circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica

Abstract

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO₂-induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO₂ on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO₂. SiO₂ induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO₂, inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO₂. circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO₂. Our study elucidated a link between SiO₂-induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

Fig. 1. Effects of SiO₂ on σ-1R expression and collagen production in HPF-a

a Representative western blot showing SiO₂-induced σ-1R upregulation in a time-dependent manner. b Densitometric analysis of σ-1R from five experiments; * p < 0.05 vs the control group. c SiO₂ upregulated collagen I and collagen III expression levels in a time-dependent manner. d Densitometric analysis of collagen I and collagen III from five experiments; * p < 0.05 vs the control group. e Representative immunocytochemical images showing that SiO₂ increased σ-1R and collagen III expression levels in HPF-a, which was attenuated by pretreatment of BD1047 (10 μM)

Fig. 2. Involvement of σ-1R in functional changes induced by SiO₂ in HPF-a

a MTT assay showing that the SiO₂-induced increase in cell viability was abolished by a 1-h pretreatment with BD1047 (10 μM); n = 5; *p <0.05  vs the control group at the corresponding time point. b Hoechst 33342 staining demonstrating that apoptosis induced by SiO2 was attenuated by pretreatment of BD1047(10 μM) in HPF-a; n = 5; *p < 0.05 vs the control group, ^# p < 0.05 vs the SiO₂ group. c BrdU labeling assay demonstrating that proliferation induced by SiO₂ was attenuated by pretreatment of BD1047(10 μM) in HPF-a; n = 5; *p < 0.05 vs the control group, ^# p < 0.05 vs the SiO₂ group. d Representative western blot showing that SiO₂-induced increased collagen I and III expression levels were attenuated by BD1047. e Densitometric analyses of collagen I and collagen III expression from five separate experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. Representative immunocytochemical images showing that the σ-1R and collagen III co-localization induced by SiO₂ was attenuated by pretreatment with BD1047. f Representative images and data of gel contraction assay demonstrating that cell activation induced by SiO₂ was attenuated by pretreatment of BD1047(10 μM) in HPF-a; n = 5; *p < 0.05 vs the control group, ^# p < 0.05 vs the SiO₂ group. g Representative images showing the SiO₂-induced increase in cell migration using a scratch assay was attenuated by pretreating HPF-a with BD1047. h Quantification of the scratch gap distances from six independent experiments; *p < 0.05 vs. the corresponding time point in the control group; ^# p < 0.05 vs the corresponding time point in the SiO₂ group. i Representative western blot showing that SiO₂-induced phosphorylation of MLC2 was attenuated by pretreatment with BD1047. j Densitometric analysis of the phosphorylation of MLC2 expression from five experiments; *p < 0.05 vs the control group

Fig. 3. SiO₂ induces ER stress in HPF-a

a Representative western blot showing that SiO₂ induced increased PERK, eIF2α, and CHOP expression. Densitometric analysis of PERK b, eIF2α c and CHOP d protein expression levels from five independent experiments; *p < 0.05 vs the control group. e Representative western blot showing that SiO₂ induced increased ATF6α and BiP expression. Densitometric analysis of ATF6α f and BiP g protein expression levels from five independent experiments; *p < 0.05 vs the control group. h Representative western blot showing that SiO₂ induced increased IRE1α expression. i Densitometric analysis of IRE1α protein expression from five independent experiments; *p < 0.05 vs the control group

Fig. 4. σ-1R is involved in ERS induced by SiO₂

a Representative western blot depicting the effect of BD1047 on PERK phosphorylation. b Densitometric analysis of PERK and p-PERK protein expression levels from five independent experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. c Representative western blot depicting the effect of BD1047 on eIF2α phosphorylation. d Densitometric analysis of eIF2α and p-eIF2α protein expression levels from five independent experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. e Representative western blot depicting the effect of BD1047 on CHOP, ATF6α and IRE1α protein expression. Densitometric analysis of CHOP f, ATF6α g, and IRE1α h protein expression levels from five independent experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group

Fig. 5. SiO₂ induced ERS-mediated fibroblast functional changes

a Representative western blot depicting the effect of salubrinal (10 μM) on σ-1R and collagen I and III expression. Densitometric analyses of σ-1R b and collagen I and III c expression levels from five separate experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. d Representative images showing that the SiO₂-induced increase in cell migration in a scratch assay was attenuated by pretreating HPF-a with salubrinal. e Quantification of the scratch gap distances from six independent experiments; *p < 0.05 vs. the corresponding time point in the control group; ^# p < 0.05 vs the corresponding time point in the SiO₂ group

Fig. 6. circHIPK2 is involved in regulating σ-1R after SiO₂exposure in HPF-a

a Bioinformatics analysis showing that circHIPK2 contains one site complementary to miR-506-3p and two miR-506-3p binding sites in σ-1R. b FISH assay showing circHIPK2 expression in HPF-a increased after SiO₂ exposure; circHIPK2 was labeled with fluorescein isothiocyanate. c qRT-PCR assay showing SiO₂ induced circHIPK2 upregulation from six independent experiments; *p < 0.05 vs the control group. d qRT-PCR assay showing SiO₂ had no effect on miR-506 expression in six independent experiments; *p < 0.05 vs the control group. e qRT-PCR assay showing siRNA knockdown of circHIPK2 inhibited circHIPK2 expression in HPF-a; n = 5. f Representative western blot depicting the effect of siRNA of circHIPK2 on σ-1R expression. g Densitometric analyses of σ-1R expression from five separate experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. h Representative western blot depicting the effect of siRNA of circHIPK2 on BiP expression. i Densitometric analyses of BiP expression from five separate experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group. j Representative western blot depicting the effect of siRNA of circHIPK2 on collagen I and III expression. k Densitometric analyses of collagen I and III expression from five separate experiments; *p < 0.05 vs the control group; ^# p < 0.05 vs the SiO₂ group

Fig. 7. Schematic diagram showing the mechanisms by which circHIPK2/σ-1R in macrophages mediates silica-induced pulmonary fibrosis

CircHIPK2 expression was increased in macrophages exposed to SiO₂, leading to a subsequent increase in σ-1R expression. σ-1R promoted fibroblast activation. Fibroblasts showed enhanced proliferation and migration capacities and increased collagen synthesis, which may contribute to fibrosis induced by SiO₂