FAM172A supervises ER (endoplasmic reticulum) stress‐triggered autophagy in the epidural fibrosis process

Abstract Backgrounds Lumbar laminectomy is usually utilized for lumbar disc herniation (LDH), but also causes epidural fibrosis (EF) process associated with abnormal proliferation of fibroblasts. FAM172A is associated with ER stress and cell proliferation, but its mechanism was unclear, especially in the process of EF. Methods Therefore, the regulation of FAM172A on the calcium flux and autophagy in fibroblasts were investigated by inducing ER stress with tunicamycin and upexpression or downexpression of FAM172A. The calcium flux was determined using Fluo‐3, and autophagy was examined with immunofluorescence or western blot for LC3, Beclin‐1, ATG‐5, and p62. Moreover, the apoptotic protein of Bax and Bcl‐2 was detected, too. Furthermore, the laminectomy model was constructed and then dealt with overexpression of FAM172A. Results Tunicamycin‐induced endoplasmic reticulum (ER) stress and autophagy process in fibroblasts were associated with the calcium flux regulated by FAM172A, especially in EF cells. Besides, tunicamycin induced autophagy and suppressed cell apoptosis of fibroblasts. Furthermore, FAM72A repressed the proliferation of fibroblasts and the process of EF in the laminectomy model through the mediation of the autophagy process. Conclusions Tunicamycin‐induced endoplasmic reticulum (ER) stress in fibroblasts was associated with calcium flux mediated by FAM172A. FAM72A participated in the autophagy regulation of fibroblasts and maybe the key interaction regulator of apoptosis and autophagy in fibroblasts, especially for epidural scar cells.

autophagosome generates after edges fusion of membrane, 7 then the formation of autolysosome combined autophagosome with lysosome. 8 But, the recent mechanism of autophagosome biogenesis needs to be explored, especially in the process of EF.
The current studies confirm that the plasma membrane of different cellular compartments, including mitochondria, endoplasmic reticulum (ER), and Golgi may be associated with the precursors of autophagosome membrane. 9 Moreover, the autophagic response can be induced by ER stress, 10 which is caused by activation of unfolded protein response (UPR) come from misfolded proteins accumulation in ER. 11 Meanwhile, the structure of the pre-autophagosome assembles after stimulation of ER stress. 12,13 The previous experimental data demonstrated the biological functions of FAM172A; FAM172A was a stabilizing factor for ER stress. Moreover, FAM172A played a tumor suppressor role in hepatocellular carcinoma (HCC) under its supervision on calcium flux. 14,15 Besides, the death of cells could be regulated by two processes, including autophagy and apoptosis. 16 The complex interactive regulation of the two processes between autophagy and apoptosis, which can be activated together through a variety of regulatory molecules and the stimuli of stress, even the two processes transform into each other. 17 Therefore, the mechanistic investigation on the relationship between ER stress and autophagy associated with intracellular calcium flux mediated by FAM172A was studied in the progression of EF in the laminectomy model. Moreover, the regulation of FAM172A on the calcium flux and autophagy in fibroblasts were explored by inducing ER stress with tunicamycin, and up-or downexpression of FAM172A. The calcium flux was determined, and autophagy was examined based on the markers of autophagy. The ketamine (100 mg/kg body weight) was used to conduct anesthesia with intraperitoneal injection for the laminectomy model; the dura mater of the L1 vertebral plate was exposed. Then, the spinous process and vertebral plate of L1 were removed with rongeur forceps. To induce overexpression of FAM172A, the recombinant adenovirus vector was used to transfer the FAM172A gene, which was injected into the tail vein of the mice model.
At the end of the experiment, mice were over anesthetized with isoflurane (0.5 ml/min), until heartbeat and breathing stopped in the entire process but without pain. The unified approach was adopted to hand over animal carcasses.

| Cell culture
Primary cells were directly isolated from living tissues of mices. In this experiment, fibroblasts were obtained from the living normal fiber tissues (FTs) of rat control (n = 6), and the living epidural scar tissues (STs) were isolated from rats which underwent laminectomies (n = 6), respectively. Then, these fibroblasts were cultured in vitro.
The obtained tissues were firstly cleaned with D-Hanks and Hanks solution to remove blood stains on the surface of cells and the attached connective tissues. After a repeated clean process, a scalpel was used to cut the tissues into several small pieces about 1 mm 3 in size, then was transferred into a small beaker contained with Hanks solution, Secondly, the above tissues were performed to digest and separate small pieces into cell clusters and scattered single cells with digestive enzymes, trypsin, and collagenase. Finally, the cell suspensions were incubated, and the cells were counted with a counting plate. The number of cells was adjusted to (2$5) Â 10 5 cells/ml with the culture medium, then cells were dealt with a CO 2 incubator for 5% CO 2 , at 37 C. After 3$5 days, the primary cultured cells generally adhered to the wall of the bottle, then stretch and start to grow. Furthermore, the new medium was added into the original culture medium, then cells were continued to culture for 2$3 days, the medium was changed. At 7$14 days, the subcultured cells filled the wall of the bottle. The treatment with tunicamycin in the cell experiment was 48 h.
The shRNA plasmids of the pSilencer 2.1-neo vector were used to interfere the expression of FAM172A, and the RNAi-targeting sequences for FAM172A were confirmed by the previous studies. 14,15 nation was performed based on the secondary HRP antibody using exposed film.

