The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells

Abstract

Objectives: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells.

Material and methods: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues.

Results: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin.

Conclusions: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.

Keywords: RhoA/ROCK; actomyosin cytoskeleton; compression stress; intervertebral disc degeneration; myosin II.

FIGURE 1

Compression stress induced the senescence of human NP cells. The human NP cells were cultured under 1.0 MPa compression for 0, 12, 24 and 36 h. (A, B) The level of senescent cells in different time was assessed by SA‐β‐gal staining. Scale bar: 50 μm. (C, D) mRNA levels of the ECM remodelling proteinases (MMP3, MMP13 and ADAMTS5) and ECM component (COL1A, COL2A and aggrecan) were quantified using qRT‐PCR. (E, F) The proportions of cells in each cycle were measured through flow cytometry with different time of compression stress. (G, H) CDK4, Cyclin D1, p21 and p53 expression levels in human NP cells subjected to different compression time were measured through Western blot analysis and normalized to that of GAPDH. Data were presented as the mean ± SD (n = 3). #No significance, vs control; *P < .05, vs control; **P < .01, vs control; ***P < .001, vs control

FIGURE 2

Compression stress increased the interaction of myosin IIA with actin. (A) Human NP cells untreated or treated with compression stress for 36 h were stained with myosin IIA (green), F‐actin (red) and DAPI (blue). Scale bar: 5 μm. (B) The co‐localization of myosin IIA with F‐actin was evaluated on the basis of Pearson coefficients. (C, D) Protein interaction between myosin IIA and actin was determined by co‐immunoprecipitation. Following treatment, cell lysates were immunoprecipitated with anti‐actin antibody. Isotype‐matched (IgG) served as negative control. Each precipitated sample was detected for the presence of myosin IIA and actin by immunoblot analysis using specific antibodies. Whole cell lysates prior to the immunoprecipitation served as input controls. (E, F) Cell lysates were immunoprecipitated with anti‐non‐muscle myosin IIA antibody, the next steps are described above. Data were presented as the mean ± SD (n = 3). **P < .05, vs control

FIGURE 3

Compression stress decreased the interaction of myosin IIB with actin. (A) Human NP cells untreated or treated with compression stress for 36 h were stained with myosin IIB (green), F‐actin (red) and DAPI (blue). Scale bar: 5 μm. (B) The co‐localization of myosin IIB with F‐actin was evaluated on the basis of Pearson coefficients. (C, D) Protein interaction between myosin IIB and actin was determined by co‐immunoprecipitation. Following treatment, cell lysates were immunoprecipitated with anti‐actin antibody. Isotype‐matched (IgG) served as negative control. Each precipitated sample was detected for the presence of myosin IIB and actin by immunoblot analysis using specific antibodies. Whole cell lysates prior to the immunoprecipitation served as input controls. (E, F) Cell lysates were immunoprecipitated with anti‐non‐muscle myosin IIB antibody, the next steps are described above. Data were presented as the mean ± SD (n = 3). *P < .05, vs control; **P < .01, vs control

FIGURE 4

Compression stress induced the nuclear translocation of MRTF‐A. (A, B) Nuclear expression levels of MRTF‐A in human NP cells subjected to different compression time measured through Western blot analysis and normalized to that of Lamin B. Data were presented as the mean ± SD (n = 3). *P < .05, vs control; **P < .01, vs control; ***P < .001, vs control. (C, D) Human NP cells untreated or pretreated with 20 μM CCG1423 for 2 h prior to compression stress for 36 h were stained with MRTF‐A (green) and DAPI (blue) (n = 20). Scale bar: 10 μm. (E, F) mRNA levels of the ECM remodelling proteinases (MMP3, MMP13 and ADAMTS5) and ECM component (COL1A, COL2A and aggrecan) were measured after the human NP cells were treated with CCG1423. (G, H) The proportions of cells in each cycle were measured through flow cytometry after the human NP cells were treated with CCG1423. (I, J) CDK4, Cyclin D1, p21 and p53 expression levels in different groups were measured through Western blot analysis and normalized to that of GAPDH. Data were presented as the mean ± SD (n = 3). #No significance, vs compression group; *P < .05, vs compression group; **P < .01, vs compression group

