vhl cKO and Cre-negative mice at age 4 months (Fig 1B, C). However, increased BV/TV was observed in subchonral bone from Vhl cKO mice (Fig 1D).

Figure 1. Basal cartilage characteristics in the knee joints of Cre-negative and Vhl cKO mice.

Figure 1

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(A) No gross differences were observed in the articular cartilage of knee joints between Vhl cKO and Cre-negative (Cre(−)) mice at 4-month-old (n = 5 mice each genotype, Scale bar, 200μm). Lower panel shows the higher magnification of knee joint in Vhl cKO or Cre-negative mice, respectively (Scale bar, 100μm). (B) The knee joints were analyzed by micro-CT in 4-month-old Vhl cKO and Cre-negative mice (n = 5 in each genotype). Asterisk showed increased trabecular bone mass in the Vhl cKO mice. (C) The subchondral bone density in the ROI described in (B) was comparable between Vhl cKO and Cre-negative mice. (D) Histomorphometric analyses showed that increased bone volume per tissue volume (BV/TV) in subchondral bone from Vhl cKO mice (n = 6 in each group). Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).

Aging is one of the most important risk factor for the development of OA [18]. We then analyzed spontaneous changes in the histologic features of the articular cartilage in Vhl cKO and Cre-negative mice at age of 12 months. In Cre-negative mice, greatly decreased proteoglycans but no structure breakdown was observed in the knee joints from 12-month-old compared to that in 4-month-old revealed by Safranin O staining (Fig 2A). In contrast, knee joints from 12-months-old Vhl cKO mice exhibited spontaneous cartilage erosion and more severe loss of proteoglycans in noncalcified articular cartilage (Fig 2A). The intensity of Safranin O staining in the growth plate, as an internal control, was comparable in either Vhl cKO and Cre-negative mice (Fig 2A). The score for structural alterations and proteoglycans loss were significantly higher in the medial femora and tibia from Vhl cKO mice than that in Cre-negative mice at age 12 months (Fig 2B). These results suggest that Vhl deficiency accelerated age-related articular cartilage degeneration.

Figure 2. Appearance of OA changes in Vhl cKO mice in the age-associated spontaneous OA model.

Figure 2

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(A) Representative histological feature of knee joints of 12-month-old Vhl cKO and Cre-negative mice (n = 3~5 mice each genotype, Scale bar in top panel, 200μm). The lower panel showed higher magnification views of the boxed area (Scale bar, 100μm). The arrow points to severe cartilage damage in Vhl cKO mice. Arrowhead points to the loss of proteoglycan in aged articular cartilage. (B) Summed score for cartilage structure damage and proteoglycan loss in femoral condyle and tibia plateau from 12-month-old Vhl cKO and Cre-negative mice. MFC = medial femoral condyle; MTP = medial tibial plateau. Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).

Inactivation of Vhl in adult articular chondrocytes increases severity of OA in the knee joints following DMM surgery

To explore the effect of Vhl deficiency on the progression of OA induced by injury, we performed DMM surgery in the right knee joints of Vhl cKO and Cre-negative mice. Only male mice were used in these experiments. 12 weeks after surgery, histological changes in knee joints were analyzed between Vhl cKO and Cre-negative littermates at age of 5 months. Sham-operated knee joints were normal in either Vhl cKO or Cre-negative mice (Fig 3A). In contrast, remarkable cartilage degenerations and proteoglycans loss were observed in the knee joints following DMM surgery, irrespective genotypes (Fig 3A, B). However, the responsiveness related to joint destabilization-induced OA progression in the Vhl cKO and Cre-negative littermates were significantly different. Mice lacking Vhl showed significant cartilage damage and severe loss of proteoglycans, whereas such changes were significantly milder in Cre-negative mice (Fig 3B). To quantify the severity of cartilage damage, we compared the scores of articular cartilage histologic structure in either Vhl cKO or Cre-negative mice 12 weeks after DMM surgery. The combined and maximal scores for morphological structure changes were significantly greater in the knee joints from Vhl cKO mice than that in Cre-negative mice after DMM surgery (Fig 3C). These results demonstrate that inactivation of Vhl in adult joints accelerated progression of OA induced by DMM surgery.

Figure 3. Appearance of severe OA changes in Vhl cKO mice in the surgically induced OA model.

