Mol. Cells 2023; 46(4): 231-244
Published online January 10, 2023
https://doi.org/10.14348/molcells.2023.2136
© The Korean Society for Molecular and Cellular Biology
Correspondence to : 2017250322@stu.njmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Leucine-rich repeat containing 15 (LRRC15) has been identified as a contributing factor for cartilage damage in osteoarthritis; however, its involvement in rheumatoid arthritis (RA) and the underlying mechanisms have not been well characterized. The purpose of this study was to explore the function of LRRC15 in RA-associated fibroblast-like synoviocytes (RA-FLS) and in mice with collagen-induced arthritis (CIA) and to dissect the epigenetic mechanisms involved. LRRC15 was overexpressed in the synovial tissues of patients with RA, and LRRC15 overexpression was associated with increased proliferative, migratory, invasive, and angiogenic capacities of RA-FLS and accelerated release of pro-inflammatory cytokines. LRRC15 knockdown significantly inhibited synovial proliferation and reduced bone invasion and destruction in CIA mice. Runt-related transcription factor 1 (RUNX1) transcriptionally represses LRRC15 by binding to core-binding factor subunit beta (CBF-β). Overexpression of RUNX1 significantly inhibited the invasive phenotype of RA-FLS and suppressed the expression of proinflammatory cytokines. Conversely, the effects of RUNX1 were significantly reversed after overexpression of LRRC15 or inhibition of RUNX1-CBF-β interactions. Therefore, we demonstrated that RUNX1-mediated transcriptional repression of LRRC15 inhibited the development of RA, which may have therapeutic effects for RA patients.
Keywords epigenetic, fibroblast-like synoviocytes, LRRC15, rheumatoid arthritis, RUNX1
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that predominantly affects joints (Smolen et al., 2018). RA mainly affects synovial joints, with inflammation of the synovial membrane (synovitis) characterized by angiogenesis, hyperplasia of the lining layer, and immune cell infiltration that stimulates local inflammation and, if untreated, can result in joint destruction and disability (Rivellese and Pitzalis, 2021). Progress in therapeutic approaches and understanding of RA pathogenesis have improved patient prognosis and likely remission, while partial/non-response to conventional and biologic therapies in certain patients underlines the need for new therapeutics (Sandhu and Thelma, 2022). Fibroblast-like synoviocytes (FLS) in the synovial lining develop aggressive phenotypes and are a main focus within the RA field, and the proportion of the surface marker CD90 correlates positively with the proportion of synovitis and synovial hypertrophy (Wu et al., 2021).
In the present study, we identified leucine-rich repeat containing 15 (
This study was approved by the Institutional Review Board (IRB) of Jinling Hospital, Jinling Clinical Medical College of Nanjing Medical University (IRB No. 2022DZGZR-026). Written informed consent was obtained from all participants in compliance with the Declaration of Helsinki principles (2013). Synovial tissues were collected from 20 patients with RA who underwent knee synovectomy or total knee arthroplasty between May 2019 and March 2020. RA patients fulfilled the American College of Rheumatology criteria for RA (Aletaha et al., 2010). Control synovial tissues were obtained from 10 patients who underwent arthroscopic surgery for severe joint trauma and had no other joint abnormalities or systemic diseases.
Total RNA was extracted from cells and tissues using TRIGene Reagent (GenStar BioSolutions, China), and first-strand cDNA was synthesized using the extracted total RNA as a template and a PrimeScriptTM RT Reagent Kit (Takara Biotechnology, China). qPCR analysis was conducted using the FastStart Universal SYBR Green Master (Roche Applied Science, Germany) on an ABI7500 real-time fluorescence qPCR instrument. The 2-ΔΔCt method was used for normalization to glyceraldehyde-3-phosphate dehydrogenase (
Total protein was extracted from cells using RIPA Lysis Buffer (Phygene, China) and quantified using the BCA Protein Assay Kit (Phygene). Proteins were separated using 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against LRRC15 (1:1,000, ab150376; Abcam, UK), RUNX1 (1:1,000, PA5-85543; Thermo Fisher Scientific, USA), CBF-β (1:1,000, ab231345; Abcam), and GAPDH (1:2,000, #5174; Cell Signaling Technologies, USA). After that, the membranes were further incubated with horseradish peroxidase (HRP)-coupled secondary IgG antibody (1:10,000, ab6721; Abcam) for 120 min at room temperature. Protein bands were examined using an ECL substrate kit (Abcam), and all proteins were quantified based on the internal reference band GAPDH using ImageJ software.