| Co-localization
The cells were digested with trypsin; then, single-cell suspensions were obtained. Moreover, the sterilized Petri dish was added a small amount of culture medium, then put the slide carefully into it, and drop the above single-cell suspension onto the slide drop by drop.
Finally, the dishes were placed in a 5% CO 2 incubator at 37 C. After cultured for 48 h, the slides were washed three times with PBS at 37 C for 3-5 s each time. Subsequently, the cells on the surface of these slides were fixed in 4% paraformaldehyde for 15 min and rinsed with 37 C deionized water to clean the formaldehyde. Additionally, the process of antibody incubation was conducted for immunofluorescent staining. Finally, observe under the microscope of Zeiss 510 META microscope was used to observe the immunofluorescence in cells. The concentration variation of free calcium in cells was represented by the ratio of F340/380 fluorescence intensity, which was the key factor for the cytosolic changes in Ca 2+ concentrations. 18 The image and fluorescence were obtained with the fluorescence microscope coupled to the camera, then analyzed using Fluorescence Ratio Imaging Software (version 7.0). A total of 50-100 cells were measured individually in each experiment group and were repeated three times.

| Construction of recombinant adenovirus overexpression vector of FAM172A
The information of the CDS (Coding DNA Sequence) region of the target gene FAM172A was obtained from the NCBI website, then the primers were designed, and a high-fidelity KOD enzyme (3 K mutation rate of 0%) was utilized to amplify from the template of cDNA library. Furthermore, it was cloned into the adenovirus

| Epidural fibrosis assessment
For 4 weeks after surgery, the epidural scar tissues of surgical sites were evaluated according to the adhesion amount (Adhesion grade: 0, not adherent to dura mater; 1, adherent to dura mater, easily dissected; 2, adherent to dura mater, difficulty dissected without disrupting dura matter; 3, firmly adherent to dura mater, cannot be dissected.

| Analysis of histology
Four weeks after surgery, the epidural scar tissues at the surgical sites were evaluated based on the histological analysis. The decalcified specimens of the whole L1 spinal column were fixed and sectioned.
Finally, the stained sections with H&E (Hematoxylin-eosin) and Masson's trichrome were observed using light microscopy to evaluate the degree of epidural collagen tissues and EF.
Immunohistochemistry for GRP78 and CHOP proteins was performed using the avidin-biotin-peroxidase complex method. For antigen retrieval, the sections were treated with 0.5% trypsin at 37 C for 10 min. After blocking with 1% skim milk, tissue sections were incubated with anti-GRP 78/BiP polyclonal antibody or anti-CHOP antibody overnight at 4 C, and then washed in PBS solution. Biotinylated anti-rabbit IgG was used as the secondary antibody, and the sections were visualized as previously described.

| Statistics
The data were shown as mean ± SD (standard deviation). The differences between the two groups were analyzed with the Student's ttest. Kruskal-Wallis ANOVA (Analysis of variance) was used to analyze the abnormally distributed data among groups. SPSS software (version 18.0) was utilized to perform these statistical analyses.
p < 0.05 was significant statistically. Our experimental data indicated that the key protein of the endoplasmic reticulum stress signaling pathway, CHOP, 19-21 also increased in a dose-dependent manner with the stepwise tunicamycin concentration, and its expression was more significant in FT ( Figure 1A, B, and Figure 1A, B).
In addition, the results of the intracellular calcium flux detection experiment identified that with the increased concentration of the tunicamycin, the intracellular calcium flux showed a dose-dependent enhancement, and it was more marked in FT ( Figure 1C and D). The laser confocal microscope was further used to detect the intracellular calcium current; calreticulin was used as an internal reference, which was located at the endoplasmic reticulum. 22 It indicated that the calcium current in ST was distinctly stronger than that of the normal fibrosis tissue, meanwhile the calcium current was induced by tunicamycin in the two cell lines which were both significantly increased, and the calcium flux of ST enhanced more visibly (Figure 2).