Figure 3

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(A) Representative Safranin O/fast green-stained histological sections of knee joints from Vhl cKO and Cre-negative mice 12 weeks after DMM surgery or sham operation (N = 8~10 mice each genotype). GP = growth plate. Scale bar, 200μm. (B) Higher magnification of articular cartilage in both Vhl cKO and WT mice subjected to DMM surgery. Scale bar, 100μm. The arrow indicates structural damage in the knee joints. (C) Summed and maximal histologic structure score of knee cartilage 12 weeks after DMM surgery in Vhl cKO and Cre-negative mice. MFC = medial femoral condyle; MTP = medial tibial plateau. Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).

Vhl deficiency promotes chondrocyte apoptosis in knee joints of mice with surgically induced and age-related osteoarthritis

To investigate the underlying mechanisms responsible for accelerated OA development in Vhl cKO mice, we next determined changes of articular chondrocytes in the procedure of aging and abnormal stress induced by surgery. Chondrocyte apoptosis is strongly linked to the OA development[19]. We performed TUNEL assay in the knee joint samples harvested from DMM surgery induced or age-related OA mice in both genotypes. Increased chondrocyte apoptosis in the knee joints was observed compared to Sham-operated mice in both Vhl cKO and Cre-negative mice at 3 weeks after DMM surgery (Fig 4A). A significant increase in apoptotic chondrocyte numbers were observed in the Vhl-deficient articular cartilage compared to that in Cre-negative mice after DMM surgery (Fig 4A). Moreover, the knee joints cartilage from Vhl cKO mice at the age of 12 months also displayed increased apoptotic chondrocytes compared to their littlemates controls (Fig 4B). Noticeably, TUNEL-positive cells were mainly detected in the deep zones of articular cartilage without the clear reason (Fig 4B). Result of immunostaining also showed an increased expression of Fas in articular cartilage of knee joints in Vhl cKO mice compare to that in Cre-negative littermates at the age of 12 months and in DMM model (Fig 4C and Fig S1C). It’s well established that Bcl-2 family members played an important role in the regulation of apoptosis[20]. Bcl-2 was anti-apoptotic protein which blocks the recruitment and activation of pro-apoptotic protein, such as Bax. Increased expression of Bax and decreased expression of Bcl-2 were observed in the knee joints from Vhl cKO and Cre-negative mice 3 weeks after DMM surgery determined by Real-time PCR analysis (Fig 4D). These findings support the hypothesis that inactivation of Vhl in postnatal cartilage increased susceptibility to chondrocyte apoptosis under the conditions of aging or surgically induced destabilization of the joint.

Figure 4. Vhl deficiency increased articular chondrocyte apoptosis in DMM-induced and age-associated OA models.

Figure 4

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(A) Articular chondrocyte apoptosis was analyzed by TUNEL staining in the knee joints from Vhl cKO and Cre-negative mice 3 weeks after DMM surgery or sham operation (n = 3 mice each genotype). Top panel scale bar, 100μm. The bottom panel was TUNEL-positive apoptotic cells determined by DAB staining (Scale Bar, 100μm). (B) Increased apoptotic articular chondrocytes in the knee joints from aged Vhl cKO mice relative to Cre-negative mice (n = 3 mice each genotype, two replicates each group). Scale bar, 100μm. (C) Immunohistochemical staining for Fas in aged knee joints from Vhl cKO and Cre-negative mice (n = 3 mice each genotype, two replicates each sample). Sale bar, 100μm. (D) Quantification analysis of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in the knee joints from Vhl cKO and Cre-negative mice 3 weeks after DMM surgery determined by real-time PCR. Each PCR reaction was run in triplicates and performed twice. Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).

Vhl deficiency reduces autophagy-related gene expression of LC-3 and Beclin1 in articular chondrocytes

Previous studies have reported that autophagy is a protective mechanism in normal cartilage and regulates OA-like gene expression changes through the modulation of apoptosis [21, 22]. The knee joints from mice with age-related or surgically induced OA displayed a reduced autophagy and a related increased apoptosis [21]. To determine whether autophagy changes contributes to the development of OA in the Vhl cKO mice, we examined the expression of the autophagy marker LC-3 and Beclin1 in articular cartilage chondrocytes. Consistent with previous reports, results of immunostaining showed a reduced LC-3 expression in articular chondrocytes 3 weeks after DMM surgery compared to Sham operation in either Vhl cKO or Cre-negative mice (Fig 5A). Compared to Cre-negative mice, there was less LC-3 positive chondrocytes in the knee joints of Vhl cKO mice after either DMM surgery or Sham operation (Fig 5A). In addition, we also found that there was a significant reduction of LC-3 expression in the knee joints from Vhl cKO mice compared to that in Cre-negative mice at the age of 12 months (Fig 5B). Quantitative analysis revealed that there was only 28% LC-3 positive cells in Vhl-deficient articular cartilage while 59% of that in Cre-negative mice (Fig 5B). Since conversion of LC3I to the lower migrating form LC3II has been used as an indicator of autophagy, to confirm autophagy activity after Vhl deficiency, expression of LC3II and Beclin1 protein in primary chondrocytes from Vhl cKO newborn mice were analysed by western bloting. Decreased LC3II and Beclin1 expression were observed in articular chondrocytes in the condition of 4OH-TM mediated Vhl deletion. Mammalian target of rapamycin (mTOR) was considered as autophagy repressor. We found that there was a significantly increased expression of p-mTOR in the Vhl cKO mice compare to Cre-negative mice at the age of 10 months (Fig 5D). These results demonstrate that the compromised autophagy may also contribute to the accelerated development of surgically induced or age-related OA in the Vhl cKO mice.