Synovial tissues from RA patients and normal controls were dissected, rinsed 2-3 times with phosphate-buffered saline (PBS), and minced into approximately 1 mm3 pieces. Tissue fragments were transferred to flasks containing Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a 5% CO2 incubator. When the cells reached confluence (5-7 days), they were passaged. Cells were considered FLS when a typical spindle-shaped, fibroblast-like appearance was present and the positive rate for the surface marker CD90 (Manferdini et al., 2016; Zhang et al., 2021) was >90%. FLS at passages 3-6 were used for subsequent experiments.
The DNA overexpression plasmids oe-RUNX1 and oe-CBF-β based on pEXP-RB-Mam vector and a control plasmid were purchased from Guangzhou RiboBio (China). Short hairpin RNA (shRNA) targeting
CCK8 kits (GlpBio, USA) were used to assess the proliferative capacity of RA-FLS. The cells were plated in 96-well plates at 1 × 103 cells/well, and the plates were pre-incubated in a humidified incubator for designated times (0, 24, 48, and 72 h). Next, 10 μl of CCK8 solution was added to each well of the plate, and the plates were incubated for 2 h. Cell proliferation was determined by measuring the optical density at 450 nm using a microplate reader.
The BeyoClickTM EdU-594 Cell Proliferation Assay Kit (Beyotime Biotechnology, China) was used to detect DNA synthesis by RA-FLS. The cells were plated in 96-well plates at 1 × 105 cells/well and incubated for 120 min at room temperature with a 20 μM EdU working solution. The cells were fixed with 4% neutral paraformaldehyde and treated with 0.5% Triton X-100 and Click-iT reaction mixture for 0.5 h at room temperature in the dark. The cells were then stained with 5 μg/ml Hoechst 33342 for 15 min at room temperature in the dark, and staining was detected by confocal microscopy (Carl Zeiss, Germany).
HTS Transwell®-96 Permeable Support with an 8.0 μm Pore Polyester Membrane (Corning Glass Works, USA) was used to evaluate the migration and invasion of RA-FLS. When used for invasion assays, the membranes were precoated with CeturegelTM Matrix LDEV-Free Matrigel (Yeasen Biotechnology, China). RA-FLS (1 × 105) were resuspended in serum-free medium and placed in the apical chamber of the Transwell, and the basolateral chamber was supplemented with medium containing 10% FBS. After 24 h of normal culture, cells on the upper surface of the membrane were wiped off, and the cells that migrated or invaded the lower surface of the membrane were fixed with 4% neutral paraformaldehyde and stained with crystal violet. The number of cells was observed under a microscope (Olympus Optical, Japan) to measure their ability to migrate and invade.
HUVECs (1 × 104 cells/well; ATCC, USA) were seeded in 96-well plates coated with CeturegelTM Matrix LDEV-free Matrigel and incubated at 37°C for 1 h. The supernatant of RA-FLS after different treatments was added to the culture wells of HUVECs as conditioned medium. The 96-well plates were incubated for 6 h and photographed under an inverted microscope for tube formation visualization. Tube-forming capability was quantified by counting the total number of closed polygons formed.
Pro-inflammatory cytokine concentrations in RA-FLS were measured using human interleukin (IL)-6 (K4143) and IL-1β (E4818) ELISA kits (BioVision, USA), according to the manufacturer’s protocol. Concentrations of pro-inflammatory cytokines in mouse synovial tissues were measured using mouse IL-1β (ab197742; Abcam) and IL-6 (ab222503; Abcam) kits.
