ZC3H4 promotes pulmonary brosis via an ER stress-related positive feedback loop

Pulmonary brosis is the sequela of many pulmonary diseases, such as pneumoconiosis and idiopathic pulmonary brosis. The principal characteristics of pulmonary brosis comprise myobroblast proliferation, alveolar damage and deposition of extracellular matrix components, which causes abnormal lung structure remodeling and an irreversible decline in lung function; however, the detailed mechanisms remain unclear. The current study focused on the role of ZC3H4, a new member of the zinc nger protein family, in SiO 2 -induced pulmonary brosis. The expression of ZC3H4 and broblast activation markers (COL1A1, COL3A1 and ACTA1) was measured by western blotting and immunouorescence staining after SiO 2 exposure (50 µg/cm 2 ). The functional change in broblasts was studied with a scratch assay and a 3D migration assay. The CRISPR/Cas9 system was used to explore the regulatory mechanisms of ZC3H4 in pulmonary broblast cells. of cells with a ZC3H4 double nickase to observe the downregulation of ZC3H4, but SiO2 induced increased broblast and myobroblast cell viability in MLg cells, *p< 0.05 vs. the Con-NIC of control group, #p< 0.05 vs. the Con-NIC of SiO2 group. that GFP-labeled MLg cells induced cell migration in a 2D assay after SiO2 exposure, but ZC3H4NIC silencing abolished pulmonary broblast migration. Scale H. Quantication of the gap (scratch) width was detected in a 2D nested matrix assay from ve independent experiments. *p< 0.05 vs. the Con-NIC of control group, #p< 0.05 vs. the ZC3H4-NIC of control group. at the conforming time points. The effect of CRISPR/Cas9-mediated ZC3H4NIC silencing abolished pulmonary broblast migration in a 2D nested matrix assay. *p< 0.05 vs. the Con-NIC of control group, #p< 0.05 vs. the Con-NIC of SiO2 group. Demonstrative western blotting representing the effects of CRISPR/Cas9-mediated ZC3H4NIC on pulmonary broblasts revealed that SiO2 increased the broblast activation inducers COL1A1 and ACTA1. J. Densitometric analyses of COL1A1 and ACTA1 expression levels from ve independent experiments in MLg cells. *p< 0.05 vs. the Con-NIC of control group, #p< 0.05 vs. the Con-NIC SiO2


Introduction
Silicosis is a chronic occupational disease caused by exposure to dust particles containing a high level of free silica-containing silicon dioxide (SiO 2 ), and these particles are often found in mining, quarry, metal foundries and other sandy industrial environments (1,2). Silicosis is a severe problem in developing and developed countries. Although preventive efforts have been implemented for many years, silicosis remains an incurable, fatal and disabling pulmonary disease characterized by pulmonary interstitial brosis and silicotic nodule formation (3). Silicosis progression is due to a lack of a hygienic measures and inadequate surveillance (4), and silicosis has a complex molecular and biological mechanism (5).
Additionally, the prevalence and frequency of silicosis remain very high, and currently, no effective therapies are available (6).
Despite comprehensive studies examining the toxicity of crystalline silica over the last several years, the exact etiology and particular mechanism of silicosis remain unknown. The deposition of silica particles in the alveoli of the lungs is the beginning of the pathophysiological process of silicosis. Silica particles ingested by lung cells induce an in ammatory response that produces large amounts of collagen and stimulates broblast overproliferation (7). The principal characteristics of pulmonary brosis in silicosis include broblasts, myo broblast proliferation, alveolar damage and deposition of extracellular matrix (ECM) components, which involve the abnormal remodeling of lung structure and an irreversible decline in lung function (8). Alveolar cells are the basic and essential immune barrier against a pathogenic organism and try to overcome invasion by this pathogenic organism and innate pulmonary immunity against environmental pollutants. When the free pathogenic bacteria and sand particles in the air reach the alveoli of the lung, they are triggered and removed by alveolar cells. Pulmonary broblasts pathologically indicate tissue brosis, broblast and myo broblast proliferation with elevated expression levels of alpha-smooth muscle actin (α-SMA, ACTA1), collagen I (Col I, COL1A1) and collagen III (Col III, COL3A1) and deposition of ECM components (9,10).
Mounting evidence has suggested that CCCH-type zinc nger proteins, such as ZC3H12A/MCPIP1, play an essential role in regulating cell proliferation and migration (11), modulating signaling pathways and targeting mRNA degradation (12)(13)(14). ZC3H4 is a newly found member of the CCCH-type zinc nger protein ZC3H12A (12), which has been shown to be involved in in ammation and brosis induced by SiO 2 via autophagy and endoplasmic reticulum (ER) stress (15,16). The ER plays a signi cant role during the modi cation of posttranslational transmembrane and secretory proteins. Usually, proteins are modi ed in the ER before being transferred and folded. This process is energy-dependent and sensitive to ion variations, such as in the calcium levels, oxygen balance, and glucose supply. (17). Speci c signaling pathways are activated due to ER stress, and this process is known as the unfolded protein response (UPR). Three essential proteins are involved in these signaling pathways, including inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) (18). ZC3H4associated ER stress in pulmonary broblasts has received little consideration. In the current study, we focused on the role of ZC3H4 in pulmonary broblast activation via ER stress with the involvement of the MAP kinase pathway in silicosis, which will facilitate further discoveries in the progression of pulmonary brosis, and silicosis patients may provide a treatment strategy.

