RHEB: a potential regulator of chondrocyte ... - Wiley Online Library

13 downloads 0 Views 964KB Size Report
Apr 6, 2016 - As articular cartilage has a limited ability to self-repair, successful cartilage regeneration requires clinical-grade chondrocytes with innate ...
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med 2017; 11: 2503–2515. Published online 6 April 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2148

ARTICLE

RHEB: a potential regulator of chondrocyte phenotype for cartilage tissue regeneration S. Ashraf1,2, J. Ahn1, B.-H. Cha1, J.-S. Kim1, I. Han3, H. Park2* and S.-H. Lee1* 1

Department of Biomedical Science, CHA University, Seoul, Republic of Korea School of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea 3 Department of Neurosurgery, CHA University, CHA Bundang Medical Centre, Kyeunggi-do, Republic of Korea 2

Abstract As articular cartilage has a limited ability to self-repair, successful cartilage regeneration requires clinical-grade chondrocytes with innate characteristics. However, cartilage regeneration via chondrocyte transplantation is challenging, because chondrocytes lose their innate characteristics during in vitro expansion. Here, we investigated the mechanistic underpinning of the gene Ras homologue enriched in brain (RHEB) in the control of senescence and dedifferentiation through the modulation of oxidative stress in chondrocytes, a hallmark of osteoarthritis. Serial expansion of human chondrocytes led to senescence, dedifferentiation and oxidative stress. RHEB maintained the innate characteristics of chondrocytes by regulating senescence, dedifferentiation and oxidative stress, leading to the upregulation of COL2 expression via SOX9 and the downregulation of p27 expression via MCL1. RHEB also decreased the expression of COL10. RHEB knockdown mimics decreased the expression of SOX9, COL2 and MCL1, while abrogating the suppressive function of RHEB on p27 and COL10 in chondrocytes. RHEB-overexpressing chondrocytes successfully formed cartilage tissue in vitro as well as in vivo, with increased expression of GAG matrix and chondrogenic markers. RHEB induces a distinct gene expression signature that maintained the innate chondrogenic properties over a long period. Therefore, RHEB expression represents a potentially useful mechanism in terms of cartilage tissue regeneration from chondrocytes, by which chondrocyte phenotypic and molecular characteristics can be retained through the modulation of senescence, dedifferentiation and oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd. Received 3 June 2015; Revised 14 December 2015; Accepted 22 December 2015

Keywords RHEB; chondrocyte; prolonged culture; phenotype; senescence; dedifferentiation; oxidative stress; cartilage tissue regeneration

1. Introduction Chondrocytes are primary cellular components of articular cartilage and maintain the complexity and flexibility of cartilage tissue by inducing extracellular matrix (ECM) glycoprotein expression, such as collagen type II (COL2) (Heidari et al., 2011). Due to low perfusion, cartilage tissue has limited regeneration ability, for which current medical treatments are lacking. Medications aimed to re-establish articular tissue, such as autologous chondro*Correspondence to: Soo-Hong Lee, Department of Biomedical Science, CHA University, Seoul, Republic of Korea. E-mail: [email protected]; and Hansoo Park, School of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea. E-mail: [email protected] Copyright © 2016 John Wiley & Sons, Ltd.

cyte implantation (ACI), and marrow stimulation techniques, such as abrasion arthroplasty, drilling and microfracture (Ashraf et al., 2015), have been developed. Microfracture is the most frequently used first-line treatment for symptomatic cartilage defects, but this often regenerates fibrous cartilage and not hyaline cartilage. Chondrocyte implantation resulted in better structural repair of cartilage defects than microfracture (Saris et al., 2008). Some studies have even demonstrated that ACI is associated with the formation of fibrocartilage or mixed hyaline/fibrocartilage (Bentley et al., 2003). Although chondrocyte implantation is considered promising for cartilage tissue recovery, successful cartilage regeneration requires a sufficient number of clinical-grade chondrocytes (Cha et al., 2013a, b). Chondrocyte transplantation treatment is also complicated, as it is

S. Ashraf et al.

2504

challenging to maintain the normal replicative physiological activity of human chondrocytes during in vitro culture, due to senescence, dedifferentiation and oxidative stress (Cha et al., 2013a, b; Frisbie et al., 2008). Senescent chondrocytes exhibit irreversible growth arrest and upregulated activity of senescence-associated β-galactosidase (SA-β-gal) (Hong et al., 2010). Dedifferentiation of chondrocytes is associated with phenotypic changes from a polygonal to a fibroblastic, large and flat morphology; concurrently, the dedifferentiation markers collagen type X (COL10) and collagen type I (COL1) are upregulated (von der Mark et al., 1977; Hong and Reddi, 2013). Exposure to reactive oxygen species (ROS), generated because of oxidative stress during cell passaging, results in senescent chondrocytes with a decreased survival rate (Serrano and Blasco, 2001; Brandl et al., 2011). A reduced expression of chondrocyte-specific proteins, such as COL2, proteoglycans and glycoproteins, accompanies senescence and dedifferentiation, and transplantation of these cells results in the formation of undesirable fibrous cartilage at this site (Schulze-Tanzil, 2009). Multiple genetic factors are implicated in the accumulation of oxidative stress that induces senescence and dedifferentiation, including p27, a cyclin-dependent kinase 2 (CDK2) inhibitor and a well-known mediator of senescence (Shin et al., 2002). Previously, studies in mice reported p27 expression in hypertrophic chondrocytes, suggesting that p27 may retard the differentiation and proliferation of growth plate chondrocytes. However, in p27-deficient mice, the size and width of tibiae and femora have been shown to increase (Emons et al., 2006). Certain studies in cancer cell lines have reported that myeloid cell leukaemia-1 (MCL1), an anti-apoptotic protein, prevents senescence and downregulates associated p27 expression (Bolesta et al., 2012; Hasan et al., 2013). Gene therapy combined with tissue-engineering techniques could be promising for cartilage tissue regeneration; however, it is important to first elucidate the underlying mechanisms of senescence-associated (p27), dedifferentiation (COL10) and chondrogenic (COL2) gene expression regulation. This knowledge would aid the development of novel approaches impeding senescence-associated gene expression and enhance chondrogenic gene expression, allowing prolonged in vitro culture of chondrocytes followed by successful cartilage tissue regeneration. Here, we investigated a novel role of the gene Ras homology enriched in brain (RHEB) in the modulation of senescence, dedifferentiation and oxidative stress for successful cartilage regeneration. RHEB is a member of the Ras superfamily of G-proteins that regulates cell growth, cell proliferation and differentiation (Aspuria and Tamanoi 2004; Li et al., 2004). However, RHEB has not been investigated in chondrocyte biology. Therefore, we examined the role and underlying signalling mechanisms of RHEB in chondrocytes in terms of senescence and oxidative stress. Furthermore, we investigated these chondrogenic properties for extended periods during in vitro and in vivo cartilage tissue formation. Copyright © 2016 John Wiley & Sons, Ltd.

