Korean J Physiol Pharmacol.  2023 Jul;27(4):407-416. 10.4196/kjpp.2023.27.4.407.

PSME4 determines mesenchymal stem cell fate towards cardiac commitment through YAP1 degradation

• Kim M
• Kim YS
• Ahn Y
• Eom GH
• Yoon S

• KMID: 2544142
• DOI: http://doi.org/10.4196/kjpp.2023.27.4.407

Abstract

The regeneration of myocardium following acute circulatory events remains a challenge, despite numerous efforts. Mesenchymal stem cells (MSCs) present a promising cell therapy option, but their differentiation into cardiomyocytes is a time-consuming process. Although it has been demonstrated that PSME4 degrades acetyl-YAP1, the role of PSME4 in the cardiac commitment of MSCs has not been fully elucidated. Here we reported the novel role of PSME4 in MSCs cardiac commitment. It was found that overnight treatment with apicidin in primary-cultured mouse MSCs led to rapid cardiac commitment, while MSCs from PSME4 knock-out mice did not undergo this process. Cardiac commitment was also observed using lentivirusmediated PSME4 knockdown in immortalized human MSCs. Immunofluorescence and Western blot experiments revealed that YAP1 persisted in the nucleus of PSME4 knockdown cells even after apicidin treatment. To investigate the importance of YAP1 removal, MSCs were treated with shYAP1 and apicidin simultaneously. This combined treatment resulted in rapid YAP1 elimination and accelerated cardiac commitment. However, overexpression of acetylation-resistant YAP1 in apicidin-treated MSCs impeded cardiac commitment. In addition to apicidin, the universal effect of histone deacetylase (HDAC) inhibition on cardiac commitment was confirmed using tubastatin A and HDAC6 siRNA. Collectively, this study demonstrates that PSME4 is crucial for promoting the cardiac commitment of MSCs. HDAC inhibition acetylates YAP1 and facilitates its translocation to the nucleus, where it is removed by PSME4, promoting cardiac commitment. The failure of YAP1 to translocate or be eliminated from the nucleus results in the MSCs' inability to undergo cardiac commitment.

Keyword

Heart failure; Myocardial infarction; Regenerative medicine

Figure

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  Fig. 1 Deletion of PSME4 decelerates cardiac commitment of bone-marrow mesenchymal stem cells (BM-MSCs). (A) Primary cultured mouse BM-MSCs were prepared in culture dishes, and protein alterations were visualized. Global ablation of endogenous PSME4 led to a complete loss of its protein in BM-MSCs, while YAP1 protein levels remained more abundant in correlation with the decrease in PSME4. The Western blot images shown in the figure represent the results of independently conducted experiments. Western blot analyses were performed at least three times independently, with consistent outcomes observed across all replicates. (B–D) Cardiac commitment of BM-MSCs derived from Psme4 KO mice or from WT littermates. Cells were treated with 3 µM apicidin overnight to promote cardiac commitment. Cardiac commitment determined by Nkx2.5 (B), Tnni3 (C), or GATA4 (D) was successfully induced in BM-MSCs from WT littermates, while Psme4 deletion significantly attenuated it. Each dot represents experimental data obtained from one mouse (N = 4, B to D). Bars indicate mean ± standard error of the mean. Two-way ANOVA with Tukey post-hoc was performed for statistical analysis (B to D). F/C, fold changes; KO, knock-out; WT, wild-type. ***p < 0.001.

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  Fig. 2 PSME4 knockdown decreases cardiac commitment of immortalized human mesenchymal stem cells (MSCs) by delaying the clearance of YAP1. (A) Lentivirus-mediated small hairpin (sh) RNA reduced total mRNA levels of endogenous PSME4 two days post-infection (N = 6). (B–D) Cardiac commitment in hTERT-MSCs was significantly diminished when PSME4 levels were decreased (N = 6). (E) Immunofluorescence assay to visualize the subcellular localization of YAP1 in the absence of PSME4 following apicidin treatment. The reduction of PSME4 resulted in a predominant nuclear localization of YAP1 after stimulation with apicidin (4th panels). Yellow arrows indicate instances of nuclear YAP1 (×400). (F) Western blot analysis shows that a considerable amount of total YAP1 protein persisted. The black arrow denotes the actual signal for PSME4. (G) The administration of shRNA against PSME4 did not affect transcription arrest which was induced by apicidin treatment. Bars represent mean ± standard error of the mean. Independent sample t-test (A) or two-way ANOVA with Tukey post-hoc (B to D, G) was conducted for statistical analysis. F/C, fold changes; Ctrl, control. ***p < 0.001.