Materials And Methods
Reagents Fetal bovine serum (FBS), 10x minimum essential medium (11430-030), Pen-Strep (15140-122) and Dulbecco's modi ed Eagle's medium (DMEM; 1200-046) were purchased from Thermo Fisher Scienti c (Waltham, MA, USA). GlutaMAX supplement (35050-061) and amphotericin B (BP2645) were purchased from Gibco (Thermo Fisher Scienti c). Silicon dioxide (SiO 2 ) was obtained from MilliporeSigma (S5631; Billerica, MA, USA) and because it had more than 80% particle size diameter less than 5 µm, it was selected for use via sedimentation according to stocks' law, acid hydrolyzed and sterilized by baking overnight (200°C, 16 h) to inactivate infectious endotoxins (19). Phosphate-buffered saline (PBS) was used for the suspension of crystalline silica at a nal concentration of 5 mg/ml, and for the cellular experiments, a dosage of 50 µg/cm 2 was applied. The crystalline silica dosage administered in this study was based on our previous experimental studies (19,20).

Western blot assay
The speci c protein levels in pulmonary broblasts were determined by a western blot assay. After treatment, pulmonary broblasts cultured in 24-well plates and cold PBS were washed twice, and a cell lysis solution was used to lyse cells containing proteinase inhibitors (PI). The samples were freezethawed, and total cell proteins were harvested. The protein concentration was measured with the bicinchoninic acid assay (BCA) according to the manufacturer's instructions. Samples containing protein were separated via SDS-PAGE-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat dry milk in Tris-buffed saline and Tween 20 for 1 hr at room temperature (25°C), the membranes were incubated with primary antibodies at 4°C overnight and then with secondary antibodies in 5% nonfat dry milk in Tris-buffered saline and Tween 20 for 1 hr at room temperature. To identify the intensity of immune-reactive protein bands, chemiluminescence (ECL) was used. Each western blot was repeated independently >5 times. The expression level of GAPDH was used as a reference. The protein bands were quanti ed using ImageJ v1.48 software.
Cell counting kit 8 (CCK8) Pulmonary broblasts were seeded into 96-well plates, and triplicate wells were used for a single sample (4 × 10 3 cells/well) and cultured overnight with exposure to silica at different time points (0, 1, 3, 6, 9, 12, 15, 21 and 24 hr). CCK8 reagents (10 μl/well) were administered to the wells of the plate and incubated at 37°C for 2-4 hr. After incubation, the absorbance was measured at 450 nm by a Spectra Max Microplate Reader (Molecular Devices Instruments, Inc., Sunnyvale, California, USA).

Cell migration assays
Cell migration and motility assays were performed to observe pulmonary broblast migration after crystalline silicon dioxide (SiO 2 ) exposure as described previously (21). The cell gap was measured using ImageJ software.

CRISPR/Cas9 technology
Pulmonary broblasts were quickly transfected with CRISPR/Cas9 plasmids according to the manufacturer's protocol (Santa Cruz®) to delete/upregulate ZC3H4 and observe its downstream effects.
The western blotting assay was used to determine the transfection e ciency. In brief, 24-well plates were used for cell seeding (2x10 5 cells /well), and the cells reached 40-80% con uency. The medium was changed to 200 µl fresh antibiotic-free growth medium, and solutions A and B were added as follows. For solution A, transfection reagent (1 μl) was poured into plasmid transfection medium (9 μl), and for solution B, plasmid DNA (1 μl) was poured into plasmid transfection medium (9 μl). After 5 min, solution A was poured dropwise directly into solution B; the sample was then immediately vortexed and incubated at room temperature for >20 min. The mixed solution was added dropwise to 200 μl of the medium in the 24-well plate, and the contents of the well were mixed by swirling the plate gently. The medium was added or replaced when necessary 12 hr after transfection. The pulmonary broblast cells were incubated for an additional 24-72 hr to conduct further experiments.

