SPSB1-mediated inhibition of TGF-β receptor-II impairs myogenesis in inflammation

Abstract

Background: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-β) is involved in both processes. We uncovered an increased expression of the TGF-β receptor II (TβRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TβRII signalling impairs myogenic differentiation in response to inflammation.

Methods: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-β/TβRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses.

Results: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1β-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TβRII, resulting in TβRII ubiquitination and destabilization. SPSB1 impaired TβRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice.

Conclusions: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TβRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

Keywords: Critical illness myopathy; Inflammation-induced muscle atrophy; Myogenic differentiation; SPSB1; Sepsis; TGFβ receptor II.

Figure 1

Spsb1 is upregulated in inflammation‐induced skeletal muscle atrophy. (A) Quantitative RT‐PCR (qRT‐PCR) analysis of Spsb1 from the tibialis anterior (TA) muscle in 12‐week‐old male C57BL/6J mice subjected to cecal ligation and puncture (CLP, n = 5, 24 h; n = 7, 96 h) or sham surgery (sham, n = 4, 24 h; n = 5, 96 h). (B) qRT‐PCR of SPSB1 from the vastus lateralis muscle of patients with intensive care unit‐acquired weakness (ICUAW, n = 7) compared with healthy subjects (controls, n = 12). (C) qRT‐PCR of Spsb1 from five‐day‐differentiated C2C12 myotubes (MT5) treated with TNF (10 ng/mL), IL‐1β (10 ng/mL) or IL6/IL6R (100 ng/mL) for 2 h. (D) qRT‐PCR of Spsb1 from MT5 that were pretreated with the IKK1‐inhibitor BMS‐345541 (5 μM) 60 min before TNF or IL‐1β treatment. (E) qRT‐PCR of Il1st and Spsb1 from MT5 after Il6st‐siRNA transfection and pretreatment with the JAK2 inhibitor AG490 or the STAT3 inhibitor C188–9 prior to IL‐6 treatment. (F) qRT‐PCR analysis of Spsb1 from the TA muscle of Nlrp3 knockout (KO) and Nlrp3 wildtype (WT) littermate control mice subjected to CLP (n = 7) or sham (n = 5) surgery for 96 h, as indicated. mRNA expression was normalized to Gapdh. Data in panels (A) and (D–F) were analysed with two‐way ANOVA followed by Tukey's post‐hoc test; data in panel (B) were analysed with two‐tailed Student's t‐test; data in panel (C) were analysed with one‐way ANOVA followed by Tukey's post‐hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. n.s. denotes not significant.

Figure 2

SPSB1 associates with and ubiquitinates TGF‐β receptor II and reduces its stability. (A) Co‐immunoprecipitation (Co‐IP) using lysates from C2C12 cells transfected with FLAG‐TβRII, FLAG‐TβRII‐ΔEx2 and SPSB1‐Myc. Extracts were immunoprecipitated (IP) with anti‐FLAG agarose and detected with antibodies against Myc and FLAG, as indicated. Input Western blot with anti‐Myc and anti‐FLAG antibody as indicated. (B) Immunofluorescence using anti‐FLAG antibody together with A488‐coupled secondary antibody (green) and anti‐Myc antibody together with A555‐coupled secondary antibody (red) to detect SPSB1‐Myc, FLAG‐TβRII and FLAG‐TβRII‐ΔEx2 in transfected C2C12 cells. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C) Pictogram of functional domains and mutation sites of SPSB1 constructs. (D) Co‐IP of FLAG‐SPSB1 (WT) and mutants (SPSB1‐Y129A, ‐TYAA or ‐ΔSOCS) with endogenous TβRII from MT5 C2C12 cells overexpressing GFP, SPSB1 and SPSB1 mutants. Extracts were IP with anti‐FLAG agarose and detected with anti‐TβRII antibody. Input proteins were detected with anti‐FLAG, TβRII and GAPDH antibodies as indicated. (E) COS‐7 cells were transfected with FLAG‐TβRII, SPSB1‐Myc, SPSB1‐ΔSOCS and HA‐Ub, as indicated. Cells were treated with MG132 (25 μM) or vehicle (DMSO 0.25%) 42 h post‐transfection for a further 6 h. ll cells were lysed 48 h post‐transfection and lysates were immunoprecipitated (IP) with anti‐FLAG affinity gel. Immunoblotting (IB) with indicated antibodies. (F) Cells were infected with a retrovirus encoding GFP or SPSB1 for 48 h and then treated with cycloheximide (CHX, 50 μg/mL) for indicated timepoints. Anti‐FLAG antibody shows overexpressed FLAG‐SPSB1; specific band is indicated by asterisk. GAPDH was used as loading control. Densitometric analysis; dotted line indicates 50% abundance of TβRII. N = 3 biologically independent experiments; representative blots are shown. Data are presented as mean ± standard deviation.

