Hypoxia-induced mitochondrial DNA release promotes proliferation of pulmonary arterial smooth muscle cells via activating cGAS-STING pathway

Abstract

Objective To investigate the role and possible mechanism of mitochondrial DNA (mtDNA) release in the proliferation of rat pulmonary arterial smooth muscle cells (RPASMCs) induced by hypoxia. Methods ① RPASMCs were divided into normoxia group, hypoxia 24-hour group and hypoxia 48-hour group (n=3 for each groups), and the latter 2 groups were exposed to 1%O2 for 24 or 48 h respectively. ② Cyclosporine A (CsA) was used to inhibit the mitochondrial permeability transition pore (MPTP), and then the RPASMCs were divided into normoxia, simple hypoxia 48-hour and hypoxia 48-hour+CsA groups (n=3 for each). ③ SiRNA was adopted to knockdown the expression of stimulator of interferon genes (STING), and the cells were subsequently divided into normoxia+NC, hypoxia 48-hour+NC and hypoxia 48-hour+si-STING (n=3 for each). ④ The proliferation of the cells was measured by CCK-8 assay. The expression of cyclic GMP-AMP synthase (cGAS), STING and proliferating cell nuclear antigen (PCNA) at mRNA and protein levels was detected by RT-qPCR and Western blotting, respectively. Cytosolic mtDNA release was determined by qPCR, and the level of C-X-C motif chemokine ligand 10 (CXCL10) in the culture supernatant was tested by ELISA. Results Hypoxia induced RPASMCs to release mtDNA (P < 0.01), significantly up-regulated the expression of cGAS, STING and PCNA (P < 0.05), enhanced the secretion of CXCL10 (P < 0.01), and thus promoted the proliferation of RPASMCs (0.44±0.02 vs 0.72±0.03, P < 0.05). However, inhibition of MPTP blocked mtDNA release (P < 0.01), down-regulated cGAS, STING and PCNA (P < 0.01), and decreased CXCL10 level (P < 0.01) and hypoxia-induced proliferation of RPASMCs (0.74±0.12 vs 0.43±0.06, P < 0.01). The knockdown of STING also suppressed PCNA expression (P < 0.05), reduced CXCL10 secretion (P < 0.01), and inhibited the proliferation of RPASMCs (0.68±0.03 vs 0.58±0.01, P < 0.01). Conclusion Hypoxia can induce smooth muscle cells to release mtDNA and promote the proliferation of RPASMCs, which may be related to the activation of the cGAS-STING pathway.

Keywords

• mitochondrial dna release
• cgas-sting pathway
• pulmonary arterial smooth cells
• hypoxia
• proliferation