A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes

Animals, chemicals, and strain

Two-week-old Sprague–Dawley (SD) rats of both sexes were provided by the Animal Center of Nanjing Medical University. Fetal bovine serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Zhejiang, China). DMEM-F12, trypsin, collagenase II, and type II collagen were obtained from Sigma (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was acquired from Beyotime Institute of Biotechnology (Jiangsu, China). Protein A/G beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Blocking antibodies against integrin β1 were supplied by BD Biosciences (Franklin Lakes, NJ, USA). Anti-GSK3β antibody, anti-phospho-GSK3β rabbit antibody, anti-ERK1/2 rabbit antibody, anti-phospho-ERK1/2 (Thr202/Tyr204) rabbit antibody, and HRP-goat anti-rabbit IgG were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-ILK rabbit antibody was supplied by Abcam, (Cambridge, MA, USA). HRP-conjugated GAPDH antibody was supplied by Kang Chen (Shanghai, China, KC-5G5). ECL was purchased from Thermo Scientific (Waltham, MA, USA). RNAiso Plus, PrimeScript RT Reagent Kit, and SYBR Premix Ex Taq II were acquired from TaKaRa (Shiga, Japan). ILK shRNA lentiviral particles, control shRNA lentiviral particles, and Polybrene were supplied by Genechem (Shanghai, China).

A cell culture incubator (Heraeus BB 5060), an air-tight cell culture device and reciprocating pressure pump, barrier type pressure transducer, and inverted microscope equipped with a camera system were acquired from Heraeus (Hanau, Germany), Taixing Experimental Instrument Factory (Jiangsu, China), Tianjin Plastics Research Institute (Tianjin, China), and Olympus (Tokyo, Japan), respectively.

Cell culture

Chondrocytes were harvested using the method described by Seguin and Bernier (2003). Briefly, under sterile conditions, cartilage derived from the limb joints of 2-week-old SD rats was harvested and sliced into 1 mm³ pieces. Tissues were digested with 0.25% trypsin at 37°C for 30 min, followed by digestion with 0.2% collagenase II at 37°C for 4 h. Cells were harvested by filtering with a 200-mesh filter and cultured in DMEM-F12 medium supplemented with 10% FBS in an incubator at 37°C with 5% CO₂. The cell population was purified by repeated adherence, and morphology was examined under an inverted phase-contrast microscope following staining with a collagen type II antibody according to the conventional ABC method. Second-generation cells were seeded onto glass slides (25 mm × 25 mm) coated with type II collagen at a density of 10⁵ cells/slide. Experiments were performed when cells reached 70–80% confluence.

Integrin β1 inhibitor

A blocking antibody against integrin β1 was used as a specific inhibitor of integrin β1. It was dissolved in DMEM to form a 1,000× concentrated solution, and aliquots were stored at −20°C. This concentrated solution was diluted 1000× immediately prior to use. Cells were pre-treated with blocking antibodies against integrin β1 (10 µg/mL) or an equivalent amount of DMEM for 5 h.

Construction of a periodic mechanical stress field

A periodic stress field encompassing the perfusion culture system with adjustable stress intensity and frequency was constructed by connecting a reciprocating intensifier pump to the air-tight cell culture device through a barrier-type pressure transducer, as described previously (Nong et al., 2010). In an earlier study, we showed that rat chondrocytes subjected to stress varying from 0 to 200 kPa at 0.1 Hz yielded tissue-engineered cartilage of the best quality (Yue et al., 2007; Zhang et al., 2007). Accordingly, a pressure range of 0–200 kPa and 0.1 Hz frequency were used in the current study.

ILK assay

ILK enzymatic activity was assessed according to previously reported protocols [0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM HEPES (pH 7.4), 150 mM NaCl] (Tseng et al., 2010). In brief, after immunoprecipitation of ILK with anti-ILK antibody overnight at 4°C from 250 mg of lysate, we resuspended the beads at 30°C for 30 min in 30 µL of kinase buffer containing 1 µg of recombinant substrate [glycogen synthase kinase 3β (GSK3β) fusion protein] and 200 µmol/L cold ATP. Total GSK3β protein and the phosphorylated substrate were detected by Western blot analysis with GSK3β and phospho-GSK3β antibodies, respectively.