Figure 5. Disruption of Vhl decreased autophagy-related gene expression of LC-3 and Beclin1 in articular chondrocytes.

Figure 5

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(A) Immunohistochemical staining showed that reduced LC-3 expression in articular cartilage 3 weeks after DMM surgery compared to sham-operated mice in both genotypes (n = 3 mice each genotype, two replicates each group). Vhl deficiency decreases LC-3 expression in both DMM surgery and sham-operated mice. Scale bar, 40μm. (B) Reduction in LC-3 expression in the knee joints of 12-month-old Vhl cKO mice (n = 3 mice each genotype, two replicates each group). Scale bar, 20μm. Right graph is the quantification of LC-3 positive chondrocytes in 12-month-old Vhl cKO and Cre-negative mice. Percentage of LC-3 positive cells was significantly decreased in Vhl deficient mice. Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar). (C) Western blot analysis of LC3I, LC3II and Beclin1 in cultured chondrocytes from newborn Vhl cKO mice articular cartilage treated with or without 4-OH TM for 48 hours. (D) Higher expression of p-mTOR was observed in the knee joints of 12-month-old Vhl cKO mice compared to that of Cre-negative mice (n=3). Scale Bar, 100μm.

Vhl deficiency enhanced expression of HIF-2α and downstream molecules MMP-13 and IL-6

To determine the underlying molecular changes after Vhl deletion, the expression of HIF-1α and HIF-2α and its downstream were analysed in the articular cartilage chondrocytes. The results of immunohistochemical staining confirmed efficient deletion of Vhl in articular chondrocytes after tamoxifen treatment in Vhl cKO mice (Fig 6A). In addition, we found the expression level of HIF-1α was comparable between Vhl cKO and Cre-negative chondrocytes (Fig 6A), and HIF-2α was increased in the cartilage of Vhl cKO mice compared to that in Cre-negative control mice in ageing and DMM model (Fig 6A and Fig S1A). We also isolated primary chondrocytes from newborn Vhl cKO mice and treated with or without 4OH-TM. Western blot analysis showed that Vhl deletion led to higher expression of HIF-2α but nearly comparable expression of HIF-1α (Fig 6B).

Figure 6. Deletion of Vhl in chondrocytes enhanced expression of HIF-2α and its downstream MMP-13, IL-6 and MMP-9.

Figure 6

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(A) Immunohistochemical staining for Vhl, HIF-1α and HIF-2α in the knee joints from Vhl cKO and WT mice at 10 months of age (n = 3 each group). Tamoxifen induced deletion of Vhl in adult joints leads to higher expression of HIF-2α. Scale bar, 40μm. (B) Western blot analysis of HIF-2α, HIF-1α and Vhl in cultured chondrocytes from articular cartilage of Vhl cKO mice treated with or without 4-OH TM for 48 hours. Decreased expression of Vhl and increased HIF-2α expression were observed. (C) Higher expression of MMP-13 were observed in the knee joints from Vhl cKO mice compared with that of Cre-negative mice (n=3). Scale bar, 40μm. (D) Quantification analysis of IL-6 and MMP-9 mRNA level in the knee joints from Vhl cKO and Cre-negative mice determined by real-time PCR. Each PCR reaction was run in triplicates and performed twice. Data are expressed as the mean (symbols) ± 95% confidence intervals (error bar).

The catabolic enzyme MMP-13 is the main collagenase responsible for collagen degradation and is direct target of HIF-2α [3]. Results of immunostaining showed that the expression of MMP-13 was increased in the knee joint from 10-month-old Vhl cKO mice compared to Cre-negative mice (Fig 6C). Similar result of MMP-13 was also observed in DMM model (Fig S1B). Real time PCR showed that the expression of target genes of HIF-2α such as IL-6 and Mmp9 is higher in the knee joints of Vhl cKO mice than that of Cre negative mice in DMM model (Fig 6D). These results suggest that VHL/HIF-2α pathway plays an important role in the maintaining of cartilage homeostasis.

