RGS10 inhibits proliferation and migration of pulmonary arterial smooth muscle cell in pulmonary hypertension via AKT/mTORC1 signaling

ABSTRACT

Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH.

Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model.

Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model.

Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH.”

KEYWORDS:

• RGS10
• AKT
• mTORC1
• pulmonary arterial smooth muscle cell
• pulmonary hypertension

Introduction

Pulmonary hypertension (PH) is a complex disorder characterized by pathological pulmonary artery (PA) remodeling and enhanced pulmonary vascular resistance, eventually leading to hypertrophy and failure of the right heart. There are different five most common types of PH, including pulmonary arterial hypertension (PAH), PH resulted from left-heart diseases, lung diseases/hypoxia, obstructive PAH, or PH with unclear and/or multifactorial mechanis (1). A variety of stimuli, such as hypoxia, inflammation and oxidative stress, are involved in the development of PH (2). Chronic obstructive pulmonary disease, obstructive sleep apnea and other hypoxic diseases are usually complicated with PH (3), indicating the crucial role of hypoxia in PH. Multiple mechanisms are involved in PH, among which PA remodeling is a key event. As the major component of pulmonary vessel, pulmonary arterial smooth muscle cell (PASMC) plays an important role in the development of PH. Under the stimulus of hypoxia, the PA undergoes a pathological remodeling characterized by excessive proliferation and migration of PASMC, which finally leads to PH (4). Although several molecular signaling pathway, such as endothelin-1, have been confirmed to participate in the development of PH, the symptoms and prognosis of PH have still not been eradicated (5). Therefore, elucidating novel etiology for abnormal proliferation and migration of PASMC is important to prevent PH.

Regulators of G protein signaling (RGS) proteins are negative modulators of G-protein coupled receptors by serving as GTPase activating proteins (6). Among these RGS proteins, RGS10 belongs to the D/R12 subfamily and is richly expressed in immune system and nervous system (7). RGS10 is involved in the regulation of inflammation, apoptosis, and tumor growth (7–9). The tumor-suppression effect of RGS10 results from its anti-proliferative role in different cancer cells (9). Additionally, RGS10 is also involved in pulmonary and vascular diseases. For example, RGS10 reduces lung inflammation and suppresses platelet activation and thrombogenesis (7,10). However, whether RGS10 is involved in proliferation of PASMC and development of PH remains unclear.

Previous studies demonstrate that RGS10 regulates several cellular processes via suppressing mammalian target of rapamycin (mTOR) activity (8,9). mTOR complex 1 (mTORC1) is one of the classical complexes of mTOR. Abnormal activation of mTORC1 is associated with hypoxia-induced excessive proliferation and migration of PASMC (11,12). In addition, increased mTORC1 activity is known to promote the development of PH (13,14). Therefore, it is of clinical importance to identify the role of mTORC1 in RGS10-mediated regulation of PASMC.

In the current study, we observed increased expression of RGS10 in hypoxia-challenged PASMC and Hypoxia/SU5416 PH mouse. Overexpression of RGS10 reduced the proliferation and migration of hypoxia-challenged PASMC, while silencing RGS10 further promoted hypoxia-induced proliferation and migration of PASMC. We further revealed that excessive AKT/mTORC1 activation was responsible for RGS10-mediated regulation of PASMC. Finally, we demonstrated that RGS10 could effectively relieve the initial hyperplasia of PA and right ventricular overload in vivo. Our findings support the hypothesis that RGS10 is a potential target for preventing PH via suppressing proliferation and migration of PASMC.

Materials and methods

Culture and treatment of PASMC

Primary PASMC was isolated from PAs of 8- to 10-week-old C57BL/6J mice and incubated at 37°C in humidified 5% CO2 atmosphere as our previous study described (11). PASMC in normoxia group was incubated under the condition of 74% N₂, 21% O₂ and 5% CO₂ at 37°C, while PASMC in hypoxia group was incubated under the condition of 92% N₂, 3% O₂ and 5% CO₂ at 37°C. Adenovirus carrying Rgs10 gene (Ad-Rgs10) (3 pfu/cell) and the adenovirus carrying control empty vector (Ad-Con; 3 pfu/cell) were obtained from the manufacturer (Genechem; Shanghai) and transfected with PASMC for 72 h (h). PASMC was transfected with scramble siRNA, siRgs10 (Cat#1320003; Thermo Fisher, Waltham, MA) or siTsc1 (5’-GCUUUGACUCUCCCUUCUA-3’) (15) by using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA) for 8 h. PASMC was incubated with SC-79 (10 μM; MCE, Shanghai) (16) for 24 h. Rapa (100 nmol/L) was used to treat PASMC for 8 h (17).