2. Materials and methods 2.1. Human articular chondrocyte isolation and cell culture (2D and 3D) Human cartilage tissue was taken from the knee of a 62 year-old female patient during a total-knee replacement after receiving informed consent. Ethical approval was obtained from the institutional review board of Seoul St. Mary’s Hospital (IRB No. 2014–097). Chondrocytes were isolated as described previously (Cha et al., 2013a, b; Hidvegi et al., 2006). Briefly, whitecoloured, apparently normal-looking cartilage was separated from whole tissue, taking care to exclude subchondral bone. Isolated cartilage tissue, with an approximate weight of 4–5 g, was washed three times with Hank’s balanced salt solution (HBSS) buffer, chopped into small pieces with a blade and again washed three times with HBSS. Chondrocytes were isolated enzymatically from the chopped cartilage using 0.5 mg/ml collagenase type II in Dulbecco’s modified Eagle’s medium, low glucose (DMEM-LG; Gibco BRL, Gaithersburg, MD, USA) without serum after overnight incubation in humidified air with 5% v/v CO2 at 37°C. The next day, undigested tissue was separated from cells using a 40 mm filter; the cells were centrifuged, washed three times or more and resuspended in DMEM-LG. Approximately 2–3 × 10(Barbero & Martin, 2007) chondrocytes/g cartilage were obtained. Isolated chondrocytes were either cultured for expansion or cryopreserved in liquid nitrogen for further use. For two-dimensional (2D) culture, the chondrocytes were cultured in DMEM-LG supplemented with 10% v/v fetal bovine serum (FBS) and 100 U/ml penicillin– streptomycin (P/S) in humidified air, with 5% v/v CO2 at 37°C and with an initial seeding density of 2 × 10 (Aspuria & Tamanoi, 2004) cells/cm2. For three-dimensional (3D; ‘pellet’) cultures, cell suspensions were centrifuged at 1200 rpm for 5 min, and the pellets (2 × 10(Barbero & Martin, 2007) cells/pellet) were cultured for 21 days, then incubated in DMEM high-glucose medium supplemented with 10% FBS, 1% insulin–transferrin–selenium (ITS), ascorbic acid (50 μg/ml), dexamethasone (100 nM), P/S (1%) and 10 ng/ml transforming growth factor-β1 (TGFβ1), as previously described, with small modifications (Estes et al. 2010; Zheng et al., 2013).

2.2. Gene delivery using microporation transfection Human RHEB expression vectors (Accession No. NM_005614.3) were purchased from Enzynomics, Daejeon, Korea. Briefly, the human RHEB gene was cloned in the pEGFP–N1 vector, which also contained the green fluorescent protein (GFP) gene. As a control, pEGFP–N1 was used as a mock vector without the RHEB gene sequence but with GFP. For transfection of RHEB into J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

2505

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration

chondrocytes, a Microporator (Neon™, Invitrogen) was used. Briefly, passage 3 (P3) chondrocytes (1 × 10(Bentley et al., 2003)) were transfected with 5 μg plasmid DNA, using 100 μl ‘R’ buffer. Optimized microporator conditions at which the mock and RHEB vectors showed best transfection efficiency were a voltage of 1700 V for 20 ms with one pulse, and a voltage of 1200 V for 30 ms with one pulse, respectively. Transfection efficiency was evaluated by GFP expression, using an Eclipse 55i microscope (Nikon, Kanagawa, Japan), and quantified using a FACSCalibur system (BD, San Jose, CA, USA) 24 h after transfection.

2.3. RHEB knockdown To knock down RHEB, duplex small interfering RNA (siRNA) was purchased from Origene Technologies (Rockville, MD, USA). P3 chondrocytes were transfected with 20 and 40 nM siRNA, using lipofectamine RNAi Max reagent (Invitrogen, USA) according to the manufacturer’s instructions, along with control siRNA. After siRNA treatment, chondrocytes were incubated for 48 h for testing by PCR and 72 h for western blotting.

2.4. Separation of cytoplasmic and nuclear proteins P3 chondrocytes were transfected with mock or RHEB vectors and cultured for 48 h prior to cell fractionation and protein isolation. Cytoplasmic and nuclear protein extracts were separated using a Biovision Nuclear/cytoplasmic Fractionation kit (Milpitas, USA), according to the manufacture’s protocol.