Thirty-five 8-week-old male DBA/1J mice were purchased from Beijing HFK Bioscience (China). The mice were fed a normal rodent diet and provided water
Thirty mice were induced with CIA, and the remaining five mice were injected with an equal volume of PBS as a control. In CIA mice, bovine type II collagen (CII; Chondrex, USA) was emulsified in an equal volume of complete Freund’s adjuvant (Chondrex). Each mouse was intradermally injected at the tail root with 100 μl of an emulsion containing 100 μg of CII. Twenty-one days after the initial immunization, the mice received a booster injection of 100 μg CII emulsified with an equal volume of incomplete Freund’s adjuvant (Chondrex). After the second immunization of CIA mice (day 21), 30 mice were divided into the following six groups (n = 5 each): CIA, adeno-associated viruses (AAV)-NC, AAV-shLRRC15, AAV-RUNX1, AAV-RUNX1 + AAV-LRRC15, and AAV-RUNX1 + RO5-3335 groups.
The AAV used in animal experiments was purchased from VectorBuilder and had a virus titer of 2 × 1011 GC/ml. After the second immunization of CIA mice (day 21), each mouse subjected to AAV treatment was administered 50 μl of each AAV through the tail vein. Mice undergoing RO5-3335 treatment were administered 300 mg/kg/day RO5-3335 for 30 days starting on day 21.
The severity of arthritis was scored every seven days from day 21 to day 56. Arthritis was monitored using the Arthritis Index scoring system described as follows (Bevaart et al., 2010): 0, normal; 1, swelling of one joint (wrist/ankle or digit); 2, swelling of > two joints; 3, swelling of all joints; and 4, swelling of all joints and ankylosis. A maximum score of 4 was reached per paw, resulting in a maximum score of 16 per mouse. At the end of the experiment (day 56), all mice were euthanized by sodium pentobarbital overdose (150 mg/kg, intraperitoneally), and bone, joint, and synovial tissues were harvested.
The tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Then, 5-μm sections were cut using a microtome (Leica Microsystems, Germany), dewaxed, dehydrated, and subjected to antigen retrieval. Subsequently, endogenous peroxidase was removed using 3% H2O2, and the tissue sections were incubated overnight at 4°C with primary antibodies against LRRC15 (1:200, M041540; Abmart Shanghai, China), CBF-β (1:200, ab231345; Abcam), and RUNX1 (1:1,000, PA5-85543; Thermo Fisher Scientific). The sections were incubated with HRP-coupled secondary IgG antibody (1:2,000, ab6721; Abcam) for 60 min at room temperature and stained with DAB reagent. After counterstaining with hematoxylin, the sections were photographed under a microscope and the tan-positive stained areas of each section were analyzed using ImageJ.
For H&E and Safranin O-fast green staining, paraffin-embedded sections were dewaxed, sequentially dehydrated, and stained using standard protocols (Beijing Solarbio Life Sciences, China). Finally, photographs were obtained under a light microscope to analyze pathological changes involving the mouse tissues.