Western blotting

Total protein was extracted by using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai). Western blotting analysis was performed as previously described (18). Protein samples were loaded on sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After being blocked by 5% bovine serum albumin, the membranes were incubated corresponding primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 h at room temperature before being exposed. The primary antibodies against p-AKT^Thr308, AKT, p-S6^Ser235/236, S6, p-4EBP1^Thr37/46, 4EBP1, p-mTOR^Ser2448, mTOR, proliferating cell nuclear antigen (PCNA), and GAPDH purchased from Cell Signaling Technology (Danvers, MA). The antibody against RGS10 was purchased from Solarbio (Shanghai).

Immunofluorescence

Ki-67 immunofluorescence of PASMC was performed as a previous study described (19). Briefly, PASMC was prepared according to the established protocol (11). After fixing, membrane breaking and blocking, slides of PASMC were incubated in dark with the primary antibody against Ki-67 (1: 1000; Cell Signaling Technology, Danvers, MA) overnight at 4°C and followed by incubation in dark with Alexa Fluor 488-conjugated goat anti-rabbit (1:3000; Molecular Probes Inc., Eugene, OR) for 1 h. DAPI (5 mg/ml; VECTOR Labs, Burlingame, CA) staining was conducted in the room temperature for 5 s. Representative images were captured by using an immunofluorescent microscopy (Leica MPS 60; Wetzlar, HD).

Wound healing assay

Wound healing assay was conducted as previous study described (20). Briefly, pretreated (siRNA or adenovirus transfection and hypoxia exposure for 24 h) PASMC was cultured and grown to 80–90% confluency. After serum-deprived for 12 h, a scratch was made in PASMC by using a pipette tip. Representative images were captured at time points of 0 h and 12 h later to measure the rate of migration.

Mice

C57BL/6J mice were purchased from Dashuo Animal Science and Technology (Chengdu, Sichuan). The mice were housed as following described: periodic air changes, 12-h light/dark cycle and free access to food and water. All animal experiments in the current study were performed in accordance with the Institutional Animal Care and Use Committee and the Ethic Committee of The General Hospital of Western Theater Command (Chengdu, Sichuan). Hypoxia/SU5416 PH model, which imitates clinical PH due to hypoxia, was established as previous study described (21). Briefly, 8-week-old mice received subcutaneous injection of SU5416 (20 mg/kg; Sigma, St Louis, MI) once a week. These mice were then kept under the condition of chronic hypoxia (10% O₂) for 3 weeks. Mice in normoxia group received subcutaneous injection of equivalent DMSO and housed with room air. Another mouse PH model was established by using MCT. Briefly, mice were intraperitoneally injected with MCT (60 mg/kg) once a week for 4 weeks to induce PH, which imitates clinical PH due to lung diseases (22). Simultaneously, Ad-Rgs10 (10 (11) pfu/mL; 50 μL) or Ad-Con was injected via the tail vein once a week until MCT administration was finished. Before the evaluation of RVSP and RVHI, mice were firstly anesthetized with pentobarbital (30 mg/kg) by intraperitoneal injection. A pressure transducer catheter (Millar Instruments, Houston, TX) was inserted into the right ventricle to determine RVSP. Mice finally received anaesthetization of pentobarbital (100 mg/kg) and sacrificed for further experiments. For evaluation of RVHI, the heart was isolated, and the right ventricle-to-(septum + left ventricle) ratio was taken as RVHI.

H-E staining

Morphometric analyses of lung tissues were analyzed by using H-E staining as previous study described (23). Briefly, lung tissues were fixed, embedded and cut into 4-μm sections. Then, the sections were stained with hematoxylin and eosin as a previous study described (15). Finally, the sections were observed by using a microscopy.

Statistical analysis

Data are presented as mean ± S.D. All tests were two-tailed. Unpaired Student’s t-test was utilized to compare the two independent groups. One-way analysis of variance (ANOVA) was conducted for multi-comparisons with appropriate post hoc tests. P < .05 was considered statistically significant.