Figure 3

SPSB1 downregulates TGF‐β signalling by its SPRY and SOCS‐‐box domain and inhibits myogenic differentiation. (A) Five‐day‐differentiated C2C12 myotubes (MT5) transduced with GFP or SPSB1 were treated with TGF‐β (5 ng/mL) or solvent control for 5 min. Lysates were analysed by Western blot analysis with anti‐phospho Akt antibody (Ser473). Total Akt was used as control (left panel). Densitometric analysis (right panel). Data were analysed with two‐way ANOVA followed by Tukey's post‐hoc test. **P < 0.01, ***P < 0.001. (B) C2C12 cells were transduced by control GFP, SPSB1 (WT) or mutants (SPSB1‐Y129A, ‐TYAA or ‐ΔSOCS) retrovirus and differentiated for 5 days. Western blot analysis with anti‐phospho Akt antibody (Ser473) (left panel) and densitometric analysis (right panel). Total Akt was used as control. Data were analysed with one‐way ANOVA followed by Tukey's post‐hoc test. Asterisk (*) indicates significant differences between SPSB1 (wildtype or mutants as indicated) and the GFP control group, *P < 0.05, **P < 0.01, ***P < 0.001; ^# denotes a significant difference between indicated SPSB1 mutants and the SPSB1 wildtype group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. (C) O‐Propargyl‐puromycin (OP‐puro) labelling of de novo synthesized polypeptides. Scale bar, 100 μm. (D) Experimental design. (E) Protein lysates from MT5 were analysed by Western blot with anti‐FLAG and anti‐GAPDH antibody. (F) Immunofluorescent staining with anti‐Fast MyHC antibody. Nuclei were stained with DAPI (blue). GFP (green) indicates retrovirally transduced cells. Scale bar, 50 μm.

Figure 4

SPSB1 and proinflammatory cytokines inhibit myogenic differentiation. (A) Differentiation index, fusion index, and nuclei distribution in myosin positive (myosin⁺) cells were quantified from images in Figure 3 panel (F). (B) Western blot analysis of lysates from GFP and SPSB1 transduced cells that were differentiated for 5 days with indicated antibodies. GAPDH was used as loading control. Densitometric analysis is shown in the right panel. Data in panel (A; Differentiation and fusion index) and (B) were analysed with two‐tailed Student's t‐test; data in panel A (myosin⁺ cells) were analysed with two‐way ANOVA followed by Tukey post hoc test; asterisk (*) indicates a significant difference of SPSB1 (WT) or mutants compared with GFP group, *P < 0.05, **P < 0.01, ***P < 0.001; ^# indicates a significant difference of SPSB1 mutants compared with SPSB1 (WT) group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation. (C) Immunofluorescent staining of 5 days differentiated C2C12 cells treated with TNF (10 ng/mL), IL‐1β (10 ng/mL) or IL6/IL6R (100 ng/mL), as indicated, every 24 h throughout differentiation, with anti‐fast MyHC antibody. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) Differentiation index, fusion index, and nuclei distribution in myosin⁺‐cells were quantified from images in panel (C). (E) qRT‐PCR analysis of Myh1, Myh3, and Myh7. mRNA expression was normalized to Gapdh. Data in panel (D; Differentiation and fusion index) were analysed with two‐tailed Student's t‐test; data in panel (D; myosin⁺‐cells) and (E) were analysed with two‐way ANOVA followed by Tukey post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation.