The ability of RUNX1 to enrich the
The
Total protein in the cells was extracted using RIPA Lysis Buffer (Phygene) and centrifuged at 14,000 ×
Statistical analyses were performed using Prism 8.0 (Graphpad Software, USA). All results are presented as mean ± SD of at least three experiments. Statistical significance was determined using unpaired Student’s
The dataset GSE77298, containing 16 microarrays of end-stage RA synovial biopsies and 7 microarrays of synovial biopsies from individuals without joint disease, was selected from the GEO database to screen for differentially expressed genes in RA (Fig. 1A). Among them,
We isolated FLS from synovial tissues of patients with RA and controls (named RA-FLS and Control-FLS therein). We detected a much higher expression of LRRC15 in RA-FLS than in Control-FLS using RT-qPCR and western blotting (Figs. 2A and 2B). Subsequently, we transfected three shRNAs of
Remarkably, we observed a reduction in the migratory and invasive abilities of RA-FLS in the presence of sh-
RA mouse models were induced by CIA, and our preliminary experiments confirmed that an AAV titer of 2 × 1011 GC/ml was able to successfully infect the lung, liver, and synovial tissues of mice after 30 days (Supplementary Fig. S1). CIA mice were treated with AAV-shLRRC15 or AAV-NC (Fig. 4A). We observed a significant increase in the arthritis score in CIA mice compared to that in control mice, while AAV-shLRRC15 treatment reduced the arthritis score in CIA mice (Fig. 4B). The synovial tissues of CIA mice exhibited significantly enhanced expression of LRRC15, whereas AAV-shLRRC15 treatment decreased LRRC15 expression in the synovial tissues of mice, as evidenced by immunohistochemistry (Fig. 4C). Elevated levels of IL-6 and IL-1β in the synovial tissues of CIA mice were detected by ELISA, whereas the levels of these pro-inflammatory cytokines were significantly reduced after inhibition of LRRC15 (Fig. 4D). H&E staining of CIA mice showed pathological changes, including synovial hyperplasia and pannus formation, which were alleviated by inhibition of LRRC15 (Fig. 4E). We found that CIA mice exhibited erosion and destruction of articular bone and cartilage by staining with Safranin O-fast green, and loss of LRRC15 ameliorated such pathological changes in tissues (Fig. 4F).
To investigate the causes of LRRC15 overexpression in RA synovial tissues, we screened for upstream transcription factors responsible for the regulation of LRRC15 in hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/) and performed pathway enrichment analysis (Fig. 5A). Among the top-three enriched pathways, there was only one intersection, RUNX1 (Fig. 5B).
In the GSE77298 dataset, RUNX1 (GPL570 annotation platform No. 1563591_at) expression was downregulated in RA synovial tissues, but the difference was not statistically significant (Fig. 5C). We speculated that individual differences in patients with RA resulted in the generation of two extreme points that deviated far from the mean. Poor expression of RUNX1 was also determined in RA-FLS by RT-qPCR and western blotting (Figs. 5D and 5E). Using the ChIP-seq database Cistrome Data Browser (http://cistrome.org/db/#/), we found that there was a binding peak between RUNX1 and the
An overexpression DNA plasmid of
Further immunohistochemical staining and western blotting showed that CBF-β was slightly upregulated in RA synovial tissues and RA-FLS (Supplementary Figs. S2A and S2B). We upregulated CBF-β in RA-FLS by transfection with DNA overexpression plasmids, and we observed a decrease in LRRC15 expression upon overexpression of CBF-β using RT-qPCR and western blotting, indicating that CBF-β regulation can also affect LRRC15 expression (Supplementary Figs. S2C and S2D). Decreased protein content in immunoprecipitation experiments was detected using a Co-IP assay, indicating diminished interactions between RUNX1 and CBF-β in RA-FLS relative to Control-FLS (Supplementary Fig. S2E). It is worth noting that, considering the basal expression of RUNX1 and CBF-β in RA (only RUNX1 expression was significantly reduced in RA), the elevated LRRC15 expression and reduced interaction between RUNX1 and CBF-β in RA were mainly affected by the downregulation of RUNX1. The direct binding site of RUNX1 on the
We transfected the overexpression DNA plasmid of
CIA mice were further treated with AAV-RUNX1 in combination with RO5-3335 or AAV-LRRC15 (Fig. 7A). We observed a significant decrease in arthritis scores in AAV-RUNX1 mice compared to that in AAC-NC mice, whereas the administration of RO5-3335 or AAV-LRRC15 resulted in increased arthritis scores in mice (Fig. 7B). The expression of RUNX1 and LRRC15 was detected using immunohistochemistry (Fig. 7C). A significant increase in RUNX1 expression and a significant decline in LRRC15 expression were observed in the synovial tissues of AAV-RUNX1-administrated mice. The application of RO5-3335 or AAV-LRRC15 did not affect RUNX1 expression but significantly increased LRRC15 expression in mouse synovial tissues. Overexpression of RUNX1 also decreased IL-6 and IL-1β levels in mouse synovial tissues as detected by ELISA, while RO5-3335 or AAV-LRRC15 treatment restored the levels of pro-inflammatory cytokines (Fig. 7D). H&E staining showed that the inhibitory effect of RUNX1 on synovial hyperplasia and pannus formation was significantly reversed by RO5-3335 or AAV-LRRC15 treatment (Fig. 7E). By staining with Safranin O-fast green, we found that RUNX1 ameliorated the erosion and destruction of articular bone and cartilage in mice, whereas RO5-3335 and AAV-LRRC15 reversed this protective effect of RUNX1 on bone tissues (Fig. 7F).
