SMSCs were isolated and expanded as reported by Li et al (25). Briefly, synovial membrane tissues were harvested from the temporomandibular joints of male Sprague-Dawley rats aged 6 weeks in accordance with the guidelines approved by the Animal Committee of the Zhejiang University. The excised tissues were vigorously washed in phosphate-buffered saline (PBS), and then minced into 1 mm³ pieces and plated in a T25 culture flask with a medium consisting of high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gland Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), penicillin (100 U/ml), streptomycin (100 μg/ml) and 4 mM L-glutamine at 37°C with 5% CO2. The cells were expanded in a monolayer culture for 3–5 passages. In order to evaluate SMSC chondrogensis, cells were seeded at a density of 5×10⁴ cells/cm² in 6-well plates and cultured in chondrogenic medium composed of high glucose DMEM supplemented with 100 nmol/l dexamethasone, 50 mg/l vitamin C, 40 µg/ml proline, 50 µg/ml ascorbate-2-phosphate, 100 µg/ml pyruvate (Sigma, St. Louis, MO, USA) and 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, USA), replaced with each medium change. The culture medium was refreshed once every 2 days until day 7. The cells that were cultured in chondrogenic medium without TGF-β1 served as the control. Each experiment was repeated at least 3 times in order to verify the results.

A pharmacological inhibition study was subsequently performed to investigate the role of the RhoA/ROCK and Smad signaling pathways in TGF-β1-stimulated chondrogenesis. Specific inhibitors of RhoA (C3 transferase, 1 µM), ROCK (Y27632, 5 and 10 µM) (Cytoskeleton, Inc., Denver, CO, USA) and TGF-β type I receptor (SB431542, 5 and 10 µM) (Tocris Bioscience, Bristol, UK) were added into the chondrogenic medium. The inhibitor-containing chondrogenic medium was replenished once every 2 days.

RhoA activation by TGF-β1 was quantified using a luminescence based G-LISA RhoA activation assay biochemistry kit (Cytoskeleton). Immediately following chondrogenic stimulation in the presence of TGF-β1 for different periods of time, the cells were rinsed with ice-cold PBS, and lysed using the provided cell lysis buffer. The lysates were then clarified by centrifugation at 10,000 rpm at 4°C for 2 min. After equalizing protein concentrations in all lysed cell extracts, GTP-bound RhoA levels were determined according to the manufacturer’s instructions. Luminescence was detected as suggested by the manufacturer using a microplate spectrophotometer. The results were expressed relative to the untreated controls.

For the immunofluorescence detection of the actin cytoskeleton, the cells were grown on coverslips in 24-well plates, and then incubated with or without inhibitors of RhoA/ROCK (1 μM C3 transferase, 10 μM Y27632) and Smads (10 μM SB431542) in the presence of TGF-β1. The cells were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min. For F-actin staining, each sample was stained with fluorescein isothiocyanate (FITC)-phalloidin (Sigma) for 40 min at room temperature. Representative fluorescence images were captured using a fluorescence microscope (CX-RFL-2; Olympus, Tokyo, Japan) at ×400 magnification. The average relative intensity of F-actin/cell in each group was analyzed using the MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA).

Total cell lysates were suspended in radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing 1 mM protease inhibitor phenylmethanesulfonyl fluoride (Beyotime) and incubated on ice for 30 min. The samples were clarified by centrifugation at 12,000 rpm for 5 min at 4°C and boiled for 5 min with a sample loading buffer. The protein concentrations were determined using a commercial bicinchoninic acid protein assay kit with bovine serum albumin as the standard. Equivalent protein amounts were fractionated by 10% sodium dodecyl sulfatepolyacrylamide gels and transferred onto 0.1 μM polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Blots were blocked with 5% non-fat milk for 1 h, followed by incubation with specific primary antibodies against phosphorylated (phospho)-Smad2, phospho-Smad3, Smad2/3 and GAPDH (diluted in 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After washing, the blots were then hybridized with specific horseradish peroxidase-conjugated secondary antibodies and visualized using an electrochemiluminescence reagent (Pierce, Rockford, IL, USA).

Total RNA was obtained using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purity and yield of the isolated RNA were monitored using a NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The first-strand cDNA was synthesized from 1 μg total RNA by reverse transcription using a SYBR PrimeScript™ RT reagent kit (Takara Bio, Inc., Dalian, China). The samples were diluted in nuclease-free water and stored at −20°C prior to quantitative real-time polymerase chain reaction (PCR).

Quantitative real-time RT-PCR was performed using a SYBR PrimeScript™ RT-PCR kit (Takara) in the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The sequence-specific primers used for PCR amplification are listed in Table I. The thermal cycling conditions were 1 cycle at 95°C for 30 sec, 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The melting curve analysis for each PCR reaction was generated to ensure the purity of the amplified product (data not shown). Each gene was normalized against the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels using the ΔΔCT method (normalized C_T values were expressed relative to the untreated controls) (26). The relative gene expression of each sample was shown.