Results

RGS10 alleviates hypoxia-induced proliferation and migration of PASMC

Western blotting of RGS10 showed that hypoxia stimulus led to decreased level of RGS10 in PASMC in a time-dependent manner (Figure 1a). To investigate the role of RGS10 in PASMC remodeling, we transfected PASMC with adenovirus carrying Rgs10 gene (Ad-Rgs10) and also validated the overexpression of RGS10 (Figure S1a). Ki-67 immunofluorescence staining was utilized to analyze cell proliferation. Hypoxia stimulus significantly increased the ratio of Ki-67-positive PASMC as previous study demonstrated (Figure 1b) (11). Moreover, the increase in the ratio of Ki-67-positive PASMC induced by hypoxia was attenuated by Ad-Rgs10 (Figure 1b). We next explored cellular migration by using wound-healing assay. Compared to the normoxia group, we observed an accelerated rate of wound-healing in PASMC of hypoxia group (Figure 1c), whereas overexpression of RGS10 impaired the increased rate of wound-healing in hypoxia-challenged PASMC (Figure 1c). Additionally, by using small interfering RNA targeting Rgs10 (siRgs10), we observed the decrease in RGS10 expression (Figure S2a) and a further increase in proliferation and migration in hypoxia-challenged PASMC after siRgs10 incubation (Figure S2b,c). These results indicate that RGS10 attenuates hypoxia-induced proliferation and migration of PASMC.

Figure 1. RGS10 suppresses hypoxia-induced proliferation and migration of PASMC. (a) the relative expression of RGS10 was analyzed by immunoblotting in PASMC after 0, 6, 12 or 24 h of hypoxia stimulus (post hoc for LSD test; n = 6 samples per group). PASMC was transfected with Ad-Con or Ad-Rgs10 and then received normoxia or hypoxia stimulus. (b) PASMC as above treated was stained with Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67-positive PASMC (right) were shown (post hoc for LSD test; n = 6 samples per group). Bar = 50 μm. (c) migration of PASMC as above treated was analyzed by wound healing assay. (post hoc for LSD test; n = 6 samples per group). Bar = 200 μm. Data are shown as mean ± S.D. ***P < .001 denotes statistical comparison between the two marked groups, respectively.

RGS10 suppresses proliferation and migration of PASMC via attenuating mTORC1 activity

RGS10 is known to suppress mTORC1 activity in ovarian cancer cells and podocyte cells (8,9). mTORC1 is a classical protein complex that induces proliferation and migration of PASMC (11). We thus used western blotting to measure the activity of mTORC1 in PASMC. S6 and 4E-Binding Protein 1 (4EBP1) are two major effectors of mTORC1 and thus reflect the activity of mTORC1. We found that enhanced phosphorylation of S6 and 4EBP1 in hypoxia-treated PASMC as well as mTOR phosphorylation were both significantly reduced by Ad-Rgs10 (Figures 2a and S1b). To further investigate whether the attenuated mTORC1 activity was involved in RGS10-mediated regulation of PASMC, we utilized the small interfering RNA (siRNA) targeting a classical negative regulator of mTORC1, Tuberous sclerosis complex 1 (siTsc1) (24) to restore mTORC1 activity in RGS10-overexpressing PASMC. As expected, the phosphorylation levels of both S6, 4EBP1, and mTOR in PASMC were increased after incubation with siTsc1 (Figures 2b and S1c). The decreased Ki-67-positive area and decelerated wound-healing rate in RGS10-overexpressing PASMC were both reversed after restoring mTORC1 activity by siTsc1 (Figure 2c,d). To further confirm that mTORC1 acts downstream of RGS10 to regulation PASMC, we also used siRgs10 to incubate with PASMC to determine mTORC1 activity. We found that the phosphorylation levels of S6 and 4EBP1 in hypoxia-stimulated PASMC were further enhanced after silencing RGS10 (Figure S3a). After suppressing mTORC1 activity by rapamycin (Rapa), we found that silencing RGS10-induced proliferation and migration in hypoxia-challenged PASMC were also repressed (Figure S3b,c). Therefore, we confirmed that RGS10 suppressed proliferation and migration of PASMC by reducing mTORC1 activity.