Discussion

Our study provides in vivo evidence that inactivation of Vhl in adult articular chondrocytes accelerates cartilage destruction in the age-related and surgically induced OA model, discovering a novel function of tumor suppressor Vhl in maintaining the integrity of hypoxic articular cartilage. Specifically, deletion of Vhl in knee joints causes increased apoptosis, suppressed autophagy and accelerated degeneration of articular cartilage in knee joints. These changes are likely associated with increased expression of HIF-2α.

Mice lacking Vhl in cartilaginous elements (Vhl flox/flox;Col2a1-Cre mice) developed severe dwarfism, thus this model is not suitable for OA study. To explore the direct role of Vhl in adult joint health and avoid potential confounding effects due to abnormal development, we specifically disrupted Vhl in chondrocytes in a tamoxifen-inducible fashion using Vhl flox/flox;Col2a1-CreERT2 (Vhl cKO) mice at 2-months of age. Grossly normal histology was observed in articular cartilage in Vhl cKO mice at the age of 4 months, but knee joint from Vhl cKO mice exhibited accelerated cartilage destruction under conditions of DMM surgery or aging. These findings uncover a novel biological function of Vhl in the maintenance of joint homeostasis.

We have designed a series of experiments to understand the underlying mechanisms. It has been proposed that the accumulation of HIF-1α responsible for the increased matrix deposition in growth plate and reduced chondrocyte proliferation in mice lacking Vhl in developing cartilage [9]. In our Vhl cKO mouse model, higher expression of HIF-2α in adult articular chondrocytes was observed compared to that in Cre-negative mice, while the expression of HIF-1α was nearly comparable. It has been reported that HIFs play a specific role in regulation of MSCs differentiation into chondrocytes under hypoxic induction, and HIF-2α, but not HIF-1α, was up-regulated during chondrogenic differentiation in hypoxia [23]. Only HIF-2α protein (but not HIF-1α) was stabilized in Vhl mutant lungs compared to Cre-negative controls [24]. Consistently, the phenotype of mice lacking Vhl in articular chondrocytes in our study is similar to that of the mice with over-expression of HIF-2α in chondrocytes, both of which displayed spontaneous articular cartilage destruction [3]. These results demonstrate that the specific role of Vhl in joint homeostasis and OA development is greatly related with stabilization of HIF-2α protein. HIF-2α was reported as a catabolic regulator of osteoarthritic cartilage destruction and directly regulated several important proteinase for collagen matrix breakdown, such as MMP-13 [3]. In our Vhl cKO mice, inactivation of Vhl in articular chondrocytes resulted in up-regulation of MMP-13. In addition, IL-6 and MMP-9, other target gene of HIF-2α, were also increased in Vhl deficiency joints. Therefore, Vhl deficiency may potentially through HIF-2α and its downstream contribute to the accelerated cartilage destruction of surgically induced or age-related OA in the Vhl cKO mice.

Since chondrocyte is the major cell type present in cartilage, mechanisms regulating the number and biosynthetic activity of chrodrocytes may largely be responsible for the joint homeostasis. Substantial evidences showed that increased apoptosis of chondrocytes is associated with the cartilage degradation [20]. Autophagy is an important mechanism for the removal of dysfunctional organelles and macromolecules and maintaining cartilage homeostasis, and this function is compromised in aging cartilage [25]. In surgically induced and age-related OA model, reduced autophagy and increase in apoptosis were observed [21]. Autophagy defect occured before chondrocyte cell death during Meckel cartilage degeneration [26]. Our present study also showed that knee joints from Vhl cKO mice with age-related and surgically induced OA displayed increased numbers of apoptotic chondrocytes and decreased expression of autophagy marker LC3II and Beclin1, suggesting a novel role of Vhl in maintaining cartilage homeostasis by regulating chondrocyte survival and autophagy.

