SiO2 increases pulmonary fibroblast activation
Pulmonary fibroblasts pathologically indicate tissue fibrosis, fibroblast and myofibroblast proliferation with elevated expression levels of ACTA, COL1A1, and COL3A1 and deposition of ECM components (9, 10). Fibroblast activation markers, such as COL1A1, COL3A1 and ACTA1, were measured by an immunoblotting assay (western blot), CCK8 cell counting kit and immunofluorescence staining after SiO2 exposure (50 μg/cm2). Based on earlier experimental dosages, 50 μg/cm2 was selected for all the related experiments (19, 24-27). Western blotting results confirmed that fibroblast activation markers such as COL1A1, COL3A1 and ACTA1 were elevated in the presence of SiO2 after 3 hr (Fig. 1A-C). In addition, the cell viability of pulmonary fibroblasts increased after 12 h of addition of silica (50 µg/cm2) (Fig. 1D). Immunocytochemistry assays confirmed the upregulation of COL1A1, ACTA1 (Fig. 1E and F) and COL3A1 expression (Supplementary Fig. S1) induced by SiO2 in pulmonary fibroblasts.
SiO2increases migration in pulmonary fibroblasts
Two-dimensional (2D) and 3-dimensional (3D) nested collagen matrix assays significantly represent the cell migration ability in in vitro culture (28-30). Compared with usual scratch assays, the nested collagen matrix (3D) assay is a reliable, quick, quantitative and easy method for determining fibroblast migration and motility in 3D models (6, 28, 31, 32). Here, we provide new insights into the novel roles that pulmonary fibroblasts exposed to SiO2 were assessed via 2-dimensional and 3-dimensional nested collagen matrix gel assays, and their responses were examined in both models at the indicated time points. Although SiO2 induced fibroblast migration and proliferation differently in 2-dimensional and 3-dimensional nested collagen matrix gel systems, the peak response of fibroblasts was observed at 24 hr. The scratch assay/wound healing assay (Fig. 2 A-B) demonstrated that SiO2 significantly induced migration in MLg cells. Therefore, the schematic diagram of the 3D nested collagen matrix gel assay represents the migration of pulmonary fibroblasts after SiO2 exposure (Fig. 2C). Pulmonary fibroblasts exhibited an increase in both migration distance (Fig. 2D-E) and number of migrated cells (Fig. 2D and F).
SiO2 induces ZC3H4 in pulmonary fibroblasts
A previous study from our laboratory suggested that ZC3H4 is involved in the inflammatory stage of silicosis. To establish whether ZC3H4 is also involved in late fibrosis, MLg cells were exposed to SiO2. Western blot assays confirmed that the ZC3H4 expression level was suddenly increased after SiO2 exposure in pulmonary fibroblasts at different time points (0, 1, 3, 6, 12 and 24 hr) with a two-phase increase pattern (Fig. 3A-B). Immunocytochemistry confirmed the upregulation of ZC3H4 expression induced by SiO2 in pulmonary fibroblasts (Fig. 3C).
To simplify the role of ZC3H4 in fibroblast activation induced by SiO2, we transfected MLg cells with ZC3H4 NIC plasmids to specifically knock down ZC3H4. Western blot assays confirmed that the specific knockdown of ZC3H4 in pulmonary fibroblasts significantly inhibited the upregulation of ZC3H4 with or without SiO2 (Fig. 3D-E). In the cell viability assay, ZC3H4 knockdown attenuated the increase in cell viability induced by SiO2 (Fig. 3F), indicating that ZC3H4 mediated the proliferation induced by SiO2 in fibroblasts. Moreover, ZC3H4 NIC plasmids abolished the effect of SiO2 on the migration ability of MLg cells (Fig. 3G-H). Furthermore, the COL1A1 and ACTA1 expression levels induced by SiO2 were assessed after the specific knockdown of ZC3H4 (Fig. 3I and J), in which all upregulated markers were inhibited, confirming the role of ZC3H4 in pulmonary fibroblast activation and migration.
SiO2 induces ER stress in pulmonary fibroblasts
To depict the downstream molecular mechanism of ZC3H4 on fibroblast activation, ER stress was evaluated. The ER is a key factor for cellular activities (protein modification, folding, synthesis and transport) (33-35). Various pathological and physiological environmental factors could affect ER homeostasis, finally causing ER stress (36, 37). ER stress is responsible for pulmonary tract infections (38) and various types of lung disease (39, 40). UPRs are essential factors for maintaining homeostasis and initiating the BIP and CHOP pathways (41). In a western blot assay, we measured the ER stress markers ERN1 (inositol-requiring enzyme 1, IRE1α), BiP (binding immunoglobulin protein, BIP) and DDIT3 (C/EBP homologous protein, CHOP) in pulmonary fibroblasts after SiO2 exposure. MLg cells treated with SiO2 showed upregulation of ERN1, DDIT3 and BiP expression in a time-dependent manner with an ERN1 peak of 3 and 6 hrs, DDIT3 peak of 3 hrs and BiP peak of 12 hrs (Fig. 4 A-D). Immunocytochemistry assays were applied to confirm the colocalization of ZC3H4, BiP and DDIT3 (Fig. 4E-F). To further confirm the role of ZC3H4 in ER stress, ZC3H4 was specifically knocked down. As shown in Fig. 4G-H, ZC3H4 knockdown abolished the induction of ER stress induced by SiO2. Collectively, these results indicated that ZC3H4 is responsible for fibroblast activation induced by SiO2 via the ER stress pathway.
