RPS4XL encoded by lnc-Rps4l inhibits hypoxia-induced pyroptosis by binding HSC70 glycosylation site

Abstract

Pyroptosis is involved in pulmonary hypertension (PH); however, whether this process is regulated by long non-coding RNAs (lncRNAs) is unclear. Some lncRNAs encode peptides; therefore, whether the regulation of pyroptosis in PH depends on lncRNAs themselves or their encoded peptides needs to be explored. We aimed to characterize the role of the peptide RPS4XL encoded by lnc-Rps4l and its regulatory mechanisms during pyroptosis in PH. Transgenic mice overexpression of lnc-Rps4l was established to rescue the inhibition of hypoxia-induced pyroptosis in pulmonary artery smooth muscle cells (PASMCs). An adeno-associated virus 9 construct with a mutation in the open reading frame of lnc-Rps4l was used to verify that it could inhibit hypoxia-induced PASMCs pyroptosis through its encoded peptide RPS4XL. Glutathione S-transferase (GST) pull-down assays revealed that RPS4XL bound to HSC70, and microscale thermophoresis (MST) was performed to determine the HSC70 domain that interacted with RPS4XL. Through glycosylation site mutation, we confirmed that RPS4XL inhibited hypoxia-induced PASMCs pyroptosis by regulating HSC70 glycosylation. Our results showed that RPS4XL inhibits pyroptosis in a PH mouse model and hypoxic PASMCs by regulating HSC70 glycosylation. These results further clarify the important mechanism of vascular remodeling in PH pathology.

Keywords: HSC70; MT: Non-coding RNAs; RPS4XL; hypoxia; lnc-Rps4l; pulmonary arterial hypertension; pyroptosis.

Graphical abstract

Figure 1

Overexpression of Rps4l in vivo and in vitro inhibits hypoxia-induced pyroptosis (A) Electron micrographs of PASMCs of lung tissues in Rps4lTg and wild-type (WT) hypoxic and normoxic mice. The arrow points to the location of the cell membrane swelling and breakage (down, scale bar, 1 μm). Immunofluorescence analysis of (B) c-caspase-1, (C) NLRP3, (D) ASC in the lung tissues of Rps4lTg and WT hypoxic and normoxic mice (scale bar, 25 μm). Western blotting analysis of (E) c-caspase-1, NLRP3, ASC, IL-1β, and IL-18 in the lung tissues of Rps4lTg and WT hypoxic and normoxic mice. (F) Electron micrographs of hypoxic and normoxic PASMCs. The arrow points to the location of the cell membrane swelling and breakage (right, scale bar, 1 μm; left, scale bar, 2 μm). (G) LDH release assay in hypoxic and normoxic PASMCs transfected with OE-Rps4l or OE-NC. (H) PI staining in hypoxic and normoxic PASMCs transfected with OE-Rps4l or OE-NC (scale bar, 50 μm). (I) Flow cytometry analysis of hypoxic and normoxic PASMCs transfected with OE-Rps4l or OE-NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3). NOR, normoxia; HYP, hypoxia.

Figure 2

lnc-Rps4l-encoded peptide RPS4XL inhibits hypoxia-induced pyroptosis of PASMCs (A) Western blotting analysis of RPS4XL in hypoxic and control PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC. (B) LDH release assay using hypoxic and control PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC. (C) PI staining in hypoxic and control PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC (scale bar, 50 μm). Western blotting analysis of (D) c-caspase-1, (E) NLRP3, ASC, IL-1β, and IL-18 in hypoxic and normoxic PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC. (F) Flow cytometry analysis in hypoxic and control PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3). NOR, normoxia; HYP, hypoxia.

Figure 3

Exogenous RPS4XL treatment of PASMCs inhibits hypoxia-induced pyroptosis (A) Synthetic sequence of the exogenous peptide RPS4XL. (B) LDH release assay using PASMCs treated with 5 μg/mL, 7.5 μg/mL, or 10 μg/mL exogenous RPS4XL under hypoxia. (C) PI staining in PASMCs treated with 10 μg/mL RPS4XL under hypoxia (scale bar, 50 μm). Western blotting analysis of (D) c-caspase-1, (E) NLRP3, ASC, IL-1β, and IL-18 in PASMCs treated with 5 μg/mL, 7.5 μg/mL, or 10 μg/mL RPS4XL under hypoxia. FLAG was used as a negative control. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.0001; n ≥ 3). NOR, normoxia; HYP, hypoxia.

Figure 4

RPS4XL interacts with HSC70 (A) GO and KEGG analysis of the proteins isolated by a GST pull-down assay using RPS4XL. (B) Bioinformatics prediction of the RPS4XL interaction network. (C) Mass spectrometry of specific segments of HSC70. (D) Western blotting analysis of HSC70 expression in PASMCs transfected with the ORF-FLAG construct under normoxia after co-immunoprecipitation (coIP) using an anti-FLAG antibody (top), and RPS4XL in PASMCs under normoxia after coIP using an anti-HSC70 antibody (bottom). (E) HSC70 protein expression in hypoxic and normoxic PASMCs transfected with OE-Rps4l, Mut-Rps4l, or OE-NC. (F) Western blotting analysis of HSC70 in the lung tissues of hypoxic mice infected with serotype 9 adenovirus-associated virus (AAV9)-NC, AAV9-Rps4l, and AAV9-mut. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3). NOR, normoxia; HYP, hypoxia.

Figure 5

The functional domain of RPS4XL (A) Schematic diagram of the vector construction with the different RPS4XL domains. SWISS-MODEL schematic structural model of the different RPS4XL domains. (B) HSC70 protein expression in hypoxic and normoxic PASMCs overexpressing RPS4XL 1–41 aa, 42–104 aa, and 105–262 aa domains. (C) PI staining in hypoxic and normoxic PASMCs overexpressing the RPS4XL 1–41 aa, 42–104 aa, and 105–262 aa domains (scale bar, 100 μm). (D) LDH release assay in hypoxic and normoxic PASMCs overexpressing the RPS4XL 1–41 aa, 42–104 aa, and 105–262 aa domains. Western blotting analysis of (E) c-caspase-1, (F) NLRP3, ASC, IL-1β, and IL-18 in hypoxic and normoxic PASMCs overexpressing the RPS4XL 1–41 aa, 42–104 aa, and 105–262 aa domains. All values are represented as the mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n ≥ 3). NOR, normoxia; HYP, hypoxia; NS, no significance.

Figure 6

RPS4XL inhibits HSC70 glycosylation, which inhibits hypoxia-induced pyroptosis in PASMCs (A) Schematic diagram of the in vitro purification of the HSC70 protein domains. Microscale thermophoresis of the HSC70 1–393 aa and different concentrations of RPS4XL. (B) Bioinformatics analysis the N-glycosylation site in the 1–393 aa domain of HSC70, and schematic diagram of the HSC70 glycosylation site mutation. (C) LDH release assay in hypoxic and normoxic PASMCs transfected with OE-HSC70, Mut-HSC70, or treated with 10 μg/mL RPS4XL. (D) PI staining in hypoxic and normoxic PASMCs transfected with OE-HSC70, Mut-HSC70, or treated with 10 μg/mL RPS4XL (scale bar, 50 μm). Western blotting analysis of (E) c-caspase-1, (F) NLRP3, ASC, IL-1β, and IL-18 in hypoxic and normoxic PASMCs transfected with OE-HSC70, Mut-HSC70, or treated with 10 μg/mL RPS4XL. All values are represented as the mean ± SEM (∗p < 0.05, and ∗∗p < 0.01; n ≥ 3). NOR, normoxia; HYP, hypoxia.