Chondrocyte apoptosis is a known feature of OA and has been reported both in human OA disease and in OA animal models [19, 27]. In a guinea pig spontaneous OA model, enhanced chondrocyte apoptosis was observed at the later stage of OA development [28]. Previous studies have showed that HIFs were highly associated with chondrocyte apoptosis. HIF-1α acts as a survival factor which inhibits chondrocyte apoptosis in embryonic growth plate chondrocytes [4]. In contrast, overexpression of HIF-2α in mouse cartilage, either by intra-articular injection of Ad-Epas1 or in the context of chondrocyte-specific Epas1 transgenic mice, increased Fas-mediated chondrocyte apoptosis and cartilage destruction [6]. VHL deletion in growth plates did not display any differences in apoptotic cell death [9]. In current study, we showed that Vhl deletion in articular chondrocytes at adult stage increased chondrocyte apoptosis in age-related and surgically induced OA model. In addition, increased expression of Bax and reduced expression of Bcl-2 were found in the Vhl cKO joints3 weeks after DMM surgery. Our findings demonstrated a novel role of Vhl in maintaining the joint health and homeostasis by inhibiting apoptosis in experimental OA models. Autophagy is a cellular response to various types of stress and acts as protective mechanism in normal cartilage [21]. Activation of autophagy by rapamycin reduces cartilage destruction in experimental OA models [29]. Mechanical injury inhibits autophagy in cartilage, which may contribute to the development of OA[30]. In addition, autophagy regulates OA-related gene expression through modulation of apoptosis and ROS [22]. Previous study also showed that HIF-2α is an important suppressor of autophagy during chondrocyte maturation [31]. In our Vhl cKO mouse model, a reduction in autophagy executor LC3 was observed in the surgically induced and age-related OA models. Consistently, there was an increase in mTOR activity (p-mTOR), which is an important inhibitor of autophagy. Studies have shown that HIF-1α and HIF-2α exert differential or even opposite effects on chondrocytes and cartilage. Autophagy response is promoted by HIF-1α and antagonized by HIF-2α in chondrocytes [31, 32]. Only upregulation of HIF-2α was observed in our Vhl deficiency animal and primary chondrocytes model and which induced compromised autophagy may play an important role in the development of OA.

Taken together, increased apoptosis, suppressed autophagy and VHL/ HIF-2α signaling may largely contribute to the accelerated cartilage destruction in Vhl cKO OA model. Our results demonstrated the importance of Vhl in maintaining joint health and provided valuable information for potential OA treatment by targeting VHL/HIFs pathway.

Supplementary Material

Suppl. Fig S1. Expression of HIF-2α, MMP-13 and Fas in articular cartilage of DMM model.

(A) Immunohistochemical staining for HIF-2α in the knee joints of Cre-negative and Vhl cKO mice 3 weeks after DMM surgery (n = 3 mice each genotype). Scale bar, 40 μm. (B) Increased expression of MMP-13 in articular cartilage was observed in DMM model in both genotypes (n=3). Scale bar, 20 μm. (C) Increased expression of Fas in articular cartilage was found in DMM model in both genotypes (n=3). Scale bar, 40μm.

Acknowledgments

Grant sponsors: Special Funds for Major State Basic Research Program of China (973 Program, grant numbers: 2011CB964701), National Natural Science Foundation of China (grant numbers: 81000422).

This project was funded in part by grants from Major State Basic Research Program of China (973 Program, grant numbers: 2011CB964701) and National Natural Science Foundation of China (grant numbers: 81000422).

Footnotes

Disclosure: All authors state that they have no conflicts of interest.

Author contributions

Study design: Tujun Weng and Lin Chen.

Conduction of the Study: Tujun Weng, Yangli Xie, Lingxian Yi, Junlan Huang, Fengtao Luo, Xiaolan Du, Liang Chen, Chongyang Liu.

Data collection: Tujun Weng, Yangli Xie, Junlan Huang.

Data analysis: Tujun Weng, Yangli Xie and Lin Chen.

Data interpretation: Tujun Weng, Yangli Xie, Di Chen, Lin Chen.

Manuscript drafting: Tujun Weng, Lin Chen and Di Chen.

Manuscript revising: Lin Chen, Yangli Xie and Di Chen.

Approval of final version of the manuscript: Tujun Weng and Lin Chen. Lin Chen and Tujun Weng take responsibility for the integrity of the data analysis.

Conflict of interest

The authors declare that they have no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl. Fig S1. Expression of HIF-2α, MMP-13 and Fas in articular cartilage of DMM model.

(A) Immunohistochemical staining for HIF-2α in the knee joints of Cre-negative and Vhl cKO mice 3 weeks after DMM surgery (n = 3 mice each genotype). Scale bar, 40 μm. (B) Increased expression of MMP-13 in articular cartilage was observed in DMM model in both genotypes (n=3). Scale bar, 20 μm. (C) Increased expression of Fas in articular cartilage was found in DMM model in both genotypes (n=3). Scale bar, 40μm.

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