ZC3H4 induced ER stress via Sigmar1 in pulmonary fibroblasts
Having established the fundamental role of ZC3H4 in pulmonary fibroblast activation via ER stress in response to silica, the detailed molecular mechanism of activation of ER stress was further investigated. A previous study from our laboratory suggested that Sigmar1, a subclass of the sigma receptor family, mediated ER stress in silicosis (41). Sigmar1 is expressed in the ER with two steroid-binding domains and two transmembrane segments (42). The molecular action of Sigmar1 was previously discovered to be a ligand-regulated receptor chaperone via ER stress (43). In particular, whether ZC3H4 has a vital role in the activation of pulmonary fibroblasts via the sigmar1/ER stress pathway deserves investigation. To determine whether Sigmar1 is involved in silicosis, MLg were exposed to SiO2 to assess the Sigmar1 level. Western blotting results confirmed that the expression level of Sigmar1 was increased in the presence of SiO2 (Fig. 5A-B), which was confirmed by immunocytochemical staining (Fig. 5C). Further, specific inhibition of sigmar1 with the pharmacological agent BD1047 attenuated the upregulation of migration induced by SiO2 (Supplementary Fig. S2A-B). Furthermore, the Sigmar1 expression level was assessed after the specific knockdown of ZC3H4, in which ZC3H4-NIC suppressed sigmar1 upregulation after SiO2 exposure (Fig. 5D-E). Interestingly, the mRNA levels of ZC3H4 and Sigmar1 showed almost no change after SiO2 exposure (Supplementary Fig. S3A-B). To further understand the regulatory effect of ZC3H4 on Sigmar1, Co-IP was conducted, in which there was a direct interaction between ZC3H4 and Sigmar1 (Fig. 5F). Taken together, these results verified that ZC3H4 and Sigmar1 are involved in the pulmonary fibroblast activation induced by SiO2.
MAPK and PI3K pathways are involved in SiO2-induced ZC3H4 upregulation
To further clarify the possible regulatory mechanism on ZC3H4, the involvement of the MAPK and P13K/Akt pathways was investigated since the MAPK and P13K/Akt pathways play an essential role in cell proliferation, migration and activation (44, 45). First, the short-term effect of SiO2 on ZC3H4 was measured, in which a rapid and transient increase in ZC3H4 was observed. Then, the MAPK signaling pathway was assessed. As shown in Fig. 6C-D, phosphorylation of Mapk1, Mapk8 and Mapk14 was increased after SiO2 exposure. Moreover, Akt phosphorylation showed a slight but significant increase after SiO2 exposure (Fig. 6E-F). To assess the role of the MAPK or PI3K pathway on regulation of ZC3H4, specific pharmacological inhibitors were applied. MLg cells were pretreated with a Mapk1 (SP600125) inhibitor, Mapk8 (SB203580) inhibitor, Mapk14 (U0126) inhibitor and P13K (LY294002) inhibitor separately for 1 hr, after which SiO2 was applied. As shown in Fig. 6G-H, all inhibitors attenuated the increase in ZC3H4 and sigmar1 induced by SiO2, confirming the role of the MAPK and PI3K pathways in the regulation of ZC3H4 and Sigmar1. Furthermore, fibroblast activation markers such as COL1A1 and ACTA1, as well as the ER stress markers DDIT3 and BiP, were inhibited by pretreatment with inhibitors (Supplementary Fig. 4A-E). These results demonstrated that the MAPK and PI3K/Akt pathways play a significant role in regulation of ZC3H4.
Induction of ZC3H4 by SiO2 promotes a further increase in ZC3H4
Interestingly, SiO2 induced a two-phase increase pattern in ZC3H4 (Fig. 3A-B), in which the early increase was due to the activation of MAPK and PI3K. The mechanism of the late increase in ZC3H4 deserves to be investigated since pulmonary fibrosis is a chronic pathological process. As ZC3H4-induced ER stress may increase the UPR in the ER lumen (46), whether ER stress affect the abnormal increase in ZC3H4 needs to be clarified. To our surprise, while salubrinal inhibited the increase in ER stress markers (Supplementary Fig. 5A-B), salubrinal reversed the SiO2 effect in the upregulation of ZC3H4 in pulmonary fibroblasts (Fig. 7A-B). Furthermore, tunicamycin, a specific ER stress inducer, was applied to further explore the role of ER stress. As expected, tunicamycin induced the expression of ER stress markers in a dose-dependent manner, and ZC3H4 was also upregulated (Fig. 7. C-F). Furthermore, pulmonary fibroblasts were further exposed to tunicamycin, in which the ZC3H4 level was increased at a peak at 24 hrs, while there was no rapid increase within 6 hrs, which showed a different pattern compared with direct SiO2 exposure (Fig. 7 G-H). Immunocytochemistry assays confirmed the upregulation of ZC3H4 expression after 50 µM tunicamycin in pulmonary fibroblasts (Fig. 7 I). These results demonstrated that ER stress was involved in the upregulation and activation of ZC3H4 in pulmonary fibroblasts in the late phase, indicating that the positive feedback loop (PFL) may be involved (Fig 8). Taken together, these results demonstrate that ZC3H4/ER stress plays a significant role in the activation of pulmonary fibroblasts.