| Tunicamycin induced autophagy of cell lines and inhibited cell apoptosis regulated by FAM172A
After tunicamycin treatment, both the two cell lines, FT ( Figure 5A) and ST ( Figure 5B) occurred autophagy ( Figure 5A, B). In the meantime, the autophagy marker, LC3-II protein expression, 24 increased in a dose-dependent manner followed with the stepwise tunicamycin concentration, and it was more significant in ST ( Figure 5C, D).
Further application of laser confocal microscopy was used to detect the autophagy status of cells; the results suggested that FAM172A overexpression restrained the autophagy process of FT and ST, and the autophagy status of ST was more distinct (Figure 8). However, through prohibiting, the expression of FAM172A by interference methods promoted the autophagy process of these two cell lines (Figure 8). ER stress. [2][3][4] Moreover, ER stress was related to the cytosolic changes in the concentrations of calcium ions. 17,18 In previous work, it was found that the potent-binding activity between FAM172A protein and Ca 2+ ion and the significantly downregulated expression of FAM172A in ER stress situation induced by tunicamycin. 14 FAM172A was the key factor for the cytosolic changes of Ca 2+ concentrations, 14,15 which was related to ER stress. However, the intracellular calcium signaling pathways are referred to the variety of biological processes in cells, such as growth, apoptosis, differentiation, autophagy, and metastasis. 29 Therefore, the in-depth and F I G U R E 9 FAM172A suppressed EF associated with the apoptotic autophagy process in fibroblasts. Based on the mice model, we conducted overexpression of FAM172A in the EF process to identify its role associated with the apoptotic autophagy of fibroblasts. The HE staining was used to show typical features of scar tissues (A-D; Â200). Moreover, the autophagy marker protein LC-3 and the expression levels of its subtypes LC-3I and LC-3II were examined. In the meantime, the expression of FAM172A, the apoptosispromoting protein Bax, the apoptosis-inhibiting protein Bcl-2, the key regulatory protein complexes of autophagy Beclin-1, ATG-5, and autophagy protein p62, were evaluated in tissues of mice with WB method (E). (The number of experimental replicates was three.) comprehensive research involved in the interaction mechanism between apoptosis and autophagy must take breakthroughs on the cognition and treatment of diseases, such as scar formation and epidural fibrosis. 30 Additionally, in some cell lines, the influx of calcium monitors the ER stress. 31 Consequently, we successfully induced the endoplasmic reticulum (ER) stress with tunicamycin in fibroblasts, the two cell lines come from normal fiber tissue (FT) and epidural scar tissue (ST), respectively. Moreover, ER stress was positively correlated to the intracellular calcium flux mediated by FAM172A, especially in ST. CHOP is a homologous protein of the transcription factor C/EBP (CCAAT enhancer-binding protein) and could inhibit the transcription factor C/EBP and LAP (Liver-enriched activator protein). 19 Some cell stress, such as starvation, could induce CHOP expression, and CHOP inhibits cell cycle transition from G1 to S phase. 20 Recent studies have found that the expression level of CHOP is upregulated under ER stress, and CHOP mediates programmed cell death or apoptosis. CHOP can mediate the activation of GADD 34 during the endoplasmic reticulum stress. 21 In this work, we confirmed that CHOP showed a dose-dependent increase, followed by the increased concentration of the tunicamycin, especially in ST. Hence, our results indicated that the calcium current in ST was significantly stronger than that in FT tissue. In a word, we confirmed that tunicamycin-induced ER stress in fibroblasts is associated with calcium flux mediated by FAM172A.

| FAM172A suppressed EF associated with the apoptotic autophagy process of fibroblasts
The higher organisms all need the essential calcium for survival; the dynamic 2nd messenger calcium is versatile. 32 The endoplasmic reticulum releases calcium under excitation and activation of cells, then phosphatases and kinases that depended on calcium are activated, meanwhile numerous cellular processes are regulated, including autophagy and apoptosis. 33 Several identified regulated mechanisms of autophagy are related to calcium ions, meanwhile important to maintain cell survival and mediate life and death decisions. 32 Furthermore, through up-or downregulation of FAM172A expression, we explored whether FAM172A regulates the process of ER stress and the calcium flux in the two fibrosis cell lines. Consequently, we figured out that FAM72A participated in the regulation of the endoplasmic reticulum stress-related calcium flux in a negative role. In brief, the downregulation of FAM172A expression maybe the key pathogenic factor of the epidural fibrosis process, and it was mediated by the process of the endoplasmic reticulum stress-related calcium flux. Moreover, it was inconsistent with the previous study, 34 which results suggested that PPP1R13B regulated by circ-012091 promoted the proliferation and migration of lung fibroblasts through regulation of ERS and autophagy and played a crucial role in the development of pulmonary fibrosis in silicosis. 34 According to the different regulation methods of apoptosis and autophagy, the interaction can be roughly summarized into three types, including cooperative relationship, confrontation relationship, and promotion relationship. [35][36][37][38][39] The multiple interaction modes between autophagy and apoptosis must have common signaling pathways and regulatory proteins, which researchers call interaction regulators. 40 Wan et al. validated that the proliferation of fibroblasts was considered to be a major cause in the formation process of epidural fibrosis. Artesunate could inhibit the proliferation of fibroblasts and reduce the formation of epidural fibrosis after laminectomy. Its potential mechanism might be associated with the autophagy cascade mediated by p53/p21 waf1/cip1 pathway. Artesunate might provide a novel reagent for reducing epidural fibrosis after spinal laminectomy surgery. 41

| CONCLUSIONS
Tunicamycin-induced endoplasmic reticulum (ER) stress in fibroblasts was associated with calcium flux mediated by FAM172A. FAM72A participated in the autophagy regulation of fibroblasts. FAM172A maybe the key interaction regulator of apoptosis and autophagy in fibroblasts, especially for epidural scar cells.

ACKNOWLEDGMENTS
Not applicable.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data used to support the findings of this study are included within the article.

COMPETING INTERESTS
The authors declare that they have no competing interests.