Immunocytochemistry staining
Immuno uorescence (IF) staining, a frequently used technique in clinical diagnosis and biological research, was performed to determine the orescence effect of ZC3H4 as previously described (22).
Pulmonary broblasts were xed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. The xed samples were permeabilized for 30 min at room temperature in PBS containing 0.3% Triton X-100 and then blocked with 10% normal goat serum (NGS; Life Technologies) in PBS containing 0.3% Triton X-100 at room temperature for 2 hr. The samples were blocked and incubated overnight at 4°C with pAb diluted in PBS containing 10% NGS and 0.3% Triton X-100. PBS was used to wash the samples three times, and the samples were incubated with donkey anti-rabbit (conjugated to Alexa Fluor 488) and donkey antimouse (conjugated to Alexa Fluor 576) secondary antibodies for 2 hr at room temperature. After PBS was used for three washes, the samples were xed with mounting solution (ProLong Gold Antifade Reagent with DAPI; P36931, Life Technologies), and an EVOS FL fluorescence microscope was used to examine the prepared slides.

Immunoprecipitation (IP)
The interactions between proteins were detected by IP assays as previously described (23). Brie y, equivalent amounts of the extracted proteins were incubated with anti-ZC3H4 antibodies overnight at 4°C, and then 20 μL of protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology®, Inc.) was added for 90 min at 4°C. Then, the mixture was centrifuged (12000 × g, 3 min, 4°C), and the cell pellets were rinsed three times with RIPA lysis buffer. The cell pellets were later boiled in SDS loading buffer for 5 min. The mixture was then centrifuged (12000 × g, 3 min, 4°C). The collected supernatants were used in western blot assays to detect ZC3H4 and Sigma 1 receptor (Sigmar1).

Statistical analysis
The data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.). Unpaired numerical data were associated with an unpaired Student's t-test (2 groups) or ANOVA (2 groups). The level of signi cance was set at 0.05; values of P<0.05 indicated statistical signi cance.

SiO 2 increases migration in pulmonary broblasts
Two-dimensional (2D) and 3-dimensional (3D) nested collagen matrix assays signi cantly represent the cell migration ability in in vitro culture (28)(29)(30). Compared with usual scratch assays, the nested collagen matrix (3D) assay is a reliable, quick, quantitative and easy method for determining broblast migration and motility in 3D models (6,28,31,32). Here, we provide new insights into the novel roles that pulmonary broblasts exposed to SiO 2 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 SiO 2 induced broblast migration and proliferation differently in 2-dimensional and 3dimensional nested collagen matrix gel systems, the peak response of broblasts was observed at 24 hr.
The scratch assay/wound healing assay ( Fig. 2 A-B) demonstrated that SiO 2 signi cantly induced migration in MLg cells. Therefore, the schematic diagram of the 3D nested collagen matrix gel assay represents the migration of pulmonary broblasts after SiO 2 exposure (Fig. 2C). Pulmonary broblasts exhibited an increase in both migration distance ( Fig. 2D-E) and number of migrated cells ( Fig. 2D and F).

SiO 2 induces ZC3H4 in pulmonary broblasts
A previous study from our laboratory suggested that ZC3H4 is involved in the in ammatory stage of silicosis. To establish whether ZC3H4 is also involved in late brosis, MLg cells were exposed to SiO 2 .
Western blot assays con rmed that the ZC3H4 expression level was suddenly increased after SiO 2 exposure in pulmonary broblasts at different time points (0, 1, 3, 6, 12 and 24 hr) with a two-phase increase pattern (Fig. 3A-B). Immunocytochemistry con rmed the upregulation of ZC3H4 expression induced by SiO 2 in pulmonary broblasts (Fig. 3C).
To simplify the role of ZC3H4 in broblast activation induced by SiO 2 , we transfected MLg cells with ZC3H4 NIC plasmids to speci cally knock down ZC3H4. Western blot assays con rmed that the speci c knockdown of ZC3H4 in pulmonary broblasts signi cantly inhibited the upregulation of ZC3H4 with or without SiO 2 ( Fig. 3D-E). In the cell viability assay, ZC3H4 knockdown attenuated the increase in cell viability induced by SiO 2 (Fig. 3F), indicating that ZC3H4 mediated the proliferation induced by SiO 2 in broblasts. Moreover, ZC3H4 NIC plasmids abolished the effect of SiO 2 on the migration ability of MLg cells ( Fig. 3G-H). Furthermore, the COL1A1 and ACTA1 expression levels induced by SiO 2 were assessed after the speci c knockdown of ZC3H4 ( Fig. 3I and J), in which all upregulated markers were inhibited, con rming the role of ZC3H4 in pulmonary broblast activation and migration.

