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. 2021 Mar 9;11(4):162. doi: 10.1007/s13205-021-02710-1

Transcription factor EB promotes rheumatoid arthritis of Sprague–Dawley rats via regulating autophagy

De Lai Xu 1, Jie Pan 1,✉
PMCID: PMC7943689  PMID: 33786279

Abstract

This study investigated the effect of autophagy-related gene transcription factor EB (TFEB) on the rheumatoid arthritis (RA) and explored whether TFEB regulated RA by autophagy. The Sprague–Dawley rats were divided into two groups (n = 6). The rats were stimulated with the mixture of the type II collagen and Freund’s adjuvant or PBS at the root of the tail. Results showed that swollen and deformed joints were discovered, the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were elevated, and hematoxylin and eosin staining showed the inflammatory cells infiltrate the synovial tissue in the RA rats, compared to the control group. Immunohistochemistry displayed that the expressions of TFEB and LC3B increased in the synovial tissues of RA rats, whereas p62 decreased. The silence of TFEB in the RA-fibroblast-like synoviocytes (RA-FLS) decreased the protein expressions of LC3B, compared to the siRNA NC group. Meanwhile, the activity of FLS was raised, whereas the levels of TNF-α and IL-6 decreased in RA-FLS with TFEB knockdown. In conclusion, our study revealed that TFEB plays a crucial role in the progress of RA by regulating autophagy, which might provide novel targets for the therapy of RA.

Keywords: Transcription factor EB, Autophagy, Rheumatoid arthritis, Inflammation

Introduction

Rheumatoid arthritis (RA), which has poor prognosis and continuously increased mortality rate, showed an increase of five million per year in China (Yu et al. 2018). RA is characterized by chronic joint inflammation, bone destruction, and motor dysfunction (Holers and Banda 2018). RA mechanisms include cytokine network dysfunction, complement activation, and infiltration of various immune cells in the synovial tissue. Reports demonstrated that the risk factors of RA include heredity, autoimmune, smoking, infection, diet, and other environmental factors (Kerlan-Candon et al. 2001; Li et al. 2013; Rajaei et al. 2015). The treatment for patients with RA includes drug treatment, immune purification, function exercise, and surgery. Given the high incidence of RA and serious complications, finding critical genes that participate in the progress of RA is of great importance.

Autophagy is an evolutionarily conserved secondary lysosomal degradation process, which plays a dual role in cell metabolism and physiological function (Choi et al. 2013; Weiskirchen and Tacke 2019). Autophagy maintains cell homeostasis under extracellular stress, whereas the continuous activation of autophagy leads to intracellular protein degradation and the necrosis or apoptosis of cells (Mizushima 2007; Narendra et al. 2008). Autophagy participates in the progression of cancers, lung injury, and diabetes (Liu et al. 2019; Zhou et al. 2020a, b). Rapamycin alleviated the development of RA by activating autophagy (Bao et al. 2020). Recently, substantial evidences showed that autophagy plays a central role in the progress of RA by interacting with chondrocytes, osteoclasts, fibroblast-like synoviocytes (FLS), and pro-inflammatory cytokines (Chadha et al. 2020). The effect of antigen-presenting cells requires autophagy, and inhibiting autophagy decreases autoimmune response. Autophagy inhibitors (chlorquine and hydroxychlorquine) suppressed the activity of T cells in patients with RA and the progress of osteoclastogenesis (Bao et al. 2020). Autophagy promoted osteoclastogenesis, the bone erosion and bone tissue degradation, which deteriorated the progress of RA. In osteoarthritis, increased autophagy protected the chondrocyte from additional apoptosis and senescence (Zheng et al. 2018). Autophagy is significantly enhanced in the synovial linings of the synovial tissues and RA-fibroblast-like synoviocytes (RA-FLS) of patients with RA (Yang et al. 2017; Zhu et al. 2017). Therefore, it is critical to investigate the mechanism of autophagy of RA-FLS in RA.

Transcription factor EB (TFEB) is one of the members of the microphthalmia transcription factor/transcription factor E (MiTF/TFE) family (Raben and Puertollano 2016). TFEB is an important transcription factor in regulating the expression of autophagy-associated proteins (Palmieri et al. 2011). Moreover, TFEB is connected with the transcription of several autophagy-related genes, such as chloride channel voltage-sensitive 7 (CLCN7), vacuolar protein sorting 18 (VPS18), and microtubule-associated protein 1 light chain 3β (MAP1LC3B). Overexpression of TFEB enhanced autophagy, whereas TFEB deficiency suppressed autophagy and caused cell death (Settembre et al. 2011; Ma et al. 2012). However, the role of TFEB in autophagy of RA remains unclear.

This study aims to investigate whether TFEB regulated the progress of RA via autophagy. We established the RA model of rats and detected the levels of TFEB and autophagy-related genes in vivo. On the other hand, synovial cells were separated from the arthritis synovial tissue in vitro. Cell vitality, apoptosis, inflammation cytokines, and the LC3B were detected to reveal the effect of TFEB in the arthritis synovial cells with TFEB knockdown.