Figure 5

SPSB1 mediated inhibition of myogenic differentiation depends on its SPRY and SOCS‐‐box domain. (A, B) C2C12 myoblasts were transduced by control GFP, SPSB1 (WT) or mutant (SPSB1‐Y129A, ‐TYAA or ‐ΔSOCS) containing retrovirus and differentiated for 5 days. (A) Immunofluorescent staining with anti‐fast MyHC antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (B) Differentiation index, Fusion index, and Nuclei distribution in each myosin⁺ cell were quantified from images in panel (A). (C, D) Primary myoblasts were transduced by control GFP, SPSB1 (WT) or mutant (SPSB1‐Y129A, ‐TYAA or ‐ΔSOCS) containing retrovirus and differentiated for 5 days. (C) Immunofluorescent staining with anti‐fast MyHC antibody (red). GFP (green) indicates retrovirally transduced cells. Scale bar, 100 μm. (D) Differentiation index, Fusion index, and Nuclei distribution in each myosin⁺ cell were quantified from images in panel (C). Data in panels (B and C; Differentiation and Fusion index), were analysed with two‐tailed Student's t‐test; data in panels (B) and (H) (Nuclei distribution in myosin⁺ cells) were analysed with two‐way ANOVA followed by Tukey's post‐hoc test; asterisk (*) indicates significant differences between SPSB1 (wildtype or mutants as indicated) and the GFP control group, *P < 0.05, **P < 0.01, ***P < 0.001; denotes a significant difference between indicated SPSB1 mutants and the SPSB1 wildtype group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation.

Figure 6

Expression of Akt restores myogenesis in SPSB1 overexpressing cells. Cells were transduced by control GFP, SPSB1, Akt‐Myr, respectively, or co‐transduced by Akt‐Myr and SPSB1 retrovirus and differentiated for 5 days. (A) Western blot analysis of anti‐phospho Akt antibody (Ser473). Total Akt was used as control. Densitometric analysis is displayed in the right panel. (B) Cells described above were incubated with OP‐puro labelling for 1 h. Red fluorescence (upper panel) corresponds to de novo synthesized polypeptides. (C) Immunofluorescent staining of above cells with anti‐Fast MyHC as primary antibody and Alexa Fluor 555 conjugated secondary antibody (red). Scale bar, 100 μm. (D) Differentiation index, Fusion index, and Distribution of nuclei in myosin positive (myosin⁺) cells were quantified from images in panel (C). (E) Western blot was performed using lysates from above cells with anti‐Fast MyHC and anti‐Slow MyHC antibody. Overexpressed Akt‐Myr and SPSB1 were detected by anti‐HA and anti‐FLAG antibody, respectively. GAPDH was used as loading control. Densitometric analysis of Western blot signals are displayed (right panel). Data in panels (A), (D; differentiation and fusion index) and (E) were analysed with one‐way ANOVA followed by Tukey's post‐hoc test; data in panel (D; myosin⁺ cells) were analysed with two‐way ANOVA followed by Tukey's post‐hoc test. Asterisk (*) indicates a significant difference between SPSB1‐, Akt‐Myr‐ or SPSB1 and Akt‐Myr‐treated groups compared with GFP control treated cells, *P < 0.05, **P < 0.01, ***P < 0.001; ^# indicates a significant difference between SPSB1‐ and SPSB1 + Akt‐Myr‐treated cells, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation.