FIGURE 5

Distinct effects of myosin IIA or IIB knockdown on compression stress‐induced human NP cells senescence. NP cells were transfected with siRNAs against myosin IIA or myosin IIB as described in experimental procedures. (A, B) Myosin IIA and IIB knockdown efficiency was shown by Western blot. (C, D) Human NP cells in different groups were stained with MRTF‐A (green) and DAPI (blue) (n = 20). Scale bar: 10 μm. (E, F) mRNA levels of the ECM remodelling proteinases (MMP3, MMP13 and ADAMTS5) and ECM component (COL1A, COL2A and aggrecan) were measured after the human NP cells were treated with siRNAs against myosin IIA or myosin IIB. (G, H) The proportions of cells in each cycle were measured through flow cytometry after the human NP cells were treated with siRNAs against myosin IIA or myosin IIB. (I, J) CDK4, Cyclin D1, p21 and p53 expression levels in different groups were measured through Western blot analysis and normalized to that of GAPDH. Data were presented as the mean ± SD (n = 3). #No significance, vs compression group; *P < .05, vs compression group; **P < .01, vs compression group; ***P < .001, vs compression group

FIGURE 6

Compression stress induced RhoA/ROCK1 pathway activation in human NP cells. The protein expression level of p‐RhoA and RhoA (A), ROCK1 and ROCK2 (B), p‐MLC and MLC (C) subjected to different compression time were measured by Western blot and analysed statistically in different groups. GAPDH was used as control. (D) Immunohistochemistry analysis of p‐RhoA, ROCK1, ROCK2 and p‐MLC in control and degeneration groups. Scale bar: 50 μm. Data were presented as the mean ± SD (n = 3). #No significance, vs control; *P < .05, vs control; **P < .01, vs control; ***P < .001, vs control

FIGURE 7

RhoA/ROCK1 pathway regulated the interaction of myosin IIA and IIB with actin in human NP cells. (A, B) Human NP cells untreated or pretreated with 20 μM Y27632 for 2 h prior to compression stress for 36 h were stained with myosin IIA or IIB (green), F‐actin (red) and DAPI (blue). Scale bar: 5 μm. The co‐localization of myosin IIA or IIB with F‐actin was evaluated on the basis of Pearson coefficients. Data were presented as the mean ± SD (n = 3). *P < .05, vs compression

FIGURE 8

Inhibition of RhoA/ROCK1 pathway attenuated compression stress‐induced human NP cells senescence. (A, B) The level of senescent cells in different groups was assessed by SA‐β‐gal staining. Scale bar: 50 μm. (C, D) Human NP cells untreated or pretreated with 20 μM Y27632 for 2 h prior to compression stress for 36 h were stained with MRTF‐A (green) and DAPI (blue) (n = 20). Scale bar: 10 μm. (E, F) mRNA levels of the ECM remodelling proteinases (MMP3, MMP13 and ADAMTS5) and ECM component (COL1A, COL2A and aggrecan) were measured after the human NP cells were treated withY27632. (G, H) The proportions of cells in each cycle were measured through flow cytometry in different groups. (I, J) CDK4, Cyclin D1, p21 and p53 expression levels in different groups were measured through Western blot analysis and normalized to that of GAPDH. Data were presented as the mean ± SD (n = 3). #No significance, vs compression group; *P < .05, vs compression group; **P < .01, vs compression group; ***P < .001, vs compression group

FIGURE 9

Schematic graph of the role of myosin IIA and IIB in compression stress‐induced senescence of NP cells. Compression stress induced the RhoA/ROCK1 pathway activation, which regulated the interaction of myosin IIA and IIB with actin. The actomyosin cytoskeleton remodelling was involved in the compression stress‐induced fibrotic phenotype mediated by MRTF‐A nuclear translocation and inhibition of proliferation in human NP cells