Methods and materials

Animals’ sources and the ethics statement

A total of 12 7-week-old male (180 ± 20 g) Sprague–Dawley rats were purchased from CLOUD-CLONE CORP (Wuhan, China). Rats were fed in a room of a specific pathogen-free grade with an unlimited supply of food and water. The methods used were according to the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, with obtained approval from the experimental Animal Management Committee of Soochow University.

RA model

The RA model of rats were established following previous protocols (Wang et al. 2020). In detail, rats were randomly divided into two groups (n = 6): the control group and RA group. First, the type II collagen (Sigma, St Louis, MO, USA) was mixed with the Freund’s adjuvant at 4 °C. Rats were subcutaneously injected with a 300 µL mixture to the caudate root. Avoiding the initial injection site, the RA group was given an equal mixture after 7 days to enhance immunity. The same amount of physiological saline was given to the control group. The ankle diameter, paw thickness, arthritis scores and body weight of rats were observed once a week from the day of booster immunization to 28 days. The paw thickness and ankle diameter of rats were detected using the vernier caliper. The arthritis score of per paw was calculated based on the Hooke Laboratories manual (He et al. 2011). And then, the blood samples were withdrawn from the retro-orbital plexus. After standing at room temperature for 1 h and centrifuging at 1000×g for 15 min, the sera were collected. Subsequently, rats were anesthetized by an intraperitoneal pentobarbital sodium (Rebiosci Biotech Co., Ltd, Shanghai, China) injection (150 mg/kg body weight), followed by cervical dislocation. Synovial tissues were collected for subsequent experiments.

Primary synovial cell culture

RA-FLS cell lines were isolated from the synovial tissues of RA rats (Lauzier et al. 2011). Synovial tissues were separated into fragments under aseptic conditions. Trypsin was used to digest tissues for 2 h at 37 °C. Subsequently, the suspension was centrifuged to obtain RA-FLS. Cells were incubated in a Dulbecco’s modified eagle medium (DMEM, 11965092, Gibco) with 10% FBS (10099-141, Gibco) and double antibody. Experiments were conducted on the RA-FLS of over generation 3.

Small interfering RNA (siRNA) transfection

Synovial cells were cultured in the incubator (Thermo Fisher Scientific, NY, USA) with 37 °C and 5% CO2. Cells were seeded in a six-well plate for 24 h, and cells account for 80% of the bottom of the plate. The control siRNA and siRNA targeted against TFEB were diluted with a serum-free medium, and then transfected to synovial cells using Lipofectamine 2000 (General Biosystems, Anhui, China), according to the manufacturer’s instruction. The final concentration of siRNA was 100 nm. After being transfected for 24 h, the interference efficiency was detected by qRT-PCR.

Cell vitality

Cell Counting Kit-8 (CCK8, Beyotime Biotechnology, Shanghai, China) assay was used to detect the activity of synovial cells. The cell suspension (100 µL) was seeded in the 96-well plates at a density of 2 × 104/mL. After incubation for 24, 48, 72, or 96 h in the incubator at 37 °C with 5% CO2, a 10 µL of CCK8 solution was added to each hole of the plate. After 1 h, the absorbance at 450 nm was measured by the Microplate Reader (Thermo Scientific, NY, USA).

Enzyme linked immunosorbent assay (ELISA)

The blood of rats was collected and kept at room temperature for 1 h, and then centrifuged at 3500×g for 10 min, and sera were acquired. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the sera and the supernatant of synovial cells were detected by Rat ELISA kits (ThermoFisher Scientific, Waltham, MA), according to the manual. The absorbance was detected at 450 nm and 570 nm by the Microplate Reader (Thermo Scientific, NY, USA).

Western blot (WB) analysis

The total protein was extracted from the FLS of RA rats by Radio-Immunoprecipitation Assay (RIPA, ThermoFisher Scientific, Waltham, MA). Moreover, the BCA protein assay kit was used to measure the concentrations (Thermo Scientific, USA). Equal proteins were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gels. Proteins were then transferred into a 0.45 μm of polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by electrotransfer. After blocking for 2 h at room temperature with blocking solutions (5% non-fat milk in Tris-buffered saline), the membranes were incubated with primary antibodies against TFEB (1:1000, AF7015, Affbiotech), LC3B (1:1000, ab48394, Abcam), or GAPDH (1:10,000, 60004-1-Lg, Proteintech) for 16 h at 4 °C. Subsequently, membranes were washed and incubated with an anti-rabbit secondary antibody (1:10,000, ab6721, Abcam) or anti-mouse secondary antibody (1:10,000, ab205719, Abcam) for 2 h at room temperature. Blots were visualized with enhanced chemiluminescent (ThermoFisher Scientific, Waltham, MA) through the Electrophoresis Gel Imaging Analysis System (Qin Xiang Scientific Instrument Co., LTD, Shanghai, China). The intensity was analyzed with the Image Lab 3.0 software.