2.5. PCR Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was synthesized using RT-PreMix (Bioneer, Daejeon, Korea). Reverse transcription–PCR (RT–PCR) was performed with PCR-PreMix and analysed by agarose (1.2%) electrophoresis. The gels were stained with ethidium bromide and DNA bands were visualized using a Gel Doc system (GDS 200D). Real-time quantitative PCR (qPCR) was performed with Power SYBR Green PCR Master Mix (Life Technologies, UK) using the Step One Plus Real-Time PCR system (AB Applied, Life Technologies). All gene expression data were normalized to the housekeeping gene, GAPDH. All primer details are shown in Table S1 (see supporting information).

by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting, as described previously (Cha et al., 2013a, b). Anti-RHEB, anti-SOX9, anti-phosphorylated S6K1, antip27KIP1 and anti-MCL1 antibodies were obtained from Abcam (Cambridge, MA, USA). The anti-phosphorylated retinoblastoma (pRB) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-COL2 and anti-Lamin A/C antibodies were purchased from Millipore (Billerica, MA, USA). The anti-GAPDH antibody was purchased from ABM (Cambridge, MA, USA). Alexa 594-conjugated goat anti-mouse and anti-rabbit IgG antibodies were purchased from Molecular Probes (Eugene, OR, USA).

2.7. SA-β-galactosidase staining A staining kit (Cell Signaling Technology) was used for SA-β-galactosidase detection. Chondrocytes were cultured for 3–4 days and subconfluent cells were stained according to the kit protocol; blue-stained cells were considered positive for senescence.

2.8. ROS detection Chondrocytes were seeded on 12 mm coverslips (Thermo Scientific, NY, USA) in 12-well plates. At 80% confluence, prewarmed (37°C) HBSS/Ca/Mg solution containing 100 nM MitoTracker Green (Invitrogen) probe and 3 mM MitoSOX Red (Invitrogen) probe was added for 15 min at 37°C. The cells were counterstained with 0.2 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) in the dark. After PBS washing, the coverslips were partially dried and placed on glass slides with mounting medium. Fluorescent images were visualized using a confocal microscope (Leica, Wetzlar, Germany). Upon oxidation by ROS (mainly superoxide anion), MitoTracker Green gave green fluorescence and MitoSOX Red gave red fluorescence. The fluorescence intensity was directly proportional to the oxidative stress or ROS level.

2.9. Cell proliferation assay A Cell Counting Kit-8 (Dojindo Laboratories, Japan) was used for cell proliferation assays. Briefly, P3 transfected chondrocytes were cultured to P6. Chondrocytes were seeded at a density of 1 × 104/cm2 and incubated for 9 days in a 24-well plate. Cell proliferation was analysed on alternate days, following the standard kit protocol.

2.6. Western blot analysis 2.10. Telomere length measurement Protein was isolated from cultured cells using radioimmunoprecipitation assay buffer (Sigma). Protein concentration was measured by bicinchoninic acid assay (Pierce). Approximately 25 μg total protein was separated Copyright © 2016 John Wiley & Sons, Ltd.

P3 chondrocytes were transfected with mock and RHEB vectors and cultured to P6. Genomic DNA was isolated using the Qiagen DNA kit (Seoul, Korea), according to J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

S. Ashraf et al.

2506

the standard procedure. Relative telomere length was measured as described previously (Cha et al., 2013a, b).

2.11. Immunocytochemistry P3 chondrocytes were grown on cell-culture slides (SPL, Life Sciences, Korea), fixed with 4% formaldehyde for 20 min and permeabilized with 0.1% Triton-X100 for 5 min. After washing, blocking solution (1% BSA) was added for 1 h. Then, anti-RHEB was added for 1 h, followed by incubation with fluorochrome-conjugated secondary antibody for 1 h in the dark. Isotype control antibody (rabbit IgG; Thermo Scientific) was run in a similar fashion to primary antibody. Isotype control antibody was used as a negative control to check the specific reactivity of anti-RHEB. Nuclei were stained with DAPI. RHEB localization was visualized under a confocal microscope.

2.12. Chondrocyte implantation and in vivo cartilage formation For in vivo cartilage generation, P3 chondrocytes were transfected with mock or RHEB vectors and cultured to P6. For each injection, chondrocytes (2 × 10(Bentley et al., 2003)) were separated and mixed with fibrin gel (Chungbuk, Korea) at a 1:1 ratio of fibrinogen:thrombin. Female athymic mice (BALb/c-nude, Orientbio, Seoul, Korea), 6 weeks old, were anaesthetized with ketamine (8 mg/kg) and xylazine (1.15 mg/kg). Chondrocytes were injected into the dorsal subcutaneous spaces of the mice. Mock-transfected chondrocytes were injected into the left sides of the mice, while RHEB-transfected chondrocytes were injected into the right side in a sample size of six mice. All mice were sacrificed 6 weeks after injection and the implants were retrieved. All procedures on animals were carried out according to guidelines of an approved protocol from CHA University Institutional Animal Care and Use Committee.

2.13. Histology and Alcian blue staining In vitro pellet culture samples and tissue specimens obtained from mice 6 weeks after implantation in the dorsal subcutaneous space were fixed with 10% formalin. After processing, the specimens were embedded in paraffin wax and 4 μm-thick sections were cut, then stained with 0.5% Alcian blue in 0.1 M HCl, pH 1.0, and counterstained with 0.05% Nuclear Fast Red (Sigma, Steinheim) in 2.6% aluminium sulphate solution. The GAG matrix formed in cartilage tissue was observed under light microscopy.

2.14. Immunohistochemistry Tissue sections were deparaffinized and subsequently incubated in xylene and ethanol for hydration. Antigens were retrieved by pepsin treatment (1 mg/ml in 10 mM Copyright © 2016 John Wiley & Sons, Ltd.