Figure 2. RGS10 inhibits hypoxia-induced proliferation and migration of PASMC via reducing mTORC1 activity. PASMC was transfected with Ad-Con or Ad-Rgs10 and then received normoxia or hypoxia stimulus. (a) the relative expression of RGS10, p-S6/S6 and p-4EBP1/4EBP1 in above-treated PASMC was analyzed by immunoblotting (post hoc for LSD test; n = 6 samples per group). (b) the relative expression of TSC1, p-S6/S6 and p-4EBP1/4EBP1 in PASMC incubated with scramble siRNA or siTsc1 were analyzed by immunoblotting (n = 6 samples per group). PASMC was transfected with Ad-Con or Ad-Rgs10 and then incubated with scramble siRNA or siTsc1 before receiving hypoxia stimulus. (c) above-treated PASMC was stained with Ki-67 (green) and DAPI (blue). Representative images (upper panel) and corresponding quantification of Ki-67-positive PASMC (lower panel) were shown (post hoc for LSD test; n = 6 samples per group). Bar = 50 μm. (d) migration of above-treated PASMC was analyzed by wound healing assay. (post hoc for LSD test; n = 6 samples per group). Bar = 200 μm. Data are shown as mean ± S.D. **P < .01 and ***P < .001 denote statistical comparison between the two marked groups, respectively.

RGS10 inhibits mTORC1 activity in hypoxia-challenged PASMC via dephosphorylation of AKT

AKT serves as a critical upstream regulator of mTORC1 in PASMC (11). To explore how RGS10 regulates mTORC1 activity in PASMC, we evaluated the phosphorylation level of AKT in hypoxia-challenged PASMC. Western blotting analysis showed that hypoxia could increase the expression of phosphorylated (p)-AKT, which was obviously ameliorated after overexpressing RGS10 in PASMC (Figure 3a). More importantly, RGS10-mediated dephosphorylation of S6 and 4EBP1 in hypoxia-treated PASMC was impaired after reactivating AKT by SC-79 (a classical agonist of AKT) (25) (Figure 3b). The phosphorylation level of AKT was also increased by SC-79 (Figure S4). In addition, we confirmed that SC-79 also abolished the inhibitory effects of RGS10 on proliferation and migration of hypoxia-stimulated PASMC (Figure 3c,d). Taken together, these data suggest that RGS10 may regulate PASMC via the AKT/mTORC1 axis.

Figure 3. AKT is required for RGS10-mediated regulation of mTORC1 in PASMC. (a) PASMC was transfected with Ad-Con or Ad-Rgs10 and then received normoxia or hypoxia stimulus. The relative expression of p-AKT/AKT in above-treated PASMC was analyzed by immunoblotting (post hoc for LSD test; n = 6 samples per group). (b) PASMC was transfected with Ad-Con or Ad-Rgs10 and then incubated with DMSO or SC-79 before receiving hypoxia stimulus. The relative expression of RGS10, p-S6/S6 and p-4EBP1/4EBP1 in above-treated PASMC was analyzed by immunoblotting (post hoc for LSD test; n = 6 samples per group). (c) above-treated PASMC was stained with Ki-67 (green) and DAPI (blue). Representative images (upper panel) and corresponding quantification of Ki-67-positive PASMC (lower panel) were shown (post hoc for LSD test; n = 6 samples per group). Bar = 50 μm. (d) migration of above-treated PASMC was analyzed by wound healing assay. (post hoc for LSD test; n = 6 samples per group). Bar = 200 μm. Data are shown as mean ± S.D. *P < .05, **P < .01 and ***P < .001 denote statistical comparison between the two marked groups, respectively.