Hematoxylin–eosin staining (H&E) and immunohistochemistry (IHC)

Synovial tissues of rats were embedded in paraffin. The paraffin was sectioned into 5 μm thickness. H&E staining was performed following the H&E Staining Kit instruction (Abcam, Cambridge, USA). For IHC staining, the paraffin section was deparaffinized and rehydrated. Paraffin sections were repaired with a citric acid antigen-repair buffer to expose the antigen site. A 3% of hydrogen peroxide was then used to block the endogenous peroxidase. Later, 5% of bovine serum albumin was utilized to block the nonspecific antigen for 0.5 h at 37 °C. Sections were incubated with primary antibodies against TFEB (1:100, Ab270604, Abcam), LC3B (1:200, Ab48394, Abcam), and p62 (1:50, Ab56416, Abcam) overnight at 4 °C. After the incubation with an HRP-conjugated secondary antibody (PAE001, CLOUD-CLONE CORP, Wuhan, China) for 0.5 h, sections were incubated with a DAB kit (C1101, CLOUD-CLONE CORP, Wuhan, China) for appropriate times. The cell nuclear was stained with hematoxylin for 5 min. Sections were observed on optical microscopes (Olympus Corporation, Japan) after they were dehydrated and sealed.

Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from synovial cells using Trizol (Invitrogen, USA). The concentration and purity of RNA were detected by the TGem spectrophotometer (Tiangen Biotech Co., Ltd., Beijing, China). The integrity of RNA was detected by Agarose gel electrophoresis. We synthesized the cDNA according to the instructions of the RevertAid First Strand cDNA Synthesis Kit manual. The cDNA product was amplified using the SYBR Green Real-Time PCR Master Mixes (ThermoFisher Scientific, Waltham, MA) on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). The relative expression levels of TFEB were calculated by the 2−ΔΔCt method. Primers used were: TFEB (forward 5-CATGTACTGTCCACCTCGG-3, reverse 5-TGTCCAGGCGCATAATGTTG-3) and GAPDH (forward 5-TGGAGAAACCTGCCAAGTATGAT-3, reverse 5-TCAAAGGTGGAAGAATGGGAGT-3).

Flow cytometry

The Annexin V-FITC/PI Apoptosis Detection Kit (C1062, Beyotime Biotechnology, Shanghai, China) was used to detect synovial cell apoptosis. The negative control siRNA or TFEB siRNA cells were cultivated in a six-well plate. After 24 h, the cells were treated with transfection complex for 24 h. The cells were then collected and resuspended by the Annexin V-FITC binding buffer. Annexin V-FITC was added and incubated for 10 min at room temperate, followed by centrifugation for 5 min at 200×g. Cells were then resuspended in a binding buffer containing PI. The Annexin V-FITC presented green fluorescence while PI showed red fluorescence by BD FACSVerseTM (BD Biosciences, CA, USA).

Statistics

Data were analyzed by SPSS 17.0 (SPSS Inc., IL, USA) and were expressed as mean ± standard deviation (SD). The Independent-Samples t-test was used to compare the difference between the two groups. A P value < 0.05 was considered as statistically significant.

Results

The pathological changes of RA rats

We established the RA model induced by collagen II in the rat. The overview of the model and the representative pictures of rat paws are shown in Fig. 1a, b. The ankle diameter, paw thickness, arthritis scores, and body weight were observed every week after the booster immunization. The results showed that the average value of the ankle diameter, paw thickness, and arthritis scores were higher in the RA group at all time points than in the NC group (Fig. 1c–e, and Table 1). However, the body weight in the RA group was lower than that in the NC group after primary immunization (Fig. 1f and Table 1). We then observed inflammatory levels of rats by detecting pro-inflammatory cytokines. As shown in the Fig. 1g, h, the levels of TNF-α (323.40 ± 44.72 vs. 265.99 ± 20.94 in the NC group) and IL-6 (180.0 ± 17.73 vs. 162.40 ± 6.74 in the NC group) significantly increased in RA rats. H&E staining results demonstrated infiltrated inflammatory cells, synovial fibroblast hyperplasia, the synovial membrane thickened, and the joint cavity narrowed in the synovial tissues of RA rats (Fig. 1i).

Fig. 1.

Fig. 1

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Pathological changes of rheumatoid arthritis (RA) rats. a The construction of the RA model of rats. b The foot swelling in rats at different time points. c–f The ankle diameter, paw thickness, arthritis score, and body weight of rats. g, h The expressions of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the sera of rats. i Hematoxylin–eosin staining of the synovial tissue of the rats. NC and RA represent the control and RA group, respectively (original magnification ×200). Data in g and h are presented as means ± SEM (n = 3, biological replications). *P < 0.05 vs. the NC group

Table 1.