HCl) at 37°C for 15 min, followed by blocking with 10% goat serum for 1 h. Primary antibodies (anti-COL2, antiRHEB and anti-HNA) were added for 18 h at 4°C in a humidified chamber. Isotype control antibodies (rabbit IgG and mouse IgG1; Thermo Scientific) were run in a similar fashion to primary antibodies. Isotype control antibodies were used as negative controls to check the specific reactivity of anti-COL2, anti-RHEB and anti-HNA antibodies. The samples were then treated with 3% hydrogen peroxide (H2O2) for 15 min. A biotinylated secondary antibody obtained from GBL Laboratories (Bothell, WA, USA) was added to the slides for 30 min, followed by streptavidin– HRP for 30 min. Colour was developed by diaminobenzidine (DAB) treatment for 5–10 min. Haematoxylin was used as a counterstain.

2.15. Statistical analysis In vitro experiments in each condition were performed in triplicate. For in vivo cartilage formation, chondrocytes were implanted into six mice. The results were statistically expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for analysis of quantitative values, and the Tukey’s post hoc test was used for all pairwise comparisons among groups. ImageJ software was used for size measurements. p < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Serial passaging of chondrocytes during in vitro culture-induced phenotypic and molecular changes Chondrocyte morphology was changed from polygonal to elongated fibroblastic-like phenotype, along with increased cell area and nuclear diameter after P4, indicating dedifferentiation. Measurement of chondrocyte size and nuclear diameter showed drastic increases of almost two-fold after P4 (Figure 1a, b; n = 20; p < 0.0001). In the SA-β-gal assay, blue staining revealed that the number of senescent chondrocytes markedly increased with increasing number of culture passages (Figure 1c). Complying with senescence and dedifferentiation, oxidative stress or ROS production also increased with serial passaging of chondrocytes, as observed by increases in green and red fluorescence intensity (Figure 1d). However, no significant amount of apoptosis was observed in chondrocytes with serial passaging (data not shown). To confirm the association of phenotypic changes or dedifferentiation with cellular processes such as senescence, and oxidative stress in chondrocytes with molecular changes, we analysed several gene markers at P2, P4, P6 and P8 (Figure 1e). With an increase in passage number, the expression of COL10, a marker of chondrocyte dedifferentiation, was sequentially increased and was highest J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration

2507

Figure 1. Serial passaging of chondrocytes during in vitro culture induces phenotypic and molecular changes. (A) Cellular morphology after the serial passaging of chondrocytes (P2, P4, P6 and P8) was determined by differential interference contrast (DIC) images; (right) graphical representation of the cell area. (B) Nucleus enlargement was observed by DAPI staining; white arrow, enlarged nucleus; graphical representation of nuclear diameter is shown to the right of DAPI staining; cell area and nuclear diameter were observed from 20 randomly selected cells, using ImageJ software. (C) Senescence was determined by senescence-associated β-galactosidase (SA-β-gal) staining; blue-coloured cells show positive staining results; percentage of senescent cells is also presented in graphical form. (D) Oxidative stress or ROS was detected by MitoTracker Green and MitoSox Red dyes; an increase in the intensity of green and red fluorescence with serial passages shows increases in the ROS production; DAPI was used for nuclear counterstaining. (E) mRNA expression was observed by reverse transcription–PCR (RT–PCR); scale bar =100 μm; n = 3; **p < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com]

at P8, while the level of COL2, a chondrocyte-specific marker, was drastically reduced after P4. Interestingly, the level of SOX9, a well-known transcription factor for COL2, also gradually decreased after P4 and was Copyright © 2016 John Wiley & Sons, Ltd.

markedly lower at P8. The expression of senescenceassociated p27 was also upregulated with increasing passage number. However, expression of MCL1, a negative regulator of senescence and p27 downregulated with J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

2508

increasing passages. Surprisingly, we observed a drastic downregulation of RHEB mRNA after P4 (Figure 1e). This implies that the RHEB gene in chondrocytes may be a critical marker associated with chondrogenic characteristics.

3.2. Oxidative stress induces senescence and ROS production similar to prolonged culture ROS production was increased with increasing H2O2 concentration (Figure 2a). It was observed that oxidative stress was highly associated with senescence and expression of other molecular markers. Most importantly, senescence and p27 expression directly correlated with H2O2 concentration, along with downregulation of COL2 and SOX9 expression (Figure 2b, c). At high H2O2 concentrations, RHEB mRNA expression was also decreased (Figure 2c).

3.3. RHEB knockdown downregulates expression of chondrogenic markers and upregulates expression of senescence- or dedifferentiation-associated markers The effects of RHEB on chondrocytes in terms of senescence and dedifferentiation were investigated after silencing RHEB using siRNA (siRHEB). At an early stage, it was observed that chondrocyte molecular behaviour began to change after P4 (Figure 1e). We therefore selected P3 chondrocytes and efficiently knocked down RHEB expression. Both mRNA and protein expression analysis showed gradual downregulation of RHEB with increasing concentrations of siRNA (Figure 3). Subsequently, knockdown of RHEB resulted in distinct decreases in COL2, SOX9 and MCL1 expression, in a dose-dependent manner. MCL1 is an anti-apoptotic marker, but its expression is virtually abolished in senescence (Bolesta et al., 2012). As shown in Figure 3b, with downregulation of RHEB expression, a significant reduction in COL2 but increased COL10 expression was observed (n = 3; p < 0.01); this indicates that RHEB is a key regulator of chondrogenesis and dedifferentiation of chondrocytes. The mRNA levels were reflected in the level of protein expression (Figure 3a, c). These results suggest that RHEB has strong associations with COL2, COL10, SOX9, MCL1 and p27.

3.4. Overexpression of RHEB reduces senescence, dedifferentiation and oxidative stress and increases the proliferation rate P3 chondrocytes were transfected with mock and RHEB vectors and cultured to P6. Transfection efficiency was 52.17% and 42% for mock and RHEB vectors, respectively (Figure 4a). Senescence and oxidative stress were markedly reduced in P3-RHEB-transfected chondrocytes compared to P3-mock chondrocytes. Interestingly, P6-RHEBtransfected chondrocytes resisted senescence and Copyright © 2016 John Wiley & Sons, Ltd.