SiO 2 induces ER stress in pulmonary broblasts
To depict the downstream molecular mechanism of ZC3H4 on broblast activation, ER stress was evaluated. The ER is a key factor for cellular activities (protein modi cation, folding, synthesis and transport) (33)(34)(35). Various pathological and physiological environmental factors could affect ER homeostasis, nally 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 broblasts after SiO 2 exposure. MLg cells treated with SiO 2 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 con rm the colocalization of ZC3H4, BiP and DDIT3 (Fig. 4E-F). To further con rm the role of ZC3H4 in ER stress, ZC3H4 was speci cally knocked down. As shown in Fig. 4G-H, ZC3H4 knockdown abolished the induction of ER stress induced by SiO 2 . Collectively, these results indicated that ZC3H4 is responsible for broblast activation induced by SiO 2 via the ER stress pathway.

ZC3H4 induced ER stress via Sigmar1 in pulmonary broblasts
Having established the fundamental role of ZC3H4 in pulmonary broblast 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 broblasts via the sigmar1/ER stress pathway deserves investigation. To determine whether Sigmar1 is involved in silicosis, MLg were exposed to SiO 2 to assess the Sigmar1 level. Western blotting results con rmed that the expression level of Sigmar1 was increased in the presence of SiO 2 (Fig. 5A-B), which was con rmed by immunocytochemical staining (Fig. 5C). Further, speci c inhibition of sigmar1 with the pharmacological agent BD1047 attenuated the upregulation of migration induced by SiO 2 (Supplementary Fig. S2A-B). Furthermore, the Sigmar1 expression level was assessed after the speci c knockdown of ZC3H4, in which ZC3H4-NIC suppressed sigmar1 upregulation after SiO 2 exposure (Fig. 5D-E). Interestingly, the mRNA levels of ZC3H4 and Sigmar1 showed almost no change after SiO 2 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 veri ed that ZC3H4 and Sigmar1 are involved in the pulmonary broblast activation induced by SiO 2 .

MAPK and PI3K pathways are involved in SiO 2 -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 SiO 2 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 SiO 2 exposure. Moreover, Akt phosphorylation showed a slight but signi cant increase after SiO 2 exposure (Fig. 6E-F). To assess the role of the MAPK or PI3K pathway on regulation of ZC3H4, speci c 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 SiO 2 was applied. As shown in Fig. 6G-H, all inhibitors attenuated the increase in ZC3H4 and sigmar1 induced by SiO2, con rming the role of the MAPK and PI3K pathways in the regulation of ZC3H4 and Sigmar1. Furthermore, broblast 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 signi cant role in regulation of ZC3H4.

Induction of ZC3H4 by SiO 2 promotes a further increase in ZC3H4
Interestingly, SiO 2 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 brosis 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 clari ed. To our surprise, while salubrinal inhibited the increase in ER stress markers ( Supplementary Fig. 5A-B), salubrinal reversed the SiO 2 effect in the upregulation of ZC3H4 in pulmonary broblasts ( Fig. 7A-B). Furthermore, tunicamycin, a speci c 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 broblasts 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 SiO 2 exposure (Fig. 7 G-H). Immunocytochemistry assays con rmed the upregulation of ZC3H4 expression after 50 µM tunicamycin in pulmonary broblasts (Fig. 7 I). These results demonstrated that ER stress was involved in the upregulation and activation of ZC3H4 in pulmonary broblasts 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 signi cant role in the activation of pulmonary broblasts.

