The effects of β-catenin on cardiomyogenesis via Islet-1 and MLIP ubiquitination

Abstract

Mesenchymal stem cells (MSCs) can treat myocardial injury-related diseases by differentiating into cardiomyocytes. Islet-1 plays an essential role in cardiac maturation. We have discovered that Islet-1 plays a crucial role in the histone acetylation regulation in this process. In addition, to increase GATA4/Nkx2.5 expression, Islet-1 may bind to Gcn5 and then guide Gcn5 to the GATA4/Nkx2.5 promoters, thereby facilitating the differentiation of MSCs into cardiomyocytes. Islet-1 is an important factor in the maturation of the heart. We have previously found that the pivotal factor in histone acetylation regulation in this process is Islet-1. Furthermore, Islet-1 and Gcn5 may boost GATA4/Nkx2.5 expression, which in turn promotes cardiomyocyte differentiation from MSCs. But the molecular mechanism of Islet-1 binding to GCN5 has not been elucidated. In this study, we found that the competitive binding relationship between Islet-1 and MLIP and GCN5 affected myocardial differentiation. The key enzymes of ubiquitination modification of MLIP and Islet-1 are UBE3C and WWP1, respectively. When short hairpin RNA (shRNA) was used to inhibit β-catenin expression, we found that the expression of UBE3C was upregulated, modifying MLIP ubiquitination and reducing its expression, and it upregulated Islet-1 by inhibiting the expression of WWP1. By using the chromatin immunoprecipitation (ChIP) and luciferase reporter system, we found that when MLIP binds to Islet-1, it significantly inhibits the transcriptional activity of Islet-1. In summary, our results show that decreasing β-catenin regulates the ubiquitination of Islet-1 and MLIP, affecting their expression, reducing the amount of Islet-1 binding to MLIP, and increasing the amount of binding to GCN5 in the nucleus. Therefore, the transcriptional activity of Islet-1 is significantly activated, inducing C3H10T1/2 cells to differentiate into myocytes. Further knowledge of biochemical pathways, including molecular signaling pathways, can provide more insights into the myocardial differentiation mechanism of MSCs.

Keywords: C3H10T1/2 cells; Islet-1; MLIP; cardiomyogenesis; ubiquitination; β-catenin.

Figure 1.

β-catenin affects the expression of Islet-1, GCN5, and MLIP. β-catenin was stably knocked down in C3H10T1/2 cells using a lentiviral vector and was overexpressed in C3H10T1/2 cells using a lentiviral vector. The expression of Islets, MLIP, and Gcn5 (A) in the indicated groups was detected by Western blotting. The mRNA levels of Islets (B), MLIP (C), and Gcn5 (D) in the indicated groups were measured by qPCR. The results are expressed as means ± SD of three individual experiments that, for each condition, were performed in triplicate. *p < 0.05, **p < 0.01. (A color version of this figure is available in the online journal.)

Figure 2.

β-catenin affects the binding relationship of Islet-1, GCN5, and MLIP. β-catenin was stably knocked down in C3H10T1/2 cells using a lentiviral vector. On the 7th and 14th days after obtaining the stable strain, the cell lysates were immunoprecipitated with an anti-Islet-1 antibody, an anti-MLIP antibody, or control IgG followed by the Co-IP assay to determine the interaction of Gcn5 and Islet-1 (A). After the cell extracts from the indicated groups were subjected to immunoprecipitation with the anti-MLIP antibody and immunoblotting with the anti-Islet-1 antibody, the cell lysates were immunoprecipitated with the anti-Islet-1 antibody again, and Gcn5 was detected by immunoblotting (A). Islet-1 interaction with the promoter regions of Nkx2.5 (B) and GATA4 (C) was determined by the ChIP assay in C3H10T1/2 cells. The results are expressed as mean ± SD of three individual experiments that, for each condition, were performed in triplicate. *p < 0.05, **p < 0.01. (A color version of this figure is available in the online journal.)

Figure 3.

β-catenin affects the ubiquitination of Islet-1 and MLIP. β-catenin was stably knocked down in C3H10T1/2 cells using a lentiviral vector. The half-lives of MLIP and Islet-1 were detected by pulse-chase analysis (A–C). After knocking down β-catenin for 7 days, the cells were treated with MG132 (10 μM) for 24 h. The expression of Islet-1 and MLIP (D) in the indicated groups was detected by Western blotting. The cell extracts from the indicated groups were subjected to immunoprecipitation with an anti-Islet-1 antibody (E) or anti-MLIP antibody (F), followed by immunoblotting with an anti-ubiquitin antibody. The results are expressed as the mean ± SD of three individual experiments that, for each condition, were performed in triplicate. *p < 0.05, **p < 0.01. (A color version of this figure is available in the online journal.)

Figure 4.

The E3 ubiquitin ligase WWP1 acts on Islet-1 to affect its ubiquitin modification. β-catenin was stably knocked down in C3H10T1/2 cells using a lentiviral vector. The tissue lysates were immunoprecipitated with an anti-Islet-1 antibody or control IgG followed by the Co-IP assay to determine the interaction of Islet-1 and WWP1 (A). The interaction of β-catenin with the WWP1 promoter region was detected by the ChIP assay (B). WWP1 promoter activity was measured by the luciferase assay (C). β-catenin was stably knocked down or WWP1 was overexpressed in C3H10T1/2 cells using a lentiviral vector. The expression of Islets and MLIP (D) in the indicated groups was detected by Western blotting. The results are expressed as mean ± SD of three individual experiments that, for each condition, were performed in triplicate. *p < 0.05, **p < 0.01. (A color version of this figure is available in the online journal.)

Figure 5.

The E3 ubiquitin ligase UBE3C acts on MLIP to affect its ubiquitin modification. The tissue lysates were immunoprecipitated with an anti-MLIP antibody or control IgG followed by the Co-IP assay to determine the interaction of MLIP and UBE3C (A). UBE3C promoter activity was measured by the luciferase assay (B). UBE3C expression in the indicated groups was detected by Western blotting (C). The expression of miR-34a (D) and UBE3C (E) in the indicated groups was detected by qPCR. Luciferase assay (F) and ChIP assay (H). β-catenin or UBE3C was stably knocked down in C3H10T1/2 cells using a lentiviral vector. The expression of Islets and MLIP (G) in the indicated groups was detected by Western blotting. The results are expressed as mean ± SD of three individual experiments that, for each condition, were performed in triplicate. *p < 0.05, **p < 0.01. (A color version of this figure is available in the online journal.)

Figure 6.

Mechanistic diagram of the Wnt/β-catenin pathway in pluripotency maintenance and myocardial differentiation of C3H10T1/2 cells. (A color version of this figure is available in the online journal.)