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  Fig. 3 YAP1 clearance rate correlates positively with cardiac commitment. The reverse tetracycline transactivator (rtTA) system was integrated with a lentivirus in hTERT-MSCs, and target cells were selected following two days of puromycin incubation. The rtTA system was further validated by the direct expression of green fluorescent protein one day after treatment with 2 µg/ml doxycycline, with rtTA conducted for two days. (A) shRNA against endogenous YAP1 was effectively regulated under the rtTA system. Total mRNA levels were significantly reduced with doxycycline treatment (N = 6). (B) Overnight exposure to apicidin or doxycycline substantially decreased endogenous YAP1, respectively, and the concurrent treatment of both apicidin and doxycycline resulted in significant YAP1 elimination in hTERT-MSCs. (C–E) Doxycycline treatment further enhanced apicidin-driven cardiac commitment. Cardiac commitment was further promoted when doxycycline was added along with apicidin (3rd vs. 4th lanes in C to E) (N = 6). Bars represent mean ± standard error of the mean. Two-way ANOVA and subsequent Tukey post-hoc (A, C to E). MSCs, mesenchymal stem cells; F/C, fold changes. ***p < 0.001.

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  Fig. 4 Acetylation-dependent YAP1 degradation is essential for cardiac commitment. (A, B) Western blot analysis illustrating YAP1 characteristics during apicidin-induced cardiac commitment. A plasmid encoding mouse flag-tagged Yap1 wild-type (WT) or acetylation-resistant (lysine 494/497 to alanine, hereafter referred to as 2KA) was transfected into LT3GEPIR-infected hTERT-MSCs, and successful flag-Yap1 expression was confirmed using flag antibodies. Total YAP1 levels in hTERT-MSCs were comparable among tested conditions (A). As demonstrated in B, one-day treatment with both apicidin and doxycycline nearly eliminated endogenous YAP1, while a significant amount of exogenous Yap1 remained in hTERT-MSCs even in the presence of apicidin and YAP1-targeting shRNA. Note that the acetyl-resistant YAP1 mutant, Yap1 2KA, also displayed resistance to apicidin’s acute regulation (B). (C–E) Acetyl-deficient YAP1 mutant hindered cardiac commitment. Doxycycline-treatment YAP1 further induced cardiac commitment which was enhanced by apicidin (1st vs. 2nd lanes in C to E); apicidin-induced cardiac commitment was set as 1 and fold changes were recorded. Doxycycline-mediated potentiation was significantly diminished with acetyl-deficient Yap1 mutant (5th vs. 6th lanes in C to E) (N = 6). Bars indicate mean ± standard error of the mean. Two-way ANOVA followed by Tukey post-hoc. MSCs, mesenchymal stem cells; F/C, fold changes; rtTA, reverse tetracycline transactivator; Api, apicidin; DOX, doxycycline; EV, empty vector; NS, not significant. ***p < 0.001.

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  Fig. 5 Histone deacetylase (HDAC) inhibition redundantly promotes cardiac commitment of hTERT-MSCs. (A–C) Cytosolic HDAC inhibitor, tubastatin A, demonstrates cardiac commitment capabilities comparable to apicidin. Cardiac commitment is effectively induced in a dose-dependent manner by tubastatin A, with the highest tested dose showing potential similar to apicidin (2nd vs. 4th lanes in A to C) (N = 6). Overnight treatment with tubastatin A also induces YAP1 degradation in hTERT-MSCs (D). (E–I) In addition to HDAC inhibitors, downregulation of cytosolic HDAC6 can initiate cardiac commitment. Transfection with HDAC6-targeting small interfering (si) RNA for two days reduced mRNA of HDAC6 (E, N = 6), and Western blot analysis confirms the loss of HDAC6 in hTERT-MSCs (F). Cardiac commitment is enhanced with HDAC6 reduction (G to I, N = 6), indicating that either chemical inhibition or siRNA-mediated depletion of HDAC6 is sufficient for initiating cardiac commitment in hTERT-MSCs. Data are presented as mean ± standard error of the mean. Two-way ANOVA with Tukey post-hoc (A to C) or independent sample t-test (E, G to I) was used for statistical evaluation. MSCs, mesenchymal stem cells; F/C, fold changes; Ctrl, control; AcTubulin, acetyl-tubulin; NS, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 6 Working hypothesis. PSME4 orchestrates cardiac commitment of mesenchymal stem cells (MSCs). In the maintenance of MSCs stemness, YAP1 primarily resides in the cytosol due to phosphorylation by LAT1/2 kinase (left). Upon treatment with cytosolic histone deacetylase (HDAC) inhibitors or HDAC-targeting siRNA, YAP1 acetylation is indirectly promoted, causing its relocation to the nucleus. Acetylated YAP1 temporarily triggers cardiac gene expression, initiating the process of cardiac commitment. The nuclear proteasome subunit, PSME4, identifies and degrades acetylated YAP1. Concurrently, HDAC inhibitors strongly induce p21 expression, leading to YAP1 transcriptional repression. PSME4 and p21 act synergistically to eliminate YAP1 from MSCs, a critical step for successful cardiac commitment (right).

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