Overexpression of RGS10 prevents against PH in vivo

Since RGS10 could suppress proliferation and migration of PASMC in vitro, we then validated whether RGS10 also exerted a preventive role in PH in vivo. Western blotting analysis showed that RGS10 was also downregulated in Hypoxia/SU5416 PH mouse (Figure 4a). This model mimics clinical PH resulted from hypoxia. After transfection with Ad-Rgs10, the expression of RGS10 in PA was significantly increased (Figure 4b). To explore the crosstalk of RGS10 downregulation and PH, we measured right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) to evaluate the parameters of right ventricle. Consistent with our previous study (11), Hypoxia/SU5416 PH mouse showed obviously increased RVSP and RVHI, while overexpression of RGS10 after Ad-Rgs10 transfection significantly ameliorated these abnormal parameters (Figure 4c,d). We also performed hematoxylin-eosin (H-E) staining to evaluate the effect of RGS10 on PA remodeling, which represents the critical pathological changes of PH. Vascular wall thickening and intimal hyperplasia are canonical hallmarks of PA remodeling. As shown in Figure 4e, Hypoxia/SU5416 PH mice displayed obvious vascular wall thickening and intimal hyperplasia. In addition, the expression of proliferating marker PCNA in PAs was also increased in Hypoxia/SU5416 PH mice, which was then attenuated by the external expression of RGS10 (Figure 4f). However, these pathological changes were partly alleviated after the external expression of RGS10. Additionally, we also validated the role of RGS10 in another MCT-induced mouse PH model, which mimicked PH due to lung diseases. The expression of RGS10 was also decreased in PAs of MCT-treated mice (Figure S5a). We found that the MCT-induced mice also showed increased RVSP (Figure S5b) and RVHI (Figure S5c). Interestingly, after the external expression of RGS10 in vivo by Ad-Rgs10, RVSP and RVHI were both reduced. Furthermore, H-E analysis showed that MCT-induced intimal hyperplasia within PA was also abolished by Ad-Rgs10 transfection (Figure S5d). Additionally, the phosphorylation levels of AKT, S6 and 4EBP1 were also enhanced in PAs of MCT-treated mice (Figure S6), indicating AKT/mTORC1 pathway might also be involved in RGS10-mediated regulation of PH. Therefore, RGS10 also exerts a preventive role in PH in vivo.

Figure 4. Overexpression of RGS10 ameliorates PH in vivo. (a) the relative protein level of RGS10 in dissected PAs of hypoxia/SU5416 PH mice was measured by immunoblotting (n = 6 samples per group). Hypoxia/SU5416 PH mice indicate chronic hypoxia-induced pulmonary arterial hypertension mice, which were induced by SU5416 injection and hypoxia exposure. (b) the relative expression of RGS10 in dissected PAs of mice transfected by Ad-Con or Ad-Rgs10 were analyzed by immunoblotting (n = 6 samples per group). RVSP (c) and RVHI (d) of hypoxia/SU5416 PH mice were analyzed (post hoc for LSD test; n = 7 samples per group). RVSP: right ventricular systolic pressure. RVHI: right ventricular hypertrophy index. (e) Representative H-E staining and corresponding quantification of lung tissues in control and hypoxia/SU5416 PH mice after transfection of Ad-Con or Ad-Rgs10 (post hoc for LSD test; n = 6 samples per group). The black arrows indicate PAs within lung tissues. Bar = 50 μm. (f) the relative expression of PCNA in dissected PAs of hypoxia/SU5416 PH mice transfected by Ad-Con or Ad-Rgs10 were analyzed by immunoblotting (post hoc for LSD test; n = 6 samples per group). Data are shown as mean ± S.D. ***P < .001 denotes statistical comparison between the two marked groups.

Discussion

PH is a severe cardiopulmonary syndrome that involves multiple pathological processes, among which abnormal proliferation and migration of PASMC exerts a critical role. Herein, we demonstrated that RGS10 was downregulated in hypoxia-induced PASMC. Overexpression of RGS10 in hypoxia-challenged PASMC suppressed excessive proliferation and migration of PASMC. It was also found that RGS10 could inhibit AKT/mTORC1 signaling pathway in hypoxia-induced PASMC. Activating AKT/mTORC1 abolished RGS10-mediated regulation on PASMC after hypoxia stimulus. Overexpression of RGS10 in vivo also evidently prevented against right ventricular hypertrophy and pulmonary vascular wall thickening. These findings suggest that RGS10 may be taken as a novel potential therapeutic target for PH.

RGS10 is known to selectively target G-protein coupled receptors and thus modulate the downstream-signaling pathways (10). It has been shown that RGS10 is expressed in immune system, platelets, podocytes and tumors (8,9,26). Therefore, RGS10 is associated with multiple functions, including proliferation of tumor cells, inflammation, thrombogenesis and apoptosis (7–10). There is increasing evidence indicating that RGS10 modulates proliferation in various cell types. For example, suppression of RGS10 augments the viability and proliferation of ovarian cancer cell (9), while knockdown of RGS10 inhibits the proliferation of various immune cells such as dendritic cell (27) and T cell (28). So far, few studies have explored the role of RGS10 in the proliferation and migration of PASMC. Our current study found that RGS10 was also richly expressed in PASMC and confirmed the suppressive role of RGS10 in proliferation and migration of hypoxia-challenged PASMC.