LRRC15 is involved in cell-cell and cell-matrix interactions and has been recognized as a promising anti-cancer target because of its overexpression in mesenchymal-derived tumors and cancer-associated fibroblasts in breast, head and neck, lung, and pancreatic cancers (Ray et al., 2022). To elucidate potential regulators participating in the pathogenesis of RA, we screened the GEO database and found that LRRC15 levels were much higher in synovial tissues from RA patients than in normal controls. We demonstrated that LRRC15 deficiency suppressed RA-FLS proliferation and inflammation
Classically, epigenetic mechanisms describe chemical modifications of the histone tails or directly of DNA, thereby determining which genes in a cell are expressed or silenced (Ospelt et al., 2017). Dysregulation of transcription factors and co-factors may lead to bone deformities and bone mass disorders, and understanding the mechanisms involved in the modulation of these factors may provide solutions for treating bone diseases (Chan et al., 2021). To dissect the upstream mechanism of LRRC15 overexpression in RA, we conducted a series of bioinformatic predictions. It was thus identified that RUNX1 directly binds to the
In summary, our study convincingly demonstrated that LRRC15 loss, mediated by the RUNX1/CBF-β complex, can ameliorate RA by reducing the aggressiveness of FLS, inducing pro-inflammatory cytokine secretion, synovial hyperplasia, angiogenesis, cartilage erosion, and bone destruction (Fig. 8). Further investigations are required to determine the downstream effectors of LRRC15 in RA.
H.D., X.M., and J.Z. conceived and performed experiments, wrote the manuscript, and secured funding. P.F. and L.L. performed experiments. T.G. provided expertise and feedback.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2023; 46(4): 231-244
Published online April 30, 2023 https://doi.org/10.14348/molcells.2023.2136
Copyright © The Korean Society for Molecular and Cellular Biology.
Hao Ding1,3 , Xiaoliang Mei2,3
, Lintao Li1
, Peng Fang1
, Ting Guo1
, and Jianning Zhao1,*
1Department of Orthopedics, Jinling Hospital, Jinling Clinical Medical College of Nanjing Medical University, Nanjing 211166, China, 2Department of Orthopedics, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou 225300, China, 3These authors contributed equally to this work.
Correspondence to:2017250322@stu.njmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Leucine-rich repeat containing 15 (LRRC15) has been identified as a contributing factor for cartilage damage in osteoarthritis; however, its involvement in rheumatoid arthritis (RA) and the underlying mechanisms have not been well characterized. The purpose of this study was to explore the function of LRRC15 in RA-associated fibroblast-like synoviocytes (RA-FLS) and in mice with collagen-induced arthritis (CIA) and to dissect the epigenetic mechanisms involved. LRRC15 was overexpressed in the synovial tissues of patients with RA, and LRRC15 overexpression was associated with increased proliferative, migratory, invasive, and angiogenic capacities of RA-FLS and accelerated release of pro-inflammatory cytokines. LRRC15 knockdown significantly inhibited synovial proliferation and reduced bone invasion and destruction in CIA mice. Runt-related transcription factor 1 (RUNX1) transcriptionally represses LRRC15 by binding to core-binding factor subunit beta (CBF-β). Overexpression of RUNX1 significantly inhibited the invasive phenotype of RA-FLS and suppressed the expression of proinflammatory cytokines. Conversely, the effects of RUNX1 were significantly reversed after overexpression of LRRC15 or inhibition of RUNX1-CBF-β interactions. Therefore, we demonstrated that RUNX1-mediated transcriptional repression of LRRC15 inhibited the development of RA, which may have therapeutic effects for RA patients.