Changes in ankle diameter, paw thickness, arthritis scores and body weight of NC rats and RA rats on day 1, 7, 14, 21, and 28 (mean ± standard deviation, n = 6)

RA NC
Ankle diameter 1 (mm) 10.11 ± 0.40 6.25 ± 0.19
Ankle diameter 7 (mm) 9.85 ± 0.69 7.64 ± 0.17
Ankle diameter 14 (mm) 11.64 ± 1.05 7.71 ± 0.09
Ankle diameter 21 (mm) 10.96 ± 0.65 7.52 ± 0.19
Ankle diameter 28 (mm) 10.53 ± 0.53 7.59 ± 0.15
Paw thickness 1 (mm) 9.00 ± 0.41 5.26 ± 0.13
Paw thickness 7 (mm) 8.24 ± 0.54 5.63 ± 0.13
Paw thickness 14 (mm) 9.96 ± 0.90 5.68 ± 0.06
Paw thickness 21 (mm) 9.08 ± 0.83 5.56 ± 0.06
Paw thickness 28 (mm) 8.46 ± 0.53 5.64 ± 0.05
Arthritis scores 1 0 ± 0 4 ± 0
Arthritis scores 7 0 ± 0 4 ± 0
Arthritis scores 14 0 ± 0 4 ± 0
Arthritis scores 21 0 ± 0 4 ± 0
Arthritis scores 28 0 ± 0 3.75 ± 0.46
Weight 1 (g) 312.00 ± 10.03 312.50 ± 5.21
Weight 7 (g) 350.13 ± 20.39 370.25 ± 7.50
Weight 14 (g) 363.88 ± 26.84 389.75 ± 7.17
Weight 21 (g) 392 ± 25.11 413.38 ± 12.21
Weight 28 (g) 402.88 ± 33.59 422.00 ± 10.61
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The significant changes of autophagy-related genes in RA rats

To clarify the effect of autophagy on RA, we detected expressions of autophagy markers by IHC. TFEB is an autophagy-related gene and TFEB up-regulation increases the levels of autophagy and lysosomal biogenesis (Miana et al. 2015; Cortes and La Spada 2019). The report showed that the TFEB activity is influenced by LC3B lipidation (Nakamura et al. 2020). LC3B, Beclin1, and p62 were important proteins that participated in the autophagy process. As shown in Fig. 2, expressions of TFEB and LC3B increased, whereas p62 decreased in the synovial tissues of RA rats, compared with NC rats. These results suggested that autophagy is significantly elevated in RA rats.

Fig. 2.

Fig. 2

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Changes of autophagy-related genes in RA rats. The expressions of transcription factor EB (TFEB), microtubule-associated proteins 1 light chain 3 (LC3B), and p62 in the synovial tissues by IHC (n = 3, biological replications). Original magnification ×150

TFEB affected the autophagy-related genes in the FLS of RA rats

Macrophage-like cells and FLS are final effector cells of a joint injury of RA (Zhou et al. 2020a, b). FLS, close to the articular cartilage, is frequently leveraged to reflect the status of RA rats. Autophagy participates in the migration and invasion of RA-FLS (Zhou et al. 2020a, b). Therefore, we speculated that TFEB genes control autophagy. To obtain the maximum transfection efficiency, synoviocytes were transfected with a transfection reagent containing si-TFEB 695, si-TFEB 773, and si-TFEB 1007. The result in Fig. 3a showed that the mRNA expression of TFEB is lowest in FLS while transfected with si-TFEB 773, in which the expression of TFEB was reduced up to 74%. RA-FLS in which the TFEB gene was stably knockdown, were used in the subsequent experiment. We found that the silencing of TFEB effectively suppressed the protein expressions of TFEB and LC3B (Fig. 3b–e), compared with siRNA NC. WB results revealed that TFEB and LC3B protein expressions decreased by 33.8% and 24.5% in TFEB siRNA group, respectively. These results revealed that decreased levels of autophagy were implicated in the silence of TFEB.

Fig. 3.

Fig. 3

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TFEB affected autophagy in the FLS of RA rats. a The transfection efficiency was observed by the mRNA expression of TFEB. qRT-PCR showed that si-TFEB773 was most effective and the expression of TFEB decreased by approximately 74%. b, d The protein expressions of TFEB and LC3B in the RA-fibroblast-like synoviocytes (RA-FLS) of the siRNA NC and TFEB siRNA groups were detected by western blot analysis. c, e Quantification of TFEB and LC3B in different groups revealed that the protein expressions of TFEB and LC3B reduced up to 33.8% and 24.5% in the TFEB siRNA group. Data were displayed as means ± SEM (n = 3, independent replications). *P < 0.05 vs. siRNA NC group