Transfection analysis

The shRNA targeting ILK and negative control sequences were ligated into the GV118 vector mU6-MCS-Ubi-EGFP. After the different fragments were combined with lentivirus vectors, they were transfected into 293T cells to package lentivirus particles.

Chondrocytes were divided into two treatment groups. Each group of cells was plated at a concentration of 10⁵ cells/well into six-well plates and allowed to adhere for 1 day before transfection. Next day, the cells were transfected with Lv-shRNA-ILK vector or control Lv-GFP vector in serum-free medium. Twelve hours after transfection, all cultures were changed to complete medium. Three days after transfection, GFP expression was detected by fluorescence microscopy, and the expression of ILK protein was assessed by Western blot analysis.

Proliferation studies

Proliferation was assessed by two different methods, specifically, direct cell counting and the CCK-8 assay.

Direct cell counting

Cells were trypsinized and counted according to published protocols (Chaturvedi et al., 2007). Second-generation chondrocytes were seeded onto glass slides (25 mm × 25 mm) coated with type II collagen at a density of 10⁵ cells/slide and randomly divided into different groups, each comprising six glass slides. Experiments were performed when cells had reached approximately 70–80% confluence. Chondrocytes were cultured for 3 days with or without periodic mechanical stress for 8 h per day, prior to direct cell counting. Cells were trypsinized and counted, and the cell number was determined by counting cells from each glass slide independently. Experiments were repeated five times.

CCK-8 assay

Cell proliferation was determined using CCK-8 solution according to the manufacturer's instructions (Dojindo Lab., Kumamoto, Japan) (Bae et al., 2010). Cells were added to 10 µL of CCK-8 solution in each well of five 96-well plates (n = 5) and incubated for 4 h at 37°C. The absorbance of each well was determined at 450 nm using a microplate reader.

Quantitative real-time PCR (qPCR) analysis

Total RNA was extracted using RNAiso Plus and reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit, according to the manufacturer's protocol (TaKaRa). qPCR analysis was performed with the LightCycler System (Roche Diagnostics, Basel, Switzerland) using SYBR Premix Ex Taq II, as described previously (Kloesch et al., 2012). The reaction was performed in a 20 µL mixture containing 2 µL cDNA. Each cDNA sample was amplified using specific primers (Table 1, TaKaRa) under the following cycling conditions: 30 s initial denaturation at 95°C, followed by 40 cycles at 95°C for 5 s and 60°C for 20 s. Gene expression of AGC and Col2 was normalized against that for GAPDH.

Western blot analysis

Total protein was prepared and Western blot analyses were performed as described previously (Wang et al., 2007). Total protein was prepared using RIPA buffer, and the Bradford assay was employed to determine protein concentrations. Protein samples were resolved using SDS–PAGE and transferred to nitrocellulose membranes. Following blocking for 1 h with 5% skimmed milk in TBST, membranes were incubated with antibodies (1:1,000 dilution for all antibodies) overnight at 4°C. Blots were incubated with horseradish peroxidase-conjugated secondary antibody at ambient temperature for 1 h, and color was subsequently developed with ECL. Results were scanned using a gel imaging system (UVP LLC, Upland, CA, USA) and measured using Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA).

Statistical analysis

Statistical analyses were performed using SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA), and results are expressed as mean ± standard deviation. Student's unpaired t-test and one-way analysis of variance (ANOVA), followed by post hoc Fisher's least significance difference (LSD) test, were used to determine statistical significance. A P-value <0.05 was considered significant.

Periodic mechanical stress enhances ILK activity

ILK is widely considered to be an intracellular adaptor that couples integrins modulating multiple cellular responses (Bhattacharya et al., 2015; Moraes et al., 2015). Hsu et al. (2007) reported that ultrasound stimulation significantly enhanced the catalytic activity of ILK, leading to increased COX-2 expression in chondrocytes. However, the effects of mechanical stimulation on the catalytic activity of ILK in chondrocytes were unknown. We observed that exposure to periodic mechanical stress resulted in a rapid and sustained elevation of ILK activity (Figures 1a and 1b). The coincidence between ultrasound and periodic mechanical stress in the activation of ILK in chondrocytes raised the possibility that there may be some similar regulatory mechanisms between the two stimuli. Indeed, earlier studies by Schortinghuis et al. (2004) and Feril and Kondo (2004) elucidated that ultrasound stimulation could induce cavitation to bring out some biomechanical effects in cells.

