ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1

Figures

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  Figure 1. YB-1 phosphorylation was increased in diabetic mice heart.A, schematic diagram of animal experiment procedure. C57BL/6J male mice received 60 mg/kg STZ or sodium citrate for 5 days at the age of 8 weeks and sacrificed at 34 weeks. B, the ratios of heart weight to body weight in mice with diabetes mellitus compared with controls. C, blood glucose detected in different time points during the diabetic cardiomyopathy progression. D, representative images of transverse area of cardiomyocyte detected by WGA and HE staining. In WGA staining, green: WGA, red: cTNT, and blue: DAPI. The scale bar for WGA represents 20 μm, and the scale bar for HE represents 50 μm. E and F, histological analysis of WGA and HE staining. The areas of myocyte surface were calculated by Image-Pro Plus and standardized by control group. G, representative echocardiography images (M mode). H and I, echocardiography analysis of EF and FS. J and K, hemodynamic parameters measured by the Millar cardiac catheter system showing a reduction of cardiac function. L and M, representative immunoblotting images and analyzed scatter diagram showing the protein level of cardiac hypertrophy and dysfunction markers in heart…

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  Figure 2. YB-1 phosphorylation induced YB-1 ubiquitination.A and B, Western blot images and analyzed data showed YB-1 protein expression and YB-1 phosphorylation (S102) level in H9c2 cells stimulated with high glucose (HG; 25 mM) at indicated time points. C, immunoprecipitation (IP) of YB-1 revealed a time-dependently elevated ubiquitination level of YB-1 protein in HG-challenged H9c2 cells. D, HG stimulation reduced the interaction between YB-1 and OTUB1. E, YB-1 phosphorylation aggravated YB-1 ubiquitination. H9c2 cells were infected with overexpressing YB-1 or mutant (102A/102D) lentivirus and stimulated by HG for 24 h. Mutant of 102A was used to imitate the dephosphorylation state, and mutant of 102D was used to imitate the continuous phosphorylation state. F, interaction between YB-1 and OTUB1 was suppressed by YB-1 phosphorylation. For all groups, n = 3. Analyzed data are expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01, Student’s t test. OTUB1, otubain-1; S102, serine102; YB-1, Y-box binding protein-1.

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  Figure 3. YB-1 phosphorylation was regulated by p90RSK.A and B, representative immunoblotting images and analyzed scatter diagram showed an increased phosphorylation of p90RSK in diabetic mice heart tissue compared with controls. C–E, inhibiting the activation of RSK could suppress YB-1 phosphorylation (S102) and maintain YB-1 protein level. H9c2 cells were pretreated with 100 μM SL-0101 for 30 min and stimulated by high glucose (HG) for 24 h. F, SL-0101 restored the interaction between YB-1 and OTUB1, which destroyed by HG stimulation. G, SL-0101 ameliorated HG-induced YB-1 ubiquitination. In B, for each group, n = 6; in other panels, for each group, n = 3. Analyzed data are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, compared with control group, Student’s t test or One-way ANOVA, Bonferroni comparison test. DM, diabetes mellitus; NG, normal glucose; OTUB1, otubain-1; RSK, p90 ribosomal S6 kinase; S102, serine102; YB-1, Y-box binding protein-1.

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  Figure 4. ERK promoted YB-1 phosphorylation.A and B, representative immunoblotting images and analyzed scatter diagram of protein expression and phosphorylation level of ERK in diabetes mellitus mice heart tissue. C–F, protein expression and phosphorylation level of ERK, RSK, and YB-1 in H9c2 cells interfered with high glucose (HG) and/or 10 μM U0126. G, U0126 maintained the interaction between YB-1 and OTUB1. H9c2 cells were coinfected with GFP-tagged YB-1 and FLAG-tagged OTUB1-overexpressing lentivirus, pretreated with 10 μM U0126 for 30 min, and challenged by HG for 24 h. H, U0126 reduced YB-1 ubiquitination modification in the context of HG stimulation. In B, for each group, n = 6; in other panels, for each group, n = 3. Analyzed data are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, compared with control group, Student’s t test, Bonferroni comparison test. DM, diabetes mellitus; ERK, extracellular signal-regulated kinase; NG, normal glucose; OTUB1, otubain-1; RSK, p90 ribosomal S6 kinase; YB-1, Y-box binding protein-1.

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  Figure 5. U0126 inhibited YB-1 phosphorylation and alleviated diabetic cardiomyopathy.A, animal experiment procedure. Eight-week-old male C57BL/6J mice were injected with 60 mg/kg STZ to induce diabetes mellitus, and controls received the same volume of sodium citrate. Diabetic mice were treated with 1 mg/kg U0126 or the same volume 6% DMSO in 26 weeks. All mice were sacrificed at the age of 34 weeks. B, blood glucose detected at 34 weeks in mice with different treatments. C, ratios of heart weight to body weight in mice with diverse treatments. D, representative histological images of transverse area of myocyte in mice with different treatments. For WGA staining, green: WGA, red: cTNT, and blue: DAPI. The scale bar represents 20 μm; for HE staining, the scale bar represents 50 μm. E and F, analyzed histological data showing the myocyte cell surface area in diabetic mice with or without U0126 treatment, relative to controls. G, representative echocardiography images of mice with various interventions in M mode. H and I, echocardiography analysis of EF and FS in mice with different interventions. J, representative immunoblotting images showing protein expression level of ANP, BNP, and β-MHC…

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  Figure 6. Graphical abstract. Hyperglycemia increased the MEK1/2 phosphorylation, leading to the significant elevation of the ERK1/2 phosphorylation, which in turn induces the p90RSK phosphorylation. Thereafter, the YB-1 phosphorylation (S102) was promoted by the p90RSK, and the interaction between OTUB1 and YB-1 was subsequently suppressed, resulting in the YB-1 protein ubiquitination and degradation via the proteasome. The diminished YB-1 protein level altered the transcription of related target genes and contributing to the occurrence and progression of diabetic cardiomyopathy. Pharmacological inhibition of the ERK pathway using the MEK inhibitor U0126 could relieve the process described previously, thus alleviating diabetic cardiomyopathy. ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase; OTUB1, otubain-1; RSK, p90 ribosomal S6 kinase; S102, serine102; YB-1, Y-box binding protein-1.