S. Ashraf et al.

oxidative stress, resulting in lower levels of senescent chondrocytes and ROS compared to P6-mock chondrocytes. Moreover, nuclear diameter and cell size did not increase (n = 20; p < 0.0001), indicating that P6-RHEB-transfected chondrocytes also did not dedifferentiate (Figure 4b–d). These results demonstrate that RHEB gene expression is able to maintain the chondrocyte phenotype by reducing senescence and oxidative stress. Furthermore, the proliferation potential of P6-RHEBtransfected chondrocytes was also quite high (Figure 4e; p < 0.0001).

3.5. Overexpression of RHEB increases the chondrogenic potential and reduces senescence-associated gene expression Overexpression of RHEB and its effects on other genes were investigated by molecular analysis. RHEB mRNA expression was significantly high in P3-RHEB-transfected chondrocytes compared to P3-mock-transfected chondrocytes. As described previously, P3-transfected chondrocytes were cultured to P6. Figure 4f clearly shows that the RHEB level was decreased in P6-mock chondrocytes, while it was quite stable in P6-RHEB chondrocytes (p < 0.05). RHEB-transfected chondrocytes also exhibited significantly increased expression of COL2 and SOX9 and decreased expression of COL10 at both P3 and P6. Furthermore, RHEB-transfected chondrocytes exhibited increased levels of MCL1 mRNA and decreased levels of p27 (Figure 4f). These results are consistent with the data using siRHEB presented in Figure 3. Taken together, we can conclude from the RHEB expression data that RHEB is a very important regulator for the maintenance of chondrogenic characteristics, such as phenotype, senescence, differentiation, oxidative stress and proliferation.

3.6. RHEB localizes to the nucleus and has molecular control of senescence To further investigate the cellular mechanism of RHEB in chondrocytes, we performed immunocytochemistry and found that the RHEB protein was localized not only to the cytoplasm but also to the nucleus (Figure 5a). In order to determine the functions of this protein in different locations, cytosolic and nuclear protein fractions were separated from mock- and RHEB-transfected chondrocytes. Interestingly, a higher amount of RHEB protein was found in the nucleus than in the cytoplasm, especially in RHEBoverexpressed chondrocytes. In addition, expression of pRB, another senescence-regulating protein, was much higher in the nucleus. Furthermore, RHEB overexpression led to increases in SOX9 and MCL1 levels, while p27 levels decreased (Figure 5b). As shown in Figure 5c, no significant differences were found in the telomere lengths (p = 0.273) of P6-mock and P6-RHEB-transfected chondrocytes, demonstrating that the RHEB gene plays a J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration

2509

Figure 2. Oxidative stress induces the senescence and ROS production similarly observed in prolonged culture: passage 3 chondrocytes were treated with 10, 100 and 200 μM H2O2 for 48 h. (A) Oxidative stress or ROS was detected by MitoTracker Green and MitoSox Red dyes; DAPI was used for nuclear staining. (B) Senescence was determined by SA-β-gal staining; graph represents the increasing percentage of senescent cells with increases in H2O2 concentration. (C) mRNA expression was observed by RT–PCR; scale bar =100 μm; n = 3; **p < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com] Copyright © 2016 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

2510

S. Ashraf et al.

Figure 3. Inhibition of RHEB confirmed the downregulation of expression of chondrogenic markers and upregulation of senescence or dedifferentiation associated markers. (A) Passage 3 chondrocytes were treated with increasing order of siRNA and RT–PCR was performed to determine gene expression after 48 h. (B) Expression of RHEB, COL2 and COL10 in P3-RHEB knockdown chondrocytes were also confirmed by real-time PCR (qPCR). (C) Protein expression after 72 h of siRNA treatment was observed by western blotting; n = 3; *p < 0.05, **p < 0.01

role in chondrocyte senescence through regulating the expression of cell cycle inhibitors such as RB and p27 in a telomere-independent manner.

3.7. RHEB-expressing chondrocytes show sustained chondrogenesis in vitro and in vivo We investigated whether RHEB could enhance cartilage tissue formation in vivo as well as in vitro even after prolonged culture. P3 chondrocytes were transfected with vectors harbouring mock or RHEB genes and cultured to P6. Pellets of P6-RHEB chondrocytes exhibited higher expression of GAG matrix, COL2 and RHEB than pellets of P6-mock chondrocytes. Moreover, mRNA analysis showed a much stronger expression of COL2, SOX9, MCL1 and RHEB in pellets of RHEB-expressing chondrocytes (Figure 6a, b). Furthermore, to assess in vivo cartilage tissue regeneration, P6-mock and P6-RHEB chondrocytes were injected into the dorsal area of BALB/c nude mice. Tissue harvested from mice contained mature and large-sized cartilage in the P6-RHEB group compared to the P6-mock group. Strong GAG expression and a very clear lacunar structure were observed in P6-RHEB cartilage. Moreover, immunohistochemistry revealed significantly higher expression of COL2 and RHEB in the P6-RHEB group. To validate human chondrocytes at newly formed cartilage tissue in mice, we evaluated human nuclei antigen (HNA) expression by immunohistochemistry and found expression of HNA in both groups (Figure 6c). Overall, these results demonstrated that the stable expression of RHEB in chondrocytes significantly increases both in vitro and in vivo cartilage tissue formation, even after prolonged passages in monolayer cultures.