TFEB regulated inflammation, proliferation, and apoptosis of FLS of RA rats

In the progress of RA, the abnormal proliferation of FLS causes the hyperplasia of synovial tissues, leading to the growth of pannus and bone invasion. Meanwhile, pro-inflammatory cytokines, chemokines, and matrix Metalloproteinases (MMP) released by FLS lead to inflammatory response, bone erosion, cartilage degradation, and eventual joint destruction. In our study, a genetic knockdown (si-TFEB) in synoviocytes of RA rats increased the cell viability, compared with the negative control (Fig. 4a). Although there was no significant difference, the apoptosis of RA-FLS was decreased after TFEB knockdown (Fig. 4b). Moreover, the levels of pro-inflammatory cytokines TNF-α (280.20 ± 6.90 vs. 374.27 ± 3.61 in the siRNA NC group) and IL-6 (150.47 ± 0.41 vs. 193.03 ± 1.27 in the siRNA NC group) decreased as the expression of TFEB decreased (Fig. 4c, d). The secretions of TNF-α and IL-6 were reduced up to 25.1% and 22.3% in TFEB siRNA group, respectively. The results implied TFEB regulated the development of RA by altering the inflammatory response, proliferation and apoptosis of FLS.

Fig. 4.

Fig. 4

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TFEB affected inflammation, proliferation, and apoptosis of RA-FLS, which was transfected with TFEB siRNA or siRNA NC. a The growth curve of FLS was detected by Cell Counting Kit-8 (CCK8). The proliferation of the cells under TFEB siRNA was slightly higher than that in the siRNA NC group. b The apoptotic of FLS was observed by flow cytometry. c, d The secretions of TNF-α and IL-6 were detected by ELISA. The levels of TNF-α and IL-6 were reduced up to 25.1% and 22.3% in TFEB siRNA group, separately. Data were shown as means ± SEM in Fig. 3b–d (n = 3, independent replications). *P < 0.05 vs. the siRNA NC group

Discussion

Patients with RA suffer from joint pain, deformity swelling, and stiffness for a long time, which leads to serious social and personal economic burden because of its long-term treatment. Meanwhile, studies showed that autophagy is dysfunctional in RA (Guo et al. 2018). In this study, we described the role and underlying mechanism of TFEB in autophagy and RA. First, we established the RA model rats by injecting a type II collagen at the root of the tail, and found that TFEB changes in the synovium of RA rats. More importantly, we utilized the FLS isolated from RA rats to demonstrate the mechanism of TFEB. Our results revealed that TFEB participates in the development of RA by regulating autophagy, which may contribute to the treatment of RA. We found that arthropods, fester, and the arthritis index increased in the RA rat, and the levels of inflammation increased simultaneously.

Previous studies suggested that excessive activation of autophagy was responsible for the increase in citrullinated proteins, formation of osteoclast, and proliferation of lymphocytes in the RA synovium (Sorice et al. 2016; Vomero et al. 2018). Moreover, increased autophagy exacerbated the drug resistance of patients with RA (Xu et al. 2015). Nevertheless, suppressing autophagy improved the inflammation and synovial hyperplasia (Xu et al. 2013, 2015; Kato et al. 2014). Over 30 critical genes, including the autophagy-related gene (Atg), LC3B, Beclin-1 (Becn1), and p62, participated in regulation of different stages of autophagy (Cao et al. 2016). Monocytes of the mouse with arthritis, the expression of autophagy-related proteins, including LC3B, Atg7, and Becn1, increased in the bone marrow (Laha et al. 2019). In arthritic mice, the expression of LC3B was up-regulated (Jannat et al. 2019). In our research, we found that fester, the arthritis index, and inflammation levels increased in rats, which are the features of RA. The expression of LC3B simultaneously increased and p62 decreased in synovial tissues by IHC analysis, which validated that autophagy was involved in the progression of RA.

TFEB regulated lysosome biogenesis and autophagy-related genes. TFEB showed different biological processes in various illnesses. On one hand, the activation or the nuclear translocation of TFEB augmented the diseases. Nicotine facilitated the progression of atherosclerosis by activating autophagy-lysosomal machinery via suppressing the activity of mTORC1 and promoting nuclear translocation of TFEB (Ni et al. 2020). In the periodontitis RA mouse, RA promoted the expression of pro-inflammation cytokines and autophagy proteins (TFEB and LC3) (Wei et al. 2020). On the other hand, TFEB activation contributed to the treatment of autophagy-related illness. Remote ischemic postconditioning relieved the damage in rats with chronic cerebral ischemia by activating the autophagolysosome pathway mediated by TFEB (Li et al. 2020). The activation of the TFEB pathway induced autophagy, which attenuated the inflammation in human corneal epithelial cells elicited by hyperosmotic stress (Liu et al. 2020). The inhibition of the function of TFEB and lysosomes resulted in the death of the myocardial cell (Trivedi et al. 2020). Furthermore, the interaction of FLS and proinflammatory cytokines affects the interaction of FLS with immune and non-immune cells, resulting in the activation of immune cells, and the formation of autoantibody, which may play a central role in RA pathogenesis (Mor et al. 2005). Moreover, the expression of germinal center kinase-like kinase (GLK) significantly increased in autoimmune diseases, such as systemic lupus erythematosus and experimental autoimmune encephalomyelitis (Chuang et al. 2011). GLK regulated the phosphorylation of TFEB, and therefore influenced autophagy levels (Chuang and Tan 2019). These references revealed that TFEB plays a pivotal role in the progress of autoimmune diseases. In our study, the expression of TFEB was increased in the synovial tissues of RA rats by IHC. In RA-FLS, the silencing of TFEB decreased the expression of LC3B and pro-inflammatory cytokines, which likely represented that TFEB participated in the progress of RA via autophagy and was identified as a new therapeutic target.

