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. 2019 Apr:55:38-49.
doi: 10.1016/j.pupt.2019.01.007. Epub 2019 Jan 28.

SUMOylation of Vps34 by SUMO1 promotes phenotypic switching of vascular smooth muscle cells by activating autophagy in pulmonary arterial hypertension

Yufeng Yao  1 , Hui Li  1 , Xinwen Da  1 , Zuhan He  1 , Bo Tang  1 , Yong Li  1 , Changqing Hu  1 , Chengqi Xu  1 , Qiuyun Chen  2 , Qing K Wang  3
Affiliations

SUMOylation of Vps34 by SUMO1 promotes phenotypic switching of vascular smooth muscle cells by activating autophagy in pulmonary arterial hypertension

Yufeng Yao et al. Pulm Pharmacol Ther. 2019 Apr.

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is a life-threatening disease without effective therapies. PAH is associated with a progressive increase in pulmonary vascular resistance and irreversible pulmonary vascular remodeling. SUMO1 (small ubiquitin-related modifier 1) can bind to target proteins and lead to protein SUMOylation, an important post-translational modification with a key role in many diseases. However, the contribution of SUMO1 to PAH remains to be fully characterized.

Methods: In this study, we explored the role of SUMO1 in the dedifferentiation of vascular smooth muscle cells (VSMCs) involved in hypoxia-induced pulmonary vascular remodeling and PAH in vivo and in vitro.

Results: In a mouse model of hypoxic PAH, SUMO1 expression was significantly increased, which was associated with activation of autophagy (increased LC3b and decreased p62), dedifferentiation of pulmonary arterial VSMCs (reduced α-SMA, SM22 and SM-MHC), and pulmonary vascular remodeling. Similar results were obtained in a MCT-induced PAH model. Overexpression of SUMO1 significantly increased VSMCs proliferation, migration, hypoxia-induced VSMCs dedifferentiation, and autophagy, but these effects were abolished by inhibition of autophagy by 3-MA in aortic VSMCs. Furthermore, SUMO1 knockdown reversed hypoxia-induced proliferation and migration of PASMCs. Mechanistically, SUMO1 promotes Vps34 SUMOylation and the assembly of the Beclin-1-Vps34-Atg14 complex, thereby inducing autophagy, whereas Vps34 mutation K840R reduces Vps34 SUMOylation and inhibits VSMCs dedifferentiation.

Discussion: Our data uncovers an important role of SUMO1 in VSMCs proliferation, migration, autophagy, and phenotypic switching (dedifferentiation) involved in pulmonary vascular remodeling and PAH. Targeting of the SUMO1-Vps34-autophagy signaling axis may be exploited to develop therapeutic strategies to treat PAH.

Keywords: Atg14; Beclin-1; Vps34.