Figure 7

Myogenin restores Akt signalling and protein synthesis in SPSB1 overexpressing cells. C2C12 cells were transduced by control GFP, SPSB1, Myogenin, respectively, or co‐transduced by Myogenin and SPSB1 retrovirus and differentiated for 5 days. (A) Western blot with anti‐Myc antibody. GAPDH was used as loading control. (B) qRT‐PCR analysis of Myog mRNA expression normalized to Gapdh. (C) Western blot analysis with anti‐phospho Akt antibody (Ser473). Total Akt was used as control. Densitometric analysis is shown in the right panel. (D) O‐Propargyl‐puromycin (OP‐puro) assay: Red fluorescence (upper panel) corresponds to de novo synthesized polypeptides. (E) qRT‐PCR analysis of Mymk, Mymx, Myh1, Myh3, and Myh7 from described cells. mRNA expression was normalized to Gapdh. Data were analysed with one‐way ANOVA followed by Tukey's post‐hoc test. Asterisk (*) indicates a significant difference between SPSB1‐, Myogenin‐ or SPSB1 + Myogenin‐ and GFP control groups, *P < 0.05, **P < 0.01, ***P < 0.001; ^# indicates a significant difference between SPSB1‐ and SPSB1 + Myogenin‐treated cells, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation.

Figure 8

Myogenin restores myogenesis in SPSB1 overexpressing cells. (A–C) C2C12 cells were transduced by control GFP, SPSB1, Myogenin, respectively, or co‐transduced by Myogenin and SPSB1 retrovirus and differentiated for 5 days. (A) Immunofluorescent staining of above cells with anti‐Fast MyHC as primary antibody and Alexa Fluor 555 conjugated secondary antibody (red). Scale bar, 100 μm. (B) Differentiation index, Fusion index, and Distribution of nuclei in myosin positive (myosin⁺) cells were quantified from images in panel (A). (C) Western blot was performed using lysates from above cells with anti‐Fast MyHC and anti‐Slow MyHC antibody. Overexpressed SPSB1 was detected by anti‐FLAG antibody. GAPDH was used as loading control. Densitometric analysis of Western blot signals are displayed (right panel). Data in panels (B; Differentiation and Fusion index) and (C) were analysed with one‐way ANOVA followed by Tukey's post‐hoc test; data in panel (B; myosin⁺ cells) were analysed with two‐way ANOVA followed by Tukey's post‐hoc test. Asterisk (*) indicates a significant difference between SPSB1‐, Myogenin‐ or SPSB1 + Myogenin‐ and GFP control groups, *P < 0.05, **P < 0.01, ***P < 0.001; ^# indicates a significant difference between SPSB1‐ and SPSB1 + Myogenin‐treated cells, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation. (D, E) Primary myoblasts were transduced by control GFP, SPSB1, Myogenin, respectively, or co‐transduced by Myogenin and SPSB1 retrovirus and differentiated for 5 days. (D) Immunofluorescent staining of above cells with anti‐Fast MyHC as primary antibody and Alexa Fluor 555 conjugated secondary antibody (red). GFP (green) indicates retrovirally transduced cells. Scale bar, 100 μm. (E) Differentiation index, Fusion index, and Nuclei distribution in each myosin⁺ cell were quantified from images in panel (D). Data in panel (E; Differentiation and Fusion index) were analysed with one‐way ANOVA followed by Tukey's post‐hoc test; data in panel (E; myosin⁺ cells) were analysed with two‐way ANOVA followed by Tukey's post‐hoc test. Asterisk (*) indicates a significant difference between SPSB1‐, Myogenin‐ or SPSB1 + Myogenin‐ and GFP control groups, *P < 0.05, **P < 0.01, ***P < 0.001; ^# indicates a significant difference between SPSB1‐ and SPSB1 + Myogenin‐treated cells, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001. N = 3 biologically independent experiments; data are presented as mean ± standard deviation.