In summary, our data link the function and mechanisms of TFEB to the inflammatory response and autophagy process of RA. These studies uncover the levels of TFEB, and autophagy increased in RA rats. We discovered that the silencing of TFEB inhibits inflammation, LC3 expression, and apoptosis of FLS in vitro. TFEB might participate in the progress of RA by regulating autophagy.

Conclusion

Our findings highlighted the unfavorable effect of TFEB in the progress of RA. Mechanistically, TFEB promoted worse inflammation and symptoms of RA by regulating autophagy. Our study provided further insight into the therapeutic targets of RA.

Authors’ contributions

DLX finished the experiment and wrote the manuscript. JP designed the experiment and revised the manuscript. The authors agree to publish this paper.

Funding

This work is supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJB310016), Suzhou Science Foundation (SYSD2019181), the Second Affiliated Hospital of Soochow University Science Foundation (SDFEYQN1720). We thank all the researchers involved in this project.

Data availability

All data and methods reported in the current study are accessed upon reasonable request.

Declarations

Conflict of interest

All authors declared that there was no conflict of interest.

Ethical approval

The authors declare that all of the actions were following the ethical requirements, including plagiarism, data fabrication and/or falsification, misconduct, double publication and/or submission, and so on.

References

  1. Bao J, Chen Z, Xu L, Wu L, Xiong Y. Rapamycin protects chondrocytes against IL-18-induced apoptosis and ameliorates rat osteoarthritis. Aging. 2020;12:5152–5167. doi: 10.18632/aging.102937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cao QH, Liu F, Yang ZL, Fu XH, Yang ZH, Liu Q, Wang L, Wan XB, Fan XJ. Prognostic value of autophagy related proteins ULK1, Beclin 1, ATG3, ATG5, ATG7, ATG9, ATG10, ATG12, LC3B and p62/SQSTM1 in gastric cancer. Am J Transl Res. 2016;8:3831–3847. [PMC free article] [PubMed] [Google Scholar]
  3. Chadha S, Behl T, Bungau S, Kumar A, Kaur R, Venkatachalam T, Gupta A, Kandhwal M, Chandel D. Focus on the multimodal role of autophagy in rheumatoid arthritis. Inflammation. 2020;44:1–12. doi: 10.1007/s10753-020-01324-8. [DOI] [PubMed] [Google Scholar]
  4. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013;368:651–662. doi: 10.1056/NEJMra1205406. [DOI] [PubMed] [Google Scholar]
  5. Chuang HC, Tan TH. MAP4K3/GLK in autoimmune disease, cancer and aging. J Biomed Sci. 2019;26:82. doi: 10.1186/s12929-019-0570-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chuang HC, Lan JL, Chen DY, Yang CY, Chen YM, Li JP, Huang CY, Liu PE, Wang X, Tan TH. The kinase GLK controls autoimmunity and NF-kappaB signaling by activating the kinase PKC-theta in T cells. Nat Immunol. 2011;12:1113–1118. doi: 10.1038/ni.2121. [DOI] [PubMed] [Google Scholar]
  7. Cortes CJ, La Spada AR. TFEB dysregulation as a driver of autophagy dysfunction in neurodegenerative disease: molecular mechanisms, cellular processes, and emerging therapeutic opportunities. Neurobiol Dis. 2019;122:83–93. doi: 10.1016/j.nbd.2018.05.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guo Q, Wang Y, Xu D, Nossent J, Pavlos NJ, Xu J. Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone Res. 2018;6:15. doi: 10.1038/s41413-018-0016-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. He Y, Xu Y, Zhang C, Gao X, Dykema KJ, Martin KR, Ke J, Hudson EA, Khoo SK, Resau JH, et al. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response. Sci Signal. 2011;4:ra44. doi: 10.1126/scisignal.4159ec44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holers VM, Banda NK. Complement in the initiation and evolution of rheumatoid arthritis. Front Immunol. 2018;9:1057. doi: 10.3389/fimmu.2018.01057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jannat A, John P, Bhatti A, Hayat MQ. Tomorou attenuates progression of rheumatoid arthritis through alteration in ULK-1 independent autophagy pathway in collagen induced arthritis mice model. Cell Death Discov. 2019;5:142. doi: 10.1038/s41420-019-0222-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kato M, Ospelt C, Gay RE, Gay S, Klein K. Dual role of autophagy in stress-induced cell death in rheumatoid arthritis synovial fibroblasts. Arthritis Rheumatol. 2014;66:40–48. doi: 10.1002/art.38190. [DOI] [PubMed] [Google Scholar]