Discussion
Pulmonary brosis is the sequela of many pulmonary diseases, such as idiopathic pulmonary brosis and pneumoconiosis. Pulmonary broblasts pathologically indicate tissue brosis and broblast and myo broblast proliferation (9,10). The current study principally focused on the role of ZC3H4 and sigmar1 on the proliferation, migration and activation of pulmonary broblasts exposed to SiO 2 , which may provide a target for pulmonary brosis treatment, and the detailed molecular mechanism was also investigated.
Pulmonary broblasts clinically represent the proliferation of broblasts and myo broblasts with the deposition of ECM components (9, 10). The zinc nger protein family has been shown to play a potential role in the in ammatory response and represents a downregulation of the in ammatory reaction, as well as broblast activation, such as ZC3H12A/MCPIP1 (12,13). ZC3H4 is similar to ZC3H12A, and both belong to the same family of CCCH-type zinc nger proteins (12). Interestingly, ZC3H12A may act as a molecular signal in the initiating pathway or as a functional factor to induce or inhibit in ammation, mainly depending on the time frame. The current study suggested a similar pattern of ZC3H4, in which ZC3H4 initiated ER stress as a signaling molecule in the early stage and promoted broblast migration and proliferation in the late stage, forming PFLs. PFLs are responsible for producing all-or-none responses by continuously converting graded inputs into discrete outputs (56, 57) and transforming broblasts to myo broblasts (58). Mounting evidence has shown that brosis in pneumoconiosis and IPF is a chronic pathological process in which both signaling molecules and the TGF-β pathway are involved. CCCH-type zinc nger proteins participate in PFLs to initiate brosis and maintain the brosis process, indicating a suitable target for blocking or reserving this process. This two-phase increase pattern of ZC3H4 may guide new strategies for the treatment of brosis by an early blockade of ZC3H4 initiation.
Although the comprehensive mechanism of ZC3H4 in ER stress must be clari ed, the present study recommends a novel role of ZC3H4 in ER stress. Previous studies have shown that ZC3H4 promotes epithelial-to-mesenchymal transition (EMT) via ER stress, which participates in the brosis induced by SiO 2 (16). Currently, the detailed mechanism by which ZC3H4 induces ER stress was investigated, in which ZC3H4 directly interacts with Sigmar1, which is expressed in the ER and a subclass of the sigma receptor family (42). The molecular action of Sigmar1 was previously discovered to be a ligand-regulated receptor chaperone via ER stress. Sigmar1 has been shown to be involved in broblast activation (41). Interestingly, the mRNA levels of both ZC3H4 and Sigmar1 did not show a signi cant change due to SiO 2 exposure, while these two proteins have been shown to be regulated by noncoding RNAs, such as circular RNAs (circRNAs) (15,41). circRNAs are involved in cell proliferation and transformation, and their misregulation is related to diseases and can have phenotypes in animal models (47). However, given the low abundance of most circRNAs, whether the translation of certain circRNAs is crucial for their endogenous function remains unclear. Hence, the direct interaction between ZC3H4 and sigmar1 was investigated in the current study, but it did not rule out the role of circRNA in the regulation of these two proteins. Taken together, these results veri ed that ZC3H4 and Sigmar1 were involved in the pulmonary broblast activation induced by SiO 2 .
Although the present study still focused on the activation of pulmonary broblasts, it delivered a new direction to treat silicosis. MAPK signaling pathways were responsible for the activation of transcription factors. Activation of MAPK was involved in the upstream signaling pathways for ER stress induction (48). In addition, MAPK pathways such as p38, JNK, and ERK were also stimulated by ER stress inducers and played a signi cant role in different cell signaling pathways and cell death (49)(50)(51)(52). Inositol-requiring enzyme 1 (IRE1) is responsible for the activation and stimulation of MAPK (51). After stress, broblast cells inhibit migration due to abnormal signaling pathway responses, although ER stress is responsible for lung diseases (39,40,53). Due to the rapid and transient increase in ZC3H4 after SiO 2 exposure within 6 hr, MAPK initiated the expression of ZC3H4. Recently, Ets-like protein-1 (Elk-1) has been shown to mediate ZC3H4 expression (54) in epithelial cells, while Elk-1 also induced ZC3H12A in macrophages (55), which indicates a similar upstream regulatory mechanism for the CCCH zinc nger protein family. Taken together, these results demonstrate that MAPK/ZC3H4/ER stress plays a signi cant role in the activation of pulmonary broblasts.

Conclusion
Our study revealed a link between ZC3H4 and pulmonary broblast activation via the sigmar1/ER stress pathway in silicosis; SiO 2 exposure enhanced the MAPK and P13K/Akt phosphorylation pathways with the involvement of ZC3H4. Interestingly, ZC3H4 has been shown to induce ER stress with the participation of a PFL, providing a novel therapeutic strategy for treating silicosis (Fig. 8).

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Availability of data and materials
All of the relevant raw data and materials are freely available to any scientist upon request.          Schematic diagram showing the mechanisms through which silica induced ZC3H4 and Sigmar1 expression in pulmonary broblast activation. ZC3H4 promotes pulmonary fibroblast activation with the involvement of MAPK (Mapk1, Mapk8 and Mapk14), and P13K/Akt phosphorylation pathways via sigmar1/ER stress in silicosis play a signi cant role in broblast proliferation, migration and differentiation. The interaction between ZC3H4 and Sigmar1 promotes the ER stress pathway with a PFL that enhances the proliferation, migration and differentiation abilities with the increased synthesis of collagen, which may lead to brosis induced by SiO2.