Previous studies demonstrated that suppression of RGS10 triggered mTOR pathway activation in padocyte and ovarian cancer cell (8,9). As one of the major complexes of mTOR, mTORC1 exerts multiple functions such as proliferation, growth, autophagy, etc (29,30). Excessive activation of mTORC1 has been confirmed to induce remodeling of PASMC and PH (11). However, no reports in the literature to date have raised that RGS10 modulates mTORC1 activity in PASMC. In the current study, we demonstrated that RGS10 negatively regulated mTORC1 activity in hypoxia-challenged PASMC. Additionally, silencing Tsc1, a canonical negative regulator of mTORC1 to increase mTORC1 activity abolished the suppressive effect of RGS10 on proliferation and migration of hypoxia-induced PASMC, whereas silencing RGS10-mediated enhancement of proliferation and migration in hypoxia-challenged PASMC were also abrogated by suppressing mTORC1 activity. Therefore, these data suggest that RGS10 may achive its regulation of PASMC by inhibiting mTORC1 activity.

AKT is a critical protein kinase that acts upstream of mTORC1 to regulate a variety of cellular processes such as proliferation (13,31). There is evidence that enhanced phosphorylation of AKT promotes proliferation and migration of PASMC via inducing activation of mTORC1 (11). Inhibition of AKT/mTORC1 signaling pathway is also associated with the remission of PH (13). Despite the fact that RGS10 knockdown causes enhanced phosphorylation of AKT in podocyte and ovarian cancer cell (8,9), whether AKT is essential for RGS10-mediated regulation of mTORC1 in PASMC remains unknown. In the current study, we demonstrated that RGS10 overexpression negatively regulated the AKT activity in hypoxia-induced PASMC. Treating PASMC with SC-79, an agonist of AKT phosphorylation, abolished the suppressive effect of RGS10 on mTORC1 activity, proliferation and migration of PASMC. These observations indicate that RGS10-dependent inhibition of AKT/mTORC1 suppresses hypoxia-induced proliferation and migration of PASMC.

Although RGS10 plays an appreciable role in PASMC remodeling, whether the effects of RGS10 on PASMC could be also observed in PH in vivo is unclear. These are several evidences that RGS10 is involved in cardiopulmonary diseases. For example, RGS10 ameliorates lung inflammation by reducing myeloid leukocyte infiltration (7). RGS10 also suppresses thrombogenesis via inhibiting platelet activation (10). However, the precise role of RGS10 in PH remains unrevealed. Our current results revealed that RGS10 was downregulated in mouse PH model and overexpression of RGS10 inhibited pulmonary vascular wall thickening and right ventricular hypertrophy, suggesting a preventive role of RGS10 in PH. However, the reversing effect of RGS10 on PH in vivo was far lower than that in PASMC in vitro. RGS10 overexpression could only partly reversed the pathological changes in Hypoxia/SU5416 PH mice instead of fully replicating the in vitro effect. This might result from the more complex molecular mechanism network in vivo. The single intervention of RGS10 was not enough to cure PH and might be constrained by other influencing factors. Therefore, effort on the co-regulation of RGS10 with other factors is needed in the future study.

Conclusions

Collectively, the current study identified the downregulation of RGS10 in hypoxia-challenged PASMC and Hypoxia/SU5416 PH mouse. Overexpression of RGS10 alleviated PH in vivo and suppressed hypoxia-induced excessive proliferation and migration of PASMC. The regulation of RGS10 on PASMC relied on declined AKT/mTORC1 activity. Therefore, our results highlight the preventive role of RGS10 against PH and offer a potential candidate target for PH.

Author contributions

S.H and Y.L conceived the project. S.H designed the study. S.H, C.M.Q and Y.L supervised the entire research. S.H performed most of the experimental work and conducted data analysis. Y.J.Z provided some technical supports. S.H and C.M.Q contributed to figure preparation. S.H, C.M.Q and Y.L discussed the study. S.H and Y.L organized the data and wrote the manuscript. All authors reviewed the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Incubation Project of The General Hospital of Western Theater Command (2021-XZYG-C10 to Y.L).