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Figures

Fig. 1.
Fig. 1.. Hypoxia induces pulmonary vascular remodeling and PAH in mice.
Mice were exposed to a continuous hypoxic condition for 4 weeks and characterized for the mean pulmonary artery pressure (mPAP), right ventricle/left ventricle + septum (RV/LV + S), total pulmonary resistance, and vascular remodeling in the lungs. (A) The mPAP was significantly increased in hypoxia-induced PAH mice compared with control mice under normoxia. (B) The RV/LV + S ratio was significantly increased in PAH mice compared with control mice. (C) The total pulmonary resistance was significantly increased in PAH mice compared with control mice. (D) The cardiac output was not significantly decreased in PAH mice compared with control mice. (E) The degree of pulmonary artery remodeling in PAH mice increased significantly compared with control mice. (F) Analysis of pulmonary arteries. The thickness of vessel walls was significantly increased in PAH mice compared with control mice. Fully muscularization of small pulmonary arteries was significantly increased in PAH mice compared with control mice. (n = 6/group, *P < 0.05, **P < 0.01).
Fig. 2.
Fig. 2.. Association of increased SUMO1 expression with activation of autophagy in PAH.
(A) Western blot analysis using lung tissue samples showed significantly increased expression levels of SUMO1, HIF-1α and autophagy marker LC3b, and a significantly decreased expression level of autophagy marker p62 in hypoxic PAH mice than in control mice. (B) The Western blotting data in (A) were quantified and plotted. (n = 6/group, *P < 0.05, **P < 0.01).
Fig. 3.
Fig. 3.. VSMCs phenotypic switching in hypoxic PAH mice.
(A) Real-time RT-PCR data showed that the mRNA expression levels of contractile marker genes for α–SMA, SM22 and SM-MHC were significantly reduced in hypoxic PAH mice compared with control mice. (B) Western blot analysis showed that the protein expression levels of α-SMA, SM22 and SM-MHC were significantly decreased in hypoxic PAH mice compared with control mice. (C) Western blot data in (B) were quantified and plotted. (n = 6/group, *P < 0.05, **P < 0.01). GAPDH was used as endogenous control.
Fig. 4.
Fig. 4.. Increased SUMO1 expression, induction of autophagy and phenotypic switching in a MCT-induced mouse model for PAH.
(A) The degree of pulmonary artery remodeling in MCT-treated mice increased significantly compared with control mice. (B) Analysis of pulmonary arteries. (C) Western blot analysis using lung tissue samples showing significantly increased expression levels of SUMO1, HIF-1α and autophagy marker LC3b, and a significantly decreased expression level of autophagy marker p62 in MCT-treated mice than in control mice. (D) Western blot data in (C) were quantified and plotted. (E) Western blot analysis showing that the protein expression levels of α-SMA, SM22 and SM-MHC were significantly decreased in hypoxic PAH mice compared with control mice. (F) Western blot data in (E) were quantified and plotted. (n = 6/group, *P < 0.05, **P < 0.01).
Fig. 5.
Fig. 5.. Overexpression of SUMO1 increased hypoxia-induced proliferation and migration of VSMCs.
(A) Analysis of cell proliferation of the VSMCs with the CCK-8 kit by reading the optical absorbance density at 450 nm. Hypoxia increased VSMCs proliferation. Overexpression of SUMO1 further increased VSMC proliferation. (B) Overexpression of SUMO1 increased the hypoxia-induced increase of the mRNA expression level of Ki67 (proliferation marker). (C) Flow cytometry analysis to measure the number of cells at the S phase during cell division. Hypoxia significantly increased the cell number at the S phase. Overexpression of SUMO1 further increased the number of S phase cells. (D) Scratch-wound healing cell migration assay. Hypoxia increased the migration of VSMCs, and overexpression of SUMO1 further accelerated VSMCs migration. (E) Flow cytometry analysis to measure the number of apoptotic cells. Hypoxia significantly decreased the number of apoptotic cells. Overexpression of SUMO1 further decreased the level of apoptosis. (F) Analysis of the level of apoptosis (n = 3/group, *P < 0.05, **P < 0.01).
Fig. 6.
Fig. 6.. SUMO1 regulates hypoxia-induced VSMCs phenotypic switching by activating autophagy.
(A) Overexpression of SUMO1 increased hypoxia-induced autophagy while decreased the expression levels of VSMCs contractile markers. Western blot analysis showed that hypoxia significantly decreased the expression levels of α–SMA, SM22 and SM-MHC. The effect was further enhanced by overexpression of SUMO1. SUMO1 significantly increased the expression level of LC3b and decreased the expression level of p62 induced by hypoxia. (B) Western blot data in (A) were quantified and plotted. (C) Autophagy inhibitor 3-MA diminished the effect of SUMO1 overexpression on protein expression levels of α–SMA, SM22 and SM-MHC. (D) Western blot data in (A) were quantified and plotted. (E) Autophagy inhibitor 3-MA diminished the effect of increased VSMCs proliferation induced by SUMO1 overexpression. (n = 3/group, *P < 0.05, **P < 0.01).
Fig. 7.
Fig. 7.. Knockdown of SUMO1 expression reversed hypoxia-induced proliferation, autophagy, phenotypic switching and migration of PASMCs.
(A) Analysis of cell proliferation of PASMCs with the CCK-8 kit by reading the optical absorbance density at 450 nm. Hypoxia increased PASMCs proliferation, however, the effect was reversed by SUMO1 knockdown. (B) SUMO1 knockdown reversed the hypoxia-induced increase of the mRNA expression level of Ki67 (a proliferation marker). (C) Flow cytometry analysis to measure the number of cells at the S phase during cell division. Hypoxia significantly increased the cell number at the S phase. SUMO1 knockdown reduced the effect. (D) Scratch-wound healing cell migration assay. Hypoxia increased the migration of PASMCs, however, the effect was reversed by SUMO1 knockdown. (E) Flow cytometry analysis to measure the number of apoptotic cells. Hypoxia significantly decreased the level of apoptosis, however, the effect was reversed by SUMO1 knockdown. (F) Analysis of the level of apoptosis. (G) Western blot analysis showing that hypoxia significantly decrease expression levels of α-SMA, SM22, SM-MHC and p62, and a significantly increased expression level of autophagy marker LC3b in PASMCs. However, the effects were reversed by SUMO1 knockdown. (H) Western blot data in (G) were quantified and plotted. (n = 3/group, *P < 0.05, **P < 0.01).
Fig. 8.
Fig. 8.. SUMO1 increases Vps34 SUMOylation involved in the activation of autophagy and hypoxia-induced VSMCs phenotypic switching.
(A) Western blot analysis showed that hypoxia induced binding of SUMO1 to endogenous Vps34 in VSMCs. (B) Western blot data in (A) were quantified and plotted. (C) Co-immunoprecipitation assays showed that hypoxia increased the Beclin-1/Vps34/ATG14L complex formation, and overexpression of SUMO1 further increased the complex formation Cell extracts were immunoprecipitated with anti-Beclin1 and probed with anti-Vps34, anti-Atg14L. (D) Western blot data in (C) were quantified and plotted. (E) Vps34 mutant with K840R at the SUMOylation site impaired the binding of SUMO1 to endogenous Vps34 in VSMCs. (F) Western blot data in (E) were quantified and plotted. (G) Vps34 mutant with K840R significantly increased the protein expression levels of α–SMA, SM22 and SM-MHCas compared to that with Vps34 wild type. (G) Western blot data in (H) were quantified and plotted. (n = 3/group, *P < 0.05, **P < 0.01).
Fig. 9.
Fig. 9.. A schematic working model for a novel SUMO1-regulated mechanism involved in pulmonary vascular remodeling and PAH.
In the hypoxia-induced mouse model for PAH, the expression level of SUMO1 was significantly increased, which induces Vps34 SUMOylation. Vps34 SUMOylation promotes the formation of autophagy initiation complex Beclin-1/Vps34/ATG14L, which induces activation of autophagy, resulting in pulmonary vascular remolding and PAH.
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