The IRE1α-XBP1 pathway function in hypoxia-induced pulmonary vascular remodeling, is upregulated by quercetin, inhibits apoptosis and partially reverses the effect of quercetin in PASMCs

Abstract

Hypoxia is a common cause of pulmonary vascular remodeling and endoplasmic reticulum stress (ERS). Upon ER stress, the unfolded protein response (UPR) which activates the IRE1α, PERK and ATF6 signaling pathways is activated to cope with ERS in mammalian cells; however, the role of the three UPR arms in pulmonary vascular remodeling has not been defined. The present study showed that GRP78, a marker of ERS, was upregulated in hypoxic pulmonary artery smooth muscle cells (PASMCs). Among the three arms of the UPR, the IRE1α pathway was noticeably upregulated in hypoxic PASMCs. An inhibitor of IRE1α/XBP1 pathway, 4u8c, inhibited hypoxia-induced cell proliferation and migration and increased cell apoptosis by downregulating PCNA and MMP9 and activating mitochondrial apoptosis by enhancing the expression of BAX, activating caspase-9 and caspase-3, and eventually cleaving PARP. Quercetin affects ERS in many cell types and was shown to relieve hypoxic pulmonary hypertension (HPH) in our previous study. We demonstrated that quercetin evoked excessive GRP78 expression in hypoxic PASMCs compared with hypoxia alone by evaluating the expression of GRP78. The expression of IRE1α and XBP1s, a cleavage form of XBP1u, was upregulated by quercetin in a dose-dependent manner. Pretreatment with 4u8c reversed the apoptosis-promoting effect of quercetin by inhibiting mitochondrial apoptosis. However, 4u8c amplified the effect of quercetin on proliferation and migration in hypoxic PASMCs. In conclusion, the study demonstrated that the IRE1α-XBP1 pathway is involved in the process of hypoxia-induced pulmonary vascular remodeling; 4u8c could restrain hypoxia-induced cell proliferation and migration and reverse the hypoxia-induced apoptosis arrest, while quercetin excited excessive ERS and the IRE1α pathway in hypoxic PASMCs and promoted apoptosis. Our data suggest that intervening the IRE1α-XBP1 pathway may be useful for hypoxia-induced pulmonary arterial hypertension therapy.

Keywords: ERS; Hypoxia; IRE1α; quercetin; unfolded protein response.

Figure 1

GRP78 is upregulated in hypoxic rat PASMCs. A. The purity of PASMCs was identified by αSMA immunofluorescence staining (magnification, × 200). B. GRP78 protein expression in PASMCs treated with different oxygen concentrations. C. GRP78 protein expression in PASMCs treated with the indicated times of hypoxia. D. Immunofluorescence staining of GRP78 visually verified ERS changes under hypoxia and normoxia. Data are shown as the mean ± SEM; n = 3. *P < 0.05, **P < 0.01. *compared with the normoxia group or 0 h time point; 0 h was considered to be a control point, which was compared with all of the other time points.

Figure 2

Changes in the three UPR sensors in hypoxic rat PASMCs. A, B. Representative Western blots for three UPR sensors in PASMCs exposed to hypoxia for different times, and densitometry of bands was analyzed by using ImageJ software. C. Lung tissue sections from normal and hypoxia-induced pulmonary artery hypertension rats were stained immunohistochemically for the UPR sensors: IRE1α and p-IRE1α (magnification, × 400). Data are shown as the mean ± SEM; n = 4. *P < 0.05, **P < 0.01 compared with 0 h.

Figure 3

Selective targeting of the IRE1α pathway by 4μ8c promoted PASMC apoptosis. A. XBP-1 splicing in hypoxic PASMCs treated with 4μ8c (XBP1U, unspliced form; XBP1S, spliced form). B, C. Annexin-V/PI assays were performed to assess cell apoptosis, and quantitation data of flow cytometry experiments are shown. D. Protein expression of mitochondrial apoptosis pathway members was evaluated by western blots. E. Quantification analysis of protein expression is shown. Data are shown as the mean ± SEM; n = 4. ^#P < 0.05, ^##P < 0.01 compared with hypoxia-treated PASMCs.

Figure 4

PASMC proliferation and migration were inhibited by 4u8c. A. CCK8 tests were performed to measure cell viability after 48 h of treatment, and OD values were analyzed. B, C. Expression of proteins related to proliferation and migration was evaluated. D, E. CFSE dilution analysis of PASMCs after CFSE labeling. F. Crystal violet staining of PASMCs in a transwell assay (original × 100, scale bars: 100 µm). G. The number of migrated cells was counted and analyzed. Data are shown as the mean ± SEM; n = 3. *P < 0.05, **P < 0.01 compared with the normoxia group; ^#P < 0.05, ^##P < 0.01 compared with the hypoxia group.

Figure 5

Quercetin elicited obvious ERS and selectively activated the IRE1α branch in hypoxic rat PASMCs. A. Immunofluorescence staining was performed to analyze the expression of GRP78 after quercetin treatment. B, C. The levels of UPR sensors were quantified by Western blots in PASMCs treated with quercetin and hypoxia for 48 h, the representative bands and protein quantifications are shown. Qu, quercetin. Data are shown as the mean ± SEM; n = 4. *P < 0.05, **P < 0.01 compared with normoxia group; ^#P < 0.05, ^##P < 0.01 compared with hypoxia.

Figure 6

The IRE1α pathway participated in apoptosis promoting effect of quercetin. A. Annexin-V/PI assays were performed to assess cell apoptosis, and analyses of early and late phase apoptosis are shown. B, C. Protein expression of mitochondrial apoptosis was valuated, and quantification analysis was shown. Qu, quercetin. Data are shown as the mean ± SEM; n = 4. ^#P < 0.05, ^##P < 0.01 compared with hypoxia-treated PASMCs, ^&P < 0.05, ^&&P < 0.01 compared with quercetin-treated PASMCs.

Figure 7

Quercetin and 4u8c have an additive effect on the proliferation and migration of hypoxic PASMCs. A, B. CFSE dilution analysis of PASMCs was performed by pretreating with 4u8c for 24 h, and quercetin was added for another 24 h. C, D. Protein expression related to proliferation was evaluated. E, F. Transwell assays were performed by adding 1 × 10⁴ cells per transwell chamber, pretreating cells with different doses of 4u8c for 12 h and adding quercetin (60 mM) for another 12 h in the lower chamber. The number of migrated cells was counted and analyzed. Qu, quercetin. Data are expressed as the mean ± SEM; n = 4. *P < 0.05 compared with the normoxia group, ^#P < 0.05, ^##P < 0.01 compared with hypoxia-treated PASMCs, ^&P < 0.05, ^&&P < 0.01 compared with quercetin-treated PASMCs.