4. Discussion The present study was the first systematic experimental investigation of the role of RHEB in chondrocytes. Our Copyright © 2016 John Wiley & Sons, Ltd.

results suggest that cellular responses, such as senescence, dedifferentiation and oxidative stress, can be controlled through a molecular signalling mechanism, and these findings can be applied for successful cartilage tissue regeneration. Changes in morphology, increased cell size, markedly increased nuclear diameter and higher ROS production have been directly correlated with senescence and dedifferentiation (Cha et al., 2013a, b; Barbero and Martin, 2007). Multiple factors in the prolonged culture of chondrocytes, including oxidative stress (Cha et al., 2013a, b; Loty et al., 2000; Xia et al., 2008; Akasaki et al., 2014), proinflammatory cytokines (Cristofalo et al., 2004) and matrix-degrading enzymes (Bui et al., 2012), are involved in senescence and dedifferentiation, which ultimately leads to the loss of chondrogenic properties. Dedifferentiation and other cellular changes, such as senescence and oxidative stress, are major challenges in chondrocyte expansion for application to cartilage regeneration (Philipot et al., 2014). Several methodologies have been adopted in other studies, including cytokine treatment, pellet culture and continuous expansion culture on a stretched surface, as an alternative to conventional monolayer culture systems. These were relatively effective in maintaining the phenotypic characteristics of chondrocytes with minimal passaging (Caron et al., 2012; Rosenzweig et al., 2012). Additionally, multiple pathways have been explored in the dedifferentiation of chondrocytes; e.g. the inhibition of p38 mitogen-activated protein kinase (MAPK) significantly increased COL2 and SOX9 expression, while COL1 expression was suppressed (Rosenzweig et al., 2013). Wild-type p53-inducible phosphate (Wip1) overexpression modulated the oxidative stress via regulation of P38 MAPK (Cha et al., 2013a, b), suggesting a blockade of dedifferentiation. Therefore, oxidative stress may be associated with p38 MAPK and further promotion of dedifferentiation. In our study, RHEB-overexpressed chondrocytes resisted oxidative stress; thus, RHEB may also have an association with p38 MAPK. J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration

2511

Figure 4. Overexpression of RHEB reduces senescence, dedifferentiation and oxidative stress and increases the chondrogenic potential and proliferation rate. (A) Transfection efficiency, as observed by GFP expression in microscopy and by flow cytometry. (B) Comparison of senescence was determined after the transfection of chondrocytes with mock and RHEB vectors in P3 chondrocytes: transfected cells were cultured to P6 and senescence was again observed; blue-coloured cells show positive staining; graph represents the percentage of senescent cells. (C) Differences in nuclear size were observed by DAPI staining; white arrows, enlargement of the nucleus in P6-mock-transfected cells in comparison with P6-RHEB-transfected cells; graphic representation of cell area and nuclear diameter shown in P6-mock- and P6RHEB-transfected cells. (D) ROS production was detected by MitoTracker Green and MitoSox Red dyes; DAPI was used as nuclear counterstain. (E) Chondrocyte proliferation in each group was determined by CCK assay: absorbance (450 nm) was measured on alternate days. (F) P3-transfected chondrocytes were cultured to P6 and mRNA expression was observed by qPCR; *p < 0.05 is significant to all of the other groups in this graph; scale bar =100 μm; n = 3; *p < 0.05, **p < 0.01). [Colour figure can be viewed at wileyonlinelibrary.com] Copyright © 2016 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

2512

S. Ashraf et al.

Figure 5. RHEB localizes in the nucleus and has molecular control of senescence. (A) Immunocytochemistry was performed to confirm RHEB localization in the chondrocytes after the transfection of P3 cells with (upper panel) mock vector and (middle panel) RHEB vector: RHEB strongly localizes in the nucleus, especially in the RHEB-transfected chondrocytes; isotype control antibody was also run as a negative control that showed the specificity of RHEB binding in the two upper panels; the nucleus was visualized by DAPI staining; confocal microscopy was used to observe the cells. (B) Expression of cytosolic and nuclear proteins separated by cell fractionation was analysed by western blotting. (C) Comparison of telomere length between P6-mock- and P6-RHEB-transfected # chondrocytes was measured by qPCR; scale bar =200 μm; n = 3; p = 0.273. [Colour figure can be viewed at wileyonlinelibrary.com]

To best of our knowledge, no previous study has reported a single gene that can enhance chondrogenic properties and reduce the gene activities responsible for senescence, dedifferentiation and oxidative stress in chondrocytes. Downregulation of RHEB expression in prolonged culture has revealed its striking role in chondrocyte biology. Clear downregulation of the expression of a chondrocyte-associated gene (COL2) and its master Copyright © 2016 John Wiley & Sons, Ltd.

transcription factor (SOX9) in prolonged monolayer culture has been reported previously (Hong et al., 2010; Lefebvre et al., 1997). Upregulation of p27 expression in serial passages of chondrocytes also indicates a strong molecular association with senescence (Bringold and Serrano, 2000). This type of structured shift in gene transcription may alter phenotypic properties in chondrocytes during in vitro expansion. J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration

2513

Figure 6. In vitro and in vivo articular cartilage tissue formation. P3 chondrocytes were transfected with mock and RHEB harbouring vectors and then serially cultured up to P6; P6 cells were subjected to 3D (pellet) culture for 21 days and also injected into the subdural space in nude mice for 6 weeks for cartilage tissue formation. (A) In pellet culture samples, the GAG matrix was observed as a blue colour in chondrocytes by Alcian blue staining; COL2 and RHEB expressions were observed by immunohistochemistry (IHC), using anti-COL2 and anti-RHEB antibodies. (B) Expression of mRNA in P6-mock and P6-RHEB on 3D culture were shown by RT– PCR (n = 6). (C) Cartilage formation in the formed tissue in mice was validated by Alcian blue staining; the blue colour indicates a GAG matrix in the cartilage-like extracellular matrix; COL2, RHEB and HNA expressions were observed by IHC; isotype control IHC showed negative results. (D) Schematic summary of RHEB in prolonged culture of chondrocytes for the control of phenotypic, cellular and molecular changes, followed by cartilage formation; scale bar =100 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Notably, knocking down RHEB further attenuated the chondrogenic potential, even in early-passage chondrocytes (P3). RHEB ablation clearly increased COL10 and p27 levels. In addition, expression of COL2, SOX9 and MCL1 decreased with the decrease in RHEB expression, and these changes were dose-dependent. Conversely, RHEB-expressing chondrocytes retained chondrogenic properties with stable expression of COL2 and SOX9, even in P6 chondrocytes, while downregulating COL10 and p27 expression. Surprisingly, upregulation of MCL1 expression (an anti-apoptotic member of the BCL2 family) by RHEB overexpression may in fact be a Copyright © 2016 John Wiley & Sons, Ltd.