  13. Kerlan-Candon S, Combe B, Vincent R, Clot J, Pinet V, Eliaou JF. HLA-DRB1 gene transcripts in rheumatoid arthritis. Clin Exp Immunol. 2001;124:142–149. doi: 10.1046/j.1365-2249.2001.01498.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laha D, Deb M, Das H. KLF2 (kruppel-like factor 2 [lung]) regulates osteoclastogenesis by modulating autophagy. Autophagy. 2019;15:2063–2075. doi: 10.1080/15548627.2019.1596491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lauzier A, Charbonneau M, Harper K, Jilaveanu-Pelmus M, Dubois CM. Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src. Arthritis Rheum. 2011;63:1591–1602. doi: 10.1002/art.30305. [DOI] [PubMed] [Google Scholar]
  16. Li S, Yu Y, Yue Y, Zhang Z, Su K. Microbial infection and rheumatoid arthritis. J Clin Cell Immunol. 2013;4:174. doi: 10.4172/2155-9899.1000174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li Z, Cui X, Lv H, Liu J, Di W, Jiang F, Liu Y, Cheng X. Remote ischemic postconditioning attenuates damage in rats with chronic cerebral ischemia by upregulating the autophagolysosome pathway via the activation of TFEB. Exp Mol Pathol. 2020;115:104475. doi: 10.1016/j.yexmp.2020.104475. [DOI] [PubMed] [Google Scholar]
  18. Liu W, Li CC, Lu X, Bo LY, Jin FG. Overexpression of transcription factor EB regulates mitochondrial autophagy to protect lipopolysaccharide-induced acute lung injury. Chin Med J. 2019;132:1298–1304. doi: 10.1097/CM9.0000000000000243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu Z, Chen D, Chen X, Bian F, Qin W, Gao N, Xiao Y, Li J, Pflugfelder SC, Li DQ. Trehalose induces autophagy against inflammation by activating TFEB signaling pathway in human corneal epithelial cells exposed to hyperosmotic stress. Invest Ophthalmol Vis Sci. 2020;61:26. doi: 10.1167/iovs.61.10.26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ma X, Godar RJ, Liu H, Diwan A. Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death. Autophagy. 2012;8:297–309. doi: 10.4161/auto.18658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miana M, Galan M, Martinez-Martinez E, Varona S, Jurado-Lopez R, Bausa-Miranda B, Antequera A, Luaces M, Martinez-Gonzalez J, Rodriguez C, et al. The lysyl oxidase inhibitor beta-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats. Dis Model Mech. 2015;8:543–551. doi: 10.1242/dmm.020107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mizushima N. Autophagy: process and function. Genes Dev. 2007;21:2861–2873. doi: 10.1101/gad.1599207. [DOI] [PubMed] [Google Scholar]
  23. Mor A, Abramson SB, Pillinger MH. The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clin Immunol. 2005;115:118–128. doi: 10.1016/j.clim.2004.12.009. [DOI] [PubMed] [Google Scholar]
  24. Nakamura S, Shigeyama S, Minami S, Shima T, Akayama S, Matsuda T, Esposito A, Napolitano G, Kuma A, Namba-Hamano T, et al. LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury. Nat Cell Biol. 2020;22:1252–1263. doi: 10.1038/s41556-020-00583-9. [DOI] [PubMed] [Google Scholar]
  25. Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol. 2008;183:795–803. doi: 10.1083/jcb.200809125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ni H, Xu S, Chen H, Dai Q. Nicotine modulates CTSS (Cathepsin S) synthesis and secretion through regulating the autophagy-lysosomal machinery in atherosclerosis. Arterioscler Thromb Vasc Biol. 2020;40:2054–2069. doi: 10.1161/ATVBAHA.120.314053. [DOI] [PubMed] [Google Scholar]
  27. Palmieri M, Impey S, Kang H, di Ronza A, Pelz C, Sardiello M, Ballabio A. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Hum Mol Genet. 2011;20:3852–3866. doi: 10.1093/hmg/ddr306. [DOI] [PubMed] [Google Scholar]
  28. Raben N, Puertollano R. TFEB and TFE3: linking lysosomes to cellular adaptation to stress. Annu Rev Cell Dev Biol. 2016;32:255–278. doi: 10.1146/annurev-cellbio-111315-125407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rajaei E, Mowla K, Ghorbani A, Bahadoram S, Bahadoram M, Dargahi-Malamir M. The effect of omega-3 fatty acids in patients with active rheumatoid arthritis receiving DMARDs therapy: double-blind randomized controlled trial. Glob J Health Sci. 2015;8:18–25. doi: 10.5539/gjhs.v8n7p18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Settembre C, Di Malta C, Polito VA, Garcia AM, Vetrini F, Erdin S, Erdin SU, Huynh T, Medina D, Colella P, et al. TFEB links autophagy to lysosomal biogenesis. Science. 2011;332:1429–1433. doi: 10.1126/science.1204592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sorice M, Iannuccelli C, Manganelli V, Capozzi A, Alessandri C, Lococo E, Garofalo T, Di Franco M, Bombardieri M, Nerviani A, et al. Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies. Rheumatology. 2016;55:1374–1385. doi: 10.1093/rheumatology/kew178. [DOI] [PubMed] [Google Scholar]
