Low-intensity pulsed ultrasound (LIPUS) treatment of cultured chondrocytes stimulates production of CCN family protein 2 (CCN2), a protein involved in the regeneration of articular cartilage: mechanism underlying this stimulation  T. Nishida, S. Kubota, E. Aoyama, N. Yamanaka, K.M. Lyons, M. Takigawa  Osteoarthritis and Cartilage  Volume 25, Issue 5, Pages 759-769 (May 2017) DOI: 10.1016/j.joca.2016.10.003 Copyright © 2016 Terms and Conditions

Fig. 1 Promotion of chondrogenesis-associated gene expression and CCN2 production in cultured chondrocytes treated with LIPUS. (A) After HCS-2/8 cells had reached sub-confluence, the cells were treated with LIPUS at a frequency of 3.0 MHz and an intensity of 60 mW/cm2 for 20 min. After 30 min of LIPUS treatment, total RNA was isolated, and real-time RT-PCR analysis was performed. The amounts of these mRNAs were normalized to the amount of GAPDH mRNA. Graph shows the expression level of (a) COL2a1, (b) ACAN, (c) CCN2, (d) SOX9 and (e) MMP13 with (n = 6) or without LIPUS (n = 6). The dots represent the data of two sets of experiment with independent culture dishes (n = 3). The data of COL2a1 expression is analyzed by Welch's t-test, and other gene expression data was analyzed by Student's t-test. In the all graph, the ordinate indicates the relative ratio with respect to None (ratio = 1.0), and bars represent mean and 95% confidence intervals. (B, C, and D) After HCS-2/8 cells (B), primary REC cells (C), and primary RAC cells (D) had reached confluence, they were treated with LIPUS at a frequency of 3.0 MHz and an intensity of 30, 45, or 60 mW/cm2 for 20 min. The cell lysates were prepared 5 h later, and Western blot analysis was then performed by using an anti-CCN2 and β-actin antibodies. The amount of CCN2 was determined densitometrically and these amounts were normalized to the amount of β-actin. Relative densitometry (None = 1.0) from six measurements are presented at the bottom of photographs and analyzed by one-way ANOVA. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 2 Effect of LIPUS on cortical actin organization in HCS-2/8 cells. After HCS-2/8 cells had reached sub-confluence, the cells were treated with LIPUS at a frequency of 3.0 MHz and an intensity of 60 mW/cm2 for 20 min. Then, these cells were fixed with 3.5% paraformaldehyde at 0, 30, and 60 min after LIPUS treatment, and fluorescence staining was performed by using rhodamine–phalloidin to visualize F-actin. After LIPUS treatment, F-actin was detected at the cell periphery in a time-dependent manner. The bar represents 50 μm. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 3 LIPUS-stimulated phosphorylation of ERK1/2 and p38 MAPK in HCS-2/8 cells and effect of inhibitors of ROCK, Rac1, p38 MAPK, and ERK1/2 on LIPUS-induced CCN2 production. (A) HCS-2/8 cells were grown in DMEM containing 10% FBS until they had reached confluence. Then, the cells were treated with LIPUS (3.0 MHz, 60 mW/cm2) for 20 min. After LIPUS treatment, cell lysate were prepared at 0, 5, 10, 15, 30, and 60 min; and Western blot analysis was performed with the indicated antibodies. The level of phosphorylated p38 MAPK was increased immediately after the 20-min LIPUS treatment (time 0) and then declined gradually. ERK1/2 phosphorylation levels were increased at 5 and 10 min after the LIPUS treatment. In contrast, phosphorylated forms of p38 MAPK, and ERK1/2 were only slightly detected in untreated samples. (B) After HCS-2/8 cells had reached confluence, the cells were pre-treated with Y27632 (ROCK inhibitor; 10 μM), NSC23766 (Rac1 inhibitor; 10 μM), SB203580 (p38 MAPK inhibitor; 10 μM) or PD98059 (MEK1 inhibitor; 50 μM). After 18 h, the cells were treated with LIPUS under the same conditions as in “A”. Cell lysates were prepared 5 h later, and Western blot analysis was performed with anti-CCN2 and β-actin antibodies. The amount of CCN2 was determined densitometrically and these amounts were normalized to the amount of β-actin. Relative densitometry (DMSO = 1.0) from five measurements are presented at the bottom of photographs and analyzed by one-way ANOVA. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 4 Promotion of CCN2 production in chondrocytes by LIPUS was mediated by Ca2+ influx. (A) After HCS-2/8 cells had reached sub-confluence, the cells were pre-treated with Fluo-4AM (final concentration; 3 mmol/l) in a recording medium at 37°C. After 20 min, the culture medium was replaced with recording medium without Fluo-4AM; and photographs of the cells before or after LIPUS treatment were taken under a fluorescence microscope (A-upper photographs). The same field was visualized by phase-contrast microscopy (A-lower photographs). The bar represents 50 μm. (B) After HCS-2/8 cells had reached confluence, the cells were pre-treated with ionomycin (Ca2+ ionophore; 1.4 or 14 μM) for 18 h and then exposed to LIPUS (3.0 MHz, 60 mW/cm2) for 20 min. After 5 h, the cell lysates were prepared, and Western blot analysis was performed by using an anti-CCN2 and β-actin antibodies. The amount of CCN2 was determined densitometrically and these amounts were normalized to the amount of β-actin. Relative densitometry (DMSO = 1.0) from six measurements are presented at the bottom of photographs and analyzed by one-way ANOVA. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 5 Ablation of cortical actin organization and the gene expression of Col2a1 and Acan in Ccn2-deficient chondrocytes. (A) Primary chondrocytes were isolated from the rib cage of littermates of wild-type (WT) and Ccn2-deficient (KO) mouse embryos at E18.5 and were inoculated into 35-mm culture dish. WT and KO chondrocytes were cultured until they had reached confluence. Then, total RNA was prepared from both WT and KO chondrocytes, after which quantitative RT-PCR analysis was performed with Ccn2-specific primers. The dots indicated in each sample are representative data of independent culture dishes (n = 3), which was analyzed by Student's t-test. Bars represent mean and 95% confidence intervals. (B) WT and KO chondrocytes were cultured in αMEM containing 10% FBS until they had reached sub-confluence. Then, the cells were treated with LIPUS (3.0 MHz, 60 mW/cm2) for 20 min and then fixed with 3.5% paraformaldehyde at 1 h after the LIPUS treatment. Fluorescence staining was performed by using fluorescence-labeled phalloidin to visualize F-actin. Cortical actin organization (arrowheads) was detected in WT cells treated with LIPUS, but not in the KO cells treated with LIPUS. Bar represents 250 μm. (C) WT and KO chondrocytes were treated with LIPUS (3.0 MHz, 60 mW/cm2) for 20 min, and total RNA was collected 1 h later. Real-time RT-PCR analysis was performed and these gene expression levels were normalized to that level of Gapdh. (a) Graph shows the expression level of (a) Col2a1 in WT cells treated with (n = 6) or without LIPUS (n = 6) and KO cells treated with (n = 6) or without LIPUS (n = 6), (b) Acan in WT cells treated with (n = 5) or without LIPUS (n = 6) and KO cells treated with (n = 6) or without LIPUS (n = 6), (c) Sox9 in WT cells treated with (n = 6) or without LIPUS (n = 6) and KO cells treated with (n = 6) or without LIPUS (n = 6) and (d) Mmp13 in WT cells treated with (n = 6) or without LIPUS (n = 6) and KO cells treated with (n = 6) or without LIPUS (n = 6). The dots indicated in each sample are representative data of two sets of experiments with independent culture dishes (n = 3). The data of Mmp13 expression in WT sample was analyzed by Welch's t-test, while the other gene expression data is analyzed by Student's t-test. In all the graphs, the ordinate indicate the relative ratio with respect to None (ratio = 1.0), and bars represent mean and 95% confidence intervals. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 6 Down-regulated expression of Trpv4 and Bkca channel genes in KO chondrocytes and decreased Ca2+ influx in KO chondrocytes treated with LIPUS. (A) After WT and KO chondrocytes had reached confluence, total RNA was collected; and RT-PCR analysis was performed. Right graph shows the gene expression level of Trpv4 in WT (n = 3) or KO cells (n = 3). Left graph shows the gene expression level of Bkca channel in WT (n = 6) or KO cells (n = 5). These data were analyzed by Student's t-test. In these graphs, the ordinate indicates the relative ratio with respect to sample of WT cells (ratio = 1.0), and bars represent mean and 95% confidence intervals. (B) After WT and KO chondrocytes had reached sub-confluence, they were pre-treated with Fluo-4AM (final concentration; 3 mmol/l) in a recording medium at 37°C. After 20 min, the culture medium was replaced with the recording medium without Fluo-4AM; and fluorescence signaling was acquired after LIPUS treatment for 1 min (B-upper). LIPUS-induced Ca2+ influx was decreased in KO chondrocytes compared with that in WT chondrocytes. Lower photos show phase-contrast images of the cells (B-lower). The bar represents 50 μm. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 7 Stability of Trpv4 and BKca channel mRNAs in HCS-2/8 cells treated with siRNA against CCN2. (A) HCS-2/8 cells were transfected with siRNA against CCN2 (siRNA) or control (NC) RNA (final concentration; 3 μM) by using electroporation, and the gene expression level of CCN2 was analyzed after 2 days by RT-PCR analysis. The gene expression of CCN2 was significantly decreased in HCS-2/8 cells treated with the siRNA. The amount of CCN2 mRNA was normalized to that of GAPDH mRNA. The ordinate shows the relative ratio. The dots indicated in each sample are representative data from independent culture dishes (n = 3), which was analyzed by Student's t-test. Bars represent mean and 95% confidence intervals. (B and C) Degradation of Trpv4 (B) and BKca channel (C) mRNAs in HCS-2/8 cells treated with siRNA against CCN2. HCS-2/8 cells were transfected with CCN2 siRNAs under the same conditions as indicated in “A”. After 2 days, the cells were treated with 10 μg/ml actinomycin D (Act D) for 3 or 6 h. The relative amounts of Trpv4 and BKca channel mRNAs remaining after treatment with Act D were quantified by performing RT-PCR analysis and are shown as a percentage vs the value at time 0. The dots indicated in each sample are representative data from independent culture dishes (n = 6), and these data are analyzed by ANOVA. Bars represent mean and 95% confidence intervals. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions

Fig. 8 Possible intracellular signaling pathways triggered by LIPUS treatment of chondrocytes. LIPUS stimulation promotes calcium influx via TRPV4, and the intracellular Ca2+ activates MAPKs pathways. As a result, CCN2 production is up-regulated and the gene expression levels of Col2a1 and Aggrecan, which are two major components of the cartilage matrix, are increased by the CCN2, and thus may lead to cartilage matrix accumulation. Moreover, because we have reported that CCN2 interacts with actin, it is possible that CCN2 promotes actin polymerization and that the actin polymerization affects the production of CCN2. Furthermore, LIPUS-induced CCN2 regulates the stability of TRPV4 and BKca channel mRNAs. These findings suggest that CCN2 plays an important role in the formation of a positive-feedback loop to enhance the chondrocytic phenotype. Osteoarthritis and Cartilage 2017 25, 759-769DOI: (10.1016/j.joca.2016.10.003) Copyright © 2016 Terms and Conditions