critical factor in the modulation of senescence and oxidative stress. According to Bolesta et al. (2012), MCL1 prevents senescence and loss of phosphorylation of retinoblastoma (RB) protein, followed by inhibition of ROS production. In addition, Perciavalle et al. (2012) reported that MCL1 localizes to mitochondria where ROS are generated. In our study, the mitochondrial ROS level was decreased by overexpressing RHEB, as detected by the special mitochondrial ROS-detecting dyes MitoSox Red and MitoTracker Green. Several studies have reported that MCL1 is also involved in the downregulation of p27 expression, a critical inducer of senescence (Perciavalle J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

S. Ashraf et al.

2514

et al., 2012; Chen et al., 2010). We also found upregulation of the expression of the senescence-regulating protein pRB in the nucleus of RHEB-transfected chondrocytes (Alexander and Hinds, 2001; Tamrakar et al., 2000). Evidence from previous studies suggests that senescence resistance (Figure 4b), decreases in ROS level (Figure 4d) and upregulation of MCL1 expression (Figure 4f, 5b) with RHEB overexpression in our study indicate that RHEB acts in the control of senescence and oxidative stress by downregulating p27 expression and upregulating pRB expression through MCL1. In our study, higher expression of RHEB was found in nuclear protein fractions, which could indicate that RHEB would be able to bind to nuclear genes directly or indirectly. It has previously been reported that RHEB is conserved from yeast to human, is localized to the cytoplasm and is involved in cell growth and cell cycle progression (Aspuria and Tamanoi, 2004). More studies are required to discover the role of RHEB in the chondrocyte nucleus. Further, no significant difference in telomere length between mock and RHEB-transfected chondrocytes suggested that RHEB regulates the senescence of chondrocytes irrespective of telomere length-associated replicative senescence. Progressive proliferation and limitation of senescence in RHEB-transfected chondrocytes is likely due to the higher expression of COL2, SOX9, pRB and MCL1 and lower expression of p27 and COL10. These striking results lead us to conclude that RHEB is a critical factor in regulating the chondrogenic properties through distinct functions in the cytoplasm, and in the nucleus via modulating multiple genes and proteins associated with senescence and cell cycle regulation. RHEB-transfected chondrocytes induced enhanced cartilage formation with higher expression of GAG matrix and COL2 and a well-developed lacuna structure, both in vitro (pellet culture) and in vivo (mice). Much better cartilage was generated by RHEB-transfected chondrocytes, because SOX9 plays a pivotal role in maintaining chondrogenic properties through upregulation of COL2 expression (Cha et al., 2013a, b; Lefebvre et al., 1997; Sha’ban et al., 2011). RHEB also downregulated the expression of senescence and dedifferentiation markers (COL10 and p27), which negatively regulate cartilage formation (Emons et al., 2006). HNA detection implies that human chondrocytes survived and contributed to cartilage formation after 6 weeks. It is possible that 6 weeks is too short a

time for the human cells to disappear, but it is sufficient for cartilage regeneration (Kang et al., 2007). The anti-senescence, anti-dedifferentiation and antioxidative stress functions of RHEB promise to broaden the current chondrogenic regimens for cartilage regeneration. Furthermore, regulation of RHEB expression would provide a useful strategy to control chondrogenic properties by modulating SOX9, COL2, MCL1 and p27 activity (Figure 6d).

5. Conclusions The present study is the first systematic experimental investigation of the role of RHEB in chondrocytes; a new role of RHEB in chondrocytes has emerged that attenuates the onset of senescence, dedifferentiation and oxidative stress during in vitro prolonged culture of chondrocytes, resulting in the retention of phenotypic and molecular characteristics. RHEB gene ablation and overexpression implies a regulation of COL2 expression through SOX9 and p27 inhibition via MCL1. RHEB also plays a critical role in the maintenance of proliferation, even in late-passage chondrocytes, by modulating cell cycle inhibitor p27 and dedifferentiation marker COL10. Moreover, enhanced cartilage formation by stable RHEBexpressing chondrocytes both in vitro and in vivo indicates that RHEB is a potential regulator for cartilage tissue regeneration.

Acknowledgements The authors would like to thank the National Research Foundation of Korea (NRF), the Ministry of Science, ICT and Future Planning (Grant No. NRF-2013R1A2A1A09013980 and NRF2015R1A5A1037656) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (Grant No. HR14C3484) for funding this research.

Conflict of interest The authors declare no conflicts of interest.

