TMEM182 inhibits myocardial differentiation of human iPS cells by maintaining the activated state of Wnt/β‐catenin signaling through an increase in ILK expression

Figures

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  FIGURE 1. DOX‐induced overexpression of TMEM182 did not affect the pluripotency or proliferative potential of hiPSCs. (A) Evaluation of TMEM182 expression by qPCR in hiPSCs capable of overexpressing TMEM182 with DOX treatment. Values represent the mean ± SD of three independent experiments (n = 3). DOX, doxycycline. **p < 0.01 versus −DOX treatment, by Student's t‐test. (B) Detection of HA protein in the membrane fraction of hiPSCs overexpressing TMEM182 using western blotting. GAPDH antibody was used as a marker protein for the cytosolic fraction, while EGFR and Na,K‐ATPase antibodies were used for evaluating the membrane fraction. Cyto, cytosolic fraction; Mem, membrane fraction. (C) Evaluation of NANOG and POU5F1 expressions using qPCR in hiPSCs overexpressing TMEM182. Values represent the mean ± SD of three independent experiments (n = 3). There was no significant difference between the two groups, by Student's t‐test. ns, non‐significant. (D) Evaluation of cell proliferation over 7 days in hiPSCs during TMEM182 overexpression using cell counting. Values represent the mean ± SD of eight independent experiments (n = 8). There was no significant difference in cell numbers after culture…

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  FIGURE 2. Overexpression of TMEM182 had no effect on the differentiation of hiPSCs into cardiac mesoderm, although it did inhibit their differentiation into cardiac progenitor cells (CPCs) and cardiomyocytes. (A) Myocardial differentiation protocol of hiPSCs. Days 0–1: CHIR99021 (12 μM), Days 2–4: IWP‐2 (5 μM), Days 0–16: DOX treatment (1 μg/mL). (B) Change in expression of TMEM182 with DOX treatment during myocardial differentiation was confirmed using qPCR. Values represent the mean ± SD of five independent experiments (n = 5). **p < 0.01 versus each day −DOX treatment, by one‐way ANOVA followed by the Tukey–Kramer test. −D: without DOX treatment, +D: with DOX treatment. (C) Expression of BrachyuryT (mesoderm) and MESP1 (cardiac mesoderm) during TMEM182 overexpression was confirmed using qPCR. Values represent the mean ± SD of five independent experiments (n = 5). There was no significant difference between the two groups on each day, by one‐way ANOVA followed by the Tukey–Kramer test. (D) Expression of NKX2.5, TBX5, and GATA4 (CPCs) during TMEM182 overexpression was confirmed using qPCR. Values represent the mean ± SD of five independent experiments (n = 5). **p < 0.01, *p < 0.05 versus…

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  FIGURE 3. Overexpression of TMEM182 promoted the induction of differentiation of cardiac fibroblasts during the myocardial differentiation process. (A) Change in expression of POU5F1 during TMEM182 overexpression was confirmed using qPCR. Values represent the mean ± SD of five independent experiments (n = 5). **p < 0.01 versus −DOX treatment on Day 2, ## p < 0.01 versus +DOX treatment on Day 2, by one‐way ANOVA followed by the Tukey–Kramer test. (B) Expression of CDH18 (epicardial) and POSTN and DDR2 (cardiac fibroblasts) during TMEM182 overexpression was confirmed using qPCR. Values represent the mean ± SD of five independent experiments (n = 5). **p < 0.01, *p < 0.05 versus each day −DOX treatment, by one‐way ANOVA followed by the Tukey–Kramer test. (C) Expression of COL1A1 during TMEM182 overexpression on Day 12 of differentiation was confirmed using western blotting. Quantification of COL1A1 protein expression in differentiated cells overexpressing TMEM182. Values represent the mean ± SD of three independent experiments (n = 3). **p < 0.01 versus −DOX treatment, by Student's t‐test. COL1A1, collagen type I alpha 1 chain; α‐TUB, α‐tubulin. (D) Expression of vimentin during TMEM182…

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  FIGURE 4. TMEM182 inactivates GSK‐3β via phosphorylation of AKT and promotes nuclear translocation of β‐catenin. (A) Protein expression of p‐β‐catenin (S552) and β‐catenin was confirmed in cells overexpressing TMEM182 on Day 7 of differentiation by western blotting. (B) Quantification of p‐β‐catenin (S552) and β‐catenin protein expression in differentiated cells overexpressing TMEM182. Values represent the mean ± SD of five independent experiments (n = 5). *p < 0.05 versus −DOX treatment, by Student's t‐test. (C) Immunofluorescence staining of β‐catenin and DAPI in cells overexpressing TMEM182 to observe the nuclear translocation of β‐catenin. (D) Protein expression of p‐GSK‐3β (S9), GSK‐3β, p‐AKT (S473), and AKT was confirmed in cells overexpressing TMEM182 on Day 7 of differentiation by western blotting. (E) Quantification of p‐GSK‐3β (S9), GSK‐3β, p‐AKT (S473), and AKT protein expression in differentiated cells overexpressing TMEM182. Values represent the mean ± SD, p‐GSK‐3β (S9)/GSK‐3β: n = 5, p‐AKT (S473)/AKT: n = 4. **p < 0.01 versus −DOX treatment, by Student's t‐test.

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  FIGURE 5. TMEM182 interacts with integrin‐linked kinase (ILK) and increases its expression. (A) Protein expression of ILK was confirmed in cells overexpressing TMEM182 on Day 7 of differentiation. α‐TUB, α‐tubulin. (B) Quantification of ILK protein expression in differentiated cells overexpressing TMEM182. Values represent the mean ± SD of five independent experiments (n = 5). **p < 0.01 versus −DOX treatment, by Student's t‐test. (C) Membrane fractions of hiPSCs overexpressing TMEM182‐HA in a DOX‐induced manner were immunoprecipitated by HA antibody and western blotted with the ILK and HA antibody. (D) Schematic of intracellular signaling during TMEM182‐overexpressing hiPSCs differentiation into cardiac fibroblasts. IP, immunoprecipitation.

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  FIGURE 6. Differentiation into cardiomyocytes was higher in the low TMEM182‐expression iPSC line. (A) Evaluation of TMEM182 expression by qPCR in two hiPS cell lines with different TMEM182 expression levels. Values represent the mean ± SD of three independent experiments (n = 3). *p < 0.05 versus the low‐expression cell line, by Student's t‐test. (B) Expression of NANOG and POU5F1 was confirmed using qPCR in hiPSCs with two different levels of TMEM182 expression. Values represent the mean ± SD of three independent experiments (n = 3). No significant difference was observed between the two groups by Student's t‐test. ns, non‐significant. (C) Expression of TBX5 and MYH6 was confirmed using qPCR in differentiated cells derived from hiPSCs with different expression levels of TMEM182. Values represent the mean ± SD, low: n = 5, high: n = 4. **p < 0.01 versus the low‐expression cell line, by Student's t‐test. (D) Changes in the expression of cardiomyocyte‐associated proteins during the myocardial differentiation process of hiPSCs with different expression levels of TMEM182. cTnT, cardiac muscle troponin T; PLN, phospholamban; α‐TUB, α‐tubulin. (E) Changes in TNNT2 expression were confirmed by…