ILK is required for periodic mechanical stress-initiated chondrocyte proliferation and matrix synthesis

Next, we examined whether activation of ILK mediated periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Several researchers have demonstrated that following various types of non-mechanical stimulation, ILK is essential for many cell behaviors and functions such as proliferation and development in chondrocytes (Grashoff et al., 2003; Terpstra et al., 2003). Our results revealed that 80% of chondrocytes were successfully transfected and that ILK protein exhibited a significant decrease in the Lv-shRNA-ILK group compared to the Lv-GFP group (Figures 2a and 2d). In addition, ILK prevention noticeably inhibited periodic mechanical stress-induced up-regulation of chondrocyte proliferation (Figures 3a and 3b) and matrix synthesis (Figures 3c and 3d). These results strongly suggest that ILK is a pivotal decision-making protein involved in chondrocyte functions in response to periodic mechanical stress. To the best of our knowledge, this is the first study to characterize the specific roles of ILK signaling in chondrocyte mitogenic effects following exposure to periodic mechanical stress.

Integrin β1 is essential for periodic mechanical stress-induced ILK activity

We next aimed to examine the upstream and downstream relationships between ILK and integrin β1. ILK has generally been reported to be a pivotal integrin-associated signaling protein that transmits multiple extracellular inputs into intracellular biochemical signals (Pan et al., 2014; Yang et al., 2016). In our present investigation, we found that after pretreatment with a functional blocking antibody against integrin β1, periodic mechanical stress-induced ILK activity was markedly inhibited (Figures 4a and 4b). This result revealed that integrin β1 is located upstream of ILK in this context. Similarly, Nho et al. (2005) demonstrated that in a setting of matrix-derived mechanical forces, integrin β1 inhibition would result in significant depletion of the ILK activity responsible for fibroblast viability. However, some contrasting findings have also been reported. For instance, a study by Li et al. (2013) reported that in epidermal cells exposed to hepatocyte growth factor (HGF), silencing of ILK by RNA interference significantly blocked the expression levels of integrin β1 in the process of wound healing. Furthermore, in addition to integrins, some other cell surface receptors have been shown to regulate ILK in a variety of settings (Delcommenne et al., 1998). The differences may be due to the fact that signal transduction is a complex multi-component process and that variations in cell type, stimulus, and setting would contribute to different results. Based on our results, we concluded that the intrinsic ILK catalytic activity induced by periodic mechanical stress in chondrocytes is at least partly mediated by integrin β1, but that important roles for other receptors or signaling proteins upstream of ILK cannot be excluded.

ILK modulates periodic mechanical stress-induced ERK1/2 phosphorylation

We then explored the regulatory roles of ILK in ERK1/2 phosphorylation under periodic mechanical stress. A search of the literature revealed an increasing number of publications indicating that activation of ERK1/2 is dependent on the activity of ILK when subjected to some non-mechanical stimuli in a variety of cell types (Naska et al., 2006; Tabe et al., 2007). However, the effect of ILK activation on ERK1/2 phosphorylation in mechanotransduction was poorly understood. We observed that the phosphorylation levels of ERK1/2 were obviously attenuated upon ILK inhibition by its targeted shRNA in response to periodic mechanical stress (Figures 5a and 5b). Our findings strongly imply a correlation between ILK and ERK1/2 activation in this context. In contrast, Maier et al. (2008) reported that although both ERK1/2 and ILK were activated by cyclic strain, ERK1/2 activation was not regulated by ILK in fibroblasts. One reasonable explanation for these differing results is that none of the complex signaling pathways act in isolation, and the same kinase could be regulated by various signal proteins while there is also crosstalk between pathways.

In conclusion, our study confirms that ILK is an indispensable regulator closely related to the integrin β1-ERK1/2 signal cascade responsible for chondrocyte proliferation and matrix synthesis under conditions of periodic mechanical stress.