References Akasaki Y, Alvarez-Garcia O, Saito M et al. 2014; FoxO transcription factors support oxidative stress resistance in human chondrocytes. Arthritis Rheumatol 66: 3349–3358. Alexander K, Hinds PW. 2001; Requirement for p27 (KIP1) in retinoblastoma proteinmediated senescence. Mol Cell Biol 21: 3616–3631. Ashraf S, Cha BH, Kim JS et al. 2015; Regulation of senescence associated signaling

Copyright © 2016 John Wiley & Sons, Ltd.

mechanisms in chondrocytes for cartilage tissue regeneration. Osteoarthr Cartilage 24: 196–205. Aspuria PJ, Tamanoi F. 2004; The Rheb family of GTP-binding proteins. Cell Signal 16: 1105–1112. Barbero A, Martin I. 2007; Human articular chondrocytes culture. Methods Mol Med 140: 237–247. Bentley G, Biant LC, Carrington RW et al. 2003; A prospective, randomized

comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. J Bone Joint Surg Br 85: 223–230. Bolesta E, Pfannenstiel LW, Demelash A et al. 2012; Inhibition of Mcl-1 promotes senescence in cancer cells: implications for preventing tumor growth and chemotherapy resistance. Mol Cell Biol 32: 1879–1892. Brandl A, Hartmann A, Bechmann V et al, 2011; Oxidative stress induces senescence

J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term

2515

RHEB regulates chondrocyte characteristics for cartilage tissue regeneration in chondrocytes. J Orthop Res 29: 1114–1120. Bringold F, Serrano M. 2000; Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol 35: 317–329. Bui C, Barter MJ, Scott JL et al. 2012; cAMP response element-binding (CREB) recruitment following a specific CpG demethylation leads to the elevated expression of the matrix metalloproteinase 13 in human articular chondrocytes and osteoarthritis. FASEB J 26: 3000–3011. Caron MM, Emans PJ, Coolsen MM et al. 2012; Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthr Cartilage 20: 1170–1178. Cha BH, Kim JH, Kang SW et al. 2013a; Cartilage tissue formation from dedifferentiated chondrocytes by codelivery of BMP-2 and SOX-9 genes encoding bicistronic vector. Cell Transpl 22: 1519–1528. Cha BH, Lee JS, Kim SW et al. 2013b; The modulation of the oxidative stress response in chondrocytes by Wip1 and its effect on senescence and dedifferentiation during in vitro expansion. Biomaterials 34: 2380–2388. Chen DY, Liu H, Takeda S et al. 2010; Taspase1 functions as a non-oncogene addiction protease that coordinates cancer cell proliferation and apoptosis. Cancer Res 70: 5358–5367. Cristofalo VJ, Lorenzini A, Allen RG et al. 2004; Replicative senescence: a critical review. Mech Ageing Dev 125: 827–848. Emons JA, Marino R, Nilsson O et al. 2006; The role of p27Kip1 in the regulation of growth plate chondrocyte proliferation in mice. Pediatr Res 60: 288–293. Estes BT, Diekman BO, Gimble JM et al. 2010; Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 5: 1294–1311. Frisbie DD, Bowman SM, Colhoun HA et al. 2008; Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects: results at 12 and 18 months. Osteoarthr Cartilage 16: 667–679.

Hasan SM, Sheen AD, Power AM et al. 2013; Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1. Development 140: 3118–3127. Heidari M, Tahmasebi MN, Etemad S et al. 2011; In vitro human chondrocyte culture: a modified protocol. J Sci Res 9: 102–109. Hidvegi NC, Sales KM, Izadi D et al. 2006; A low temperature method of isolating normal human articular chondrocytes. Osteoarthr Cartilage 14: 89–93. Hong E, Reddi AH. 2013; Dedifferentiation and redifferentiation of articular chondrocytes from surface and middle zones: changes in microRNAs-221/-222, -140 and -143/145 expression. Tissue Eng A 19: 1015–1022. Hong EH, Lee SJ, Kim JS et al. 2010; Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase. J Biol Chem 285: 1283–1295. Kang SW, Yoo SP, Kim BS. 2007; Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage. Biomed Mater Eng 17: 269–276. Lefebvre V, Huang W, Harley VR et al. 1997; SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro-α1(II) collagen gene. Mol Cell Biol 17: 2336–2346. Li Y, Corradetti MN, Inoki K et al. 2004; TSC2: filling the GAP in the mTor signaling pathway. Trends Biochem Sci 29: 32–38. Loty S, Sautier JM, Forest N. 2000; Phenotypic modulation of nasal septal chondrocytes by cytoskeleton modification. Biorheology 37: 117–125. Perciavalle RM, Stewart DP, Koss B et al. 2012; Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration. Nat Cell Biol 14: 575–583. Philipot D, Guérit D, Platano D et al. 2014; p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis. Arthritis Res Ther 16: R58. Rosenzweig DH, Matmati M, Khayat G et al. 2012; Culture of primary bovine chondrocytes on a continuously expanding

surface inhibits dedifferentiation. Tissue Eng A 18: 2466–2476. Rosenzweig DH, Ou SJ, Quinn TM. 2013; P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture. J Cell Mol Med 17: 508–517. Saris DB, Vanlauwe J, Victor J et al. 2008; Characterized chondrocyte implantation results in better structural repair when treating symptomatic cartilage defects of the knee in a randomized controlled trial versus microfracture. Am J Sports Med 36: 235–246. Schulze-Tanzil G. 2009; Activation and dedifferentiation of chondrocytes: implications in cartilage injury and repair. Ann Anat 191: 325–338. Serrano M, Blasco MA. 2001; Putting the stress on senescence. Curr Opin Cell Biol 13: 748–753. Sha’ban M, Cassim SO, Yahya NHM et al. 2011; Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: preliminary analysis. J Tissue Eng Regen Med 8: 32–41. Shin I, Yakes FM, Rojo F et al. 2002; PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 8: 1145–1152. Tamrakar S, Rubin E, Ludlow JW. 2000; Role of pRB dephosphorylation in cell cycle regulation. Front Biosci 5: D121–137. von der Mark K, Gauss V, von der Mark H et al. 1977; Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267: 531–532. Xia Z, Murray D, Hulley PA et al. 2008; The viability and proliferation of human chondrocytes following cryopreservation. J Bone Joint Surg Br 90: 1245–1248. Zheng YH, Xiong W, Su K et al. 2013; Multilineage differentiation of human bone marrow mesenchymal stem cells in vitro and in vivo. Exp Ther Med 5: 1576–1580.

Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site. The following supporting information may be found in the online version of this article:

Copyright © 2016 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med 2017; 11: 2503–2515. DOI: 10.1002/term