  32. Trivedi PC, Bartlett JJ, Mercer A, Slade L, Surette M, Ballabio A, Flibotte S, Hussein B, Rodrigues B, Kienesberger PC, et al. Loss of function of transcription factor EB remodels lipid metabolism and cell death pathways in the cardiomyocyte. Biochim Biophys Acta Mol Basis Dis. 2020;1866:165832. doi: 10.1016/j.bbadis.2020.165832. [DOI] [PubMed] [Google Scholar]
  33. Vomero M, Barbati C, Colasanti T, Perricone C, Novelli L, Ceccarelli F, Spinelli FR, Di Franco M, Conti F, Valesini G, et al. Autophagy and rheumatoid arthritis: current knowledges and future perspectives. Front Immunol. 2018;9:1577. doi: 10.3389/fimmu.2018.01577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang L, Zhu B, Huang J, Xiang X, Tang Y, Ma L, Yan F, Cheng C, Qiu L. Ultrasound-targeted microbubble destruction augmented synergistic therapy of rheumatoid arthritis via targeted liposomes. J Mater Chem B. 2020;8:5245–5256. doi: 10.1039/D0TB00430H. [DOI] [PubMed] [Google Scholar]
  35. Wei W, Ren J, Yin W, Ding H, Lu Q, Tan L, Deng S, Liu J, Yang Q, Wang J, et al. Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy. Cell Prolif. 2020;53:e12722. doi: 10.1111/cpr.12722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weiskirchen R, Tacke F. Relevance of autophagy in parenchymal and non-parenchymal liver cells for health and disease. Cells. 2019;8:16. doi: 10.3390/cells8010016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Xu K, Xu P, Yao JF, Zhang YG, Hou WK, Lu SM. Reduced apoptosis correlates with enhanced autophagy in synovial tissues of rheumatoid arthritis. Inflamm Res. 2013;62:229–237. doi: 10.1007/s00011-012-0572-1. [DOI] [PubMed] [Google Scholar]
  38. Xu K, Cai YS, Lu SM, Li XL, Liu L, Li Z, Liu H, Xu P. Autophagy induction contributes to the resistance to methotrexate treatment in rheumatoid arthritis fibroblast-like synovial cells through high mobility group box chromosomal protein 1. Arthritis Res Ther. 2015;17:374. doi: 10.1186/s13075-015-0892-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yang R, Zhang Y, Wang L, Hu J, Wen J, Xue L, Tang M, Liu Z, Fu J. Increased autophagy in fibroblast-like synoviocytes leads to immune enhancement potential in rheumatoid arthritis. Oncotarget. 2017;8:15420–15430. doi: 10.18632/oncotarget.14331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yu C, Li M, Duan X, Fang Y, Li Q, Wu R, Liu S, Wang Y, Wu Z, Shi X, et al. Chinese registry of rheumatoid arthritis (CREDIT): I. Introduction and prevalence of remission in Chinese patients with rheumatoid arthritis. Clin Exp Rheumatol. 2018;36:836–840. [PubMed] [Google Scholar]
  41. Zheng G, Zhan Y, Li X, Pan Z, Zheng F, Zhang Z, Zhou Y, Wu Y, Wang X, Gao W, et al. TFEB, a potential therapeutic target for osteoarthritis via autophagy regulation. Cell Death Dis. 2018;9:858. doi: 10.1038/s41419-018-0909-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhou DM, Ran F, Ni HZ, Sun LL, Xiao L, Li XQ, Li WD. Metformin inhibits high glucose-induced smooth muscle cell proliferation and migration. Aging. 2020;12:5352–5361. doi: 10.18632/aging.102955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhou W, Shen Q, Wang H, Yang J, Zhang C, Deng Z, Wu K, Zhou Y, Zeng J, Zhang Y, et al. Knockdown of YAP/TAZ inhibits the migration and invasion of fibroblast synovial cells in rheumatoid arthritis by regulating autophagy. J Immunol Res. 2020;2020:9510594. doi: 10.1155/2020/9510594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhu L, Wang H, Wu Y, He Z, Qin Y, Shen Q. The autophagy level is increased in the synovial tissues of patients with active rheumatoid arthritis and is correlated with disease severity. Mediators Inflamm. 2017;2017:7623145. doi: 10.1155/2017/7623145. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Data Availability Statement

All data and methods reported in the current study are accessed upon reasonable request.

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