All data were presented as the means ± SD. The statistical analysis of the data was performed using the SPSS 19.0 software package by one-way ANOVA. Post hoc comparisons were made using Bonferroni corrections. P<0.05 was considered to indicate a statistically significant difference.

TGF-β1 addition caused a rapid increase in RhoA activation after 1 day of stimulation, reaching its peak on day 4, but gradually decreased as the time proceeded (Fig. 1A). RhoA activation leads to the activation of Rho kinase family members. Thus, these downstream effector kinases were assessed by quantitative real-time PCR analysis with emphasis on determining ROCK1 and ROCK2 mRNA expression. The treatment of SMSCs with TGF-β1 resulted in an upregulation of the ROCK1 and ROCK2 genes (Fig. 1B and C). The expression of these genes was low on day 1, but increased as chondrogenesis proceeded, peaked after 5 days, and was sustained at relatively high levels until day 7. These results demonstrate that TGF-β1 induces the activation of the RhoA/ROCK pathway in SMSCs.

To identify the actin cytoskeletal characteristics of SMSCs, cells were treated with or without pharmacological inhibitors (see above) in addition to TGF-β1 and visualized by fluorescence microscopy using FITC-phalloidin staining. After 7 days of in vitro expansion, the untreated control cells exhibited a relatively weak cytoplasmic staining of F-actin fibers, which were arranged randomly (Fig. 2). Compared with the control cells, TGF-β1-stimulated cells showed a dramatically increased cytoplasmic staining of F-actin fibers, primarily arranged in parallel. The value expressed as an arbitrary unit also showed a dramatic increase of F-actin staining over the controls, indicating more filamentous actin formation (Fig. 3).

The cells that were treated with RhoA/ROCK inhibitors (1 µM C3 transferase or 10 µM Y27632) displayed a significant decrease in fluorescence intensity in comparison to TGF-β1, indicating a reduced number of actin fibers. By contrast, treatment with TβRI inhibitors (10 µM SB431542) did not lead to further alterations in actin fiber formation induced by TGF-β1, showing no apparent change in the F-actin intensity of the SB431542 group. This finding demonstrates that RhoA/ROCK activation plays a specific role in the control of TGF-β1-induced actin cytoskeletal reorganization.

By quantitative real-time PCR analysis, the chondrogenic potential of SMSCs was confirmed by assessing the chondrocyte-specific gene expressions of type I collagen, type II collagen and aggrecan. The mRNA levels of type I collagen, type II collagen and aggrecan significantly increased 7 days after the TGF-β1 treatment compared with the untreated controls (Fig. 4A–C). Further treatment with the ROCK inhibitors resulted in a significantly reduced transcription of the cartilage-specific genes. Specifically, TGF-β1-induced type I collagen, type II collagen and aggrecan mRNA levels decreased by 30, 44 and 51%, respectively, after treatment with 1 µM C3 transferase. When treated with 10 µM Y27632, the TGF-β1-induced gene expressions levels of type I collagen, type II collagen and aggrecan were reduced by 37, 59 and 49%, respectively.

Sox9 has been shown to be a major transcription regulator in the progression of chondrogenesis. Correlating the above observations in type I collagen, type II collagen and aggrecan expression levels, Sox9 mRNA levels were significantly upregulated in the presence of TGF-β1 (Fig. 4D). The induction of the Sox9 mRNA expression was also repressed by C3 transferase and Y27632. The gene expression levels of type I collagen, type II collagen, aggrecan and Sox9 were also affected by C3 transferase and Y27632 in the absence of TGF-β1. However, the influence of these inhibitors on gene expression was relatively minor compared with the samples cultured with TGF-β1. These results suggest the involvement of RhoA/ROCK signaling in the regulation of TGF-β1-induced chondrocyte-specific gene expression in SMSCs.

To obtain insight into the possible interaction between RhoA/ROCK and TGF-β1 signaling cascades, ROCK activation was specifically blocked with Y27632 at the final concentrations of 5 and 10 µM. Relatively high levels of phospho-Smad2 and phospho-Smad3 were maintained after 7 days of TGF-β1 treatment (Fig. 5). After adding 5 µM SB431542 to the TGF-β1 group, the Smad2 and Smad3 phosphorylation levels dramatically decreased. When Y27632 was used at 5 µM, the TGF-β1-induced phosphorylation of Smad2 and Smad3 in SMSCs was slightly reduced. In addition, treatment with 10 µM Y27632 together with TGF-β1 resulted in a marked reduction of Smad2 and Smad3 phosphorylation compared with the TGF-β1 group, which indicated effective inhibition by this pharmacological inhibitor.