Keywords: epigenetic, fibroblast-like synoviocytes, LRRC15, rheumatoid arthritis, RUNX1
Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that predominantly affects joints (Smolen et al., 2018). RA mainly affects synovial joints, with inflammation of the synovial membrane (synovitis) characterized by angiogenesis, hyperplasia of the lining layer, and immune cell infiltration that stimulates local inflammation and, if untreated, can result in joint destruction and disability (Rivellese and Pitzalis, 2021). Progress in therapeutic approaches and understanding of RA pathogenesis have improved patient prognosis and likely remission, while partial/non-response to conventional and biologic therapies in certain patients underlines the need for new therapeutics (Sandhu and Thelma, 2022). Fibroblast-like synoviocytes (FLS) in the synovial lining develop aggressive phenotypes and are a main focus within the RA field, and the proportion of the surface marker CD90 correlates positively with the proportion of synovitis and synovial hypertrophy (Wu et al., 2021).
In the present study, we identified leucine-rich repeat containing 15 (
This study was approved by the Institutional Review Board (IRB) of Jinling Hospital, Jinling Clinical Medical College of Nanjing Medical University (IRB No. 2022DZGZR-026). Written informed consent was obtained from all participants in compliance with the Declaration of Helsinki principles (2013). Synovial tissues were collected from 20 patients with RA who underwent knee synovectomy or total knee arthroplasty between May 2019 and March 2020. RA patients fulfilled the American College of Rheumatology criteria for RA (Aletaha et al., 2010). Control synovial tissues were obtained from 10 patients who underwent arthroscopic surgery for severe joint trauma and had no other joint abnormalities or systemic diseases.
Total RNA was extracted from cells and tissues using TRIGene Reagent (GenStar BioSolutions, China), and first-strand cDNA was synthesized using the extracted total RNA as a template and a PrimeScriptTM RT Reagent Kit (Takara Biotechnology, China). qPCR analysis was conducted using the FastStart Universal SYBR Green Master (Roche Applied Science, Germany) on an ABI7500 real-time fluorescence qPCR instrument. The 2-ΔΔCt method was used for normalization to glyceraldehyde-3-phosphate dehydrogenase (
Total protein was extracted from cells using RIPA Lysis Buffer (Phygene, China) and quantified using the BCA Protein Assay Kit (Phygene). Proteins were separated using 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature and incubated overnight at 4°C with primary antibodies against LRRC15 (1:1,000, ab150376; Abcam, UK), RUNX1 (1:1,000, PA5-85543; Thermo Fisher Scientific, USA), CBF-β (1:1,000, ab231345; Abcam), and GAPDH (1:2,000, #5174; Cell Signaling Technologies, USA). After that, the membranes were further incubated with horseradish peroxidase (HRP)-coupled secondary IgG antibody (1:10,000, ab6721; Abcam) for 120 min at room temperature. Protein bands were examined using an ECL substrate kit (Abcam), and all proteins were quantified based on the internal reference band GAPDH using ImageJ software.
Synovial tissues from RA patients and normal controls were dissected, rinsed 2-3 times with phosphate-buffered saline (PBS), and minced into approximately 1 mm3 pieces. Tissue fragments were transferred to flasks containing Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a 5% CO2 incubator. When the cells reached confluence (5-7 days), they were passaged. Cells were considered FLS when a typical spindle-shaped, fibroblast-like appearance was present and the positive rate for the surface marker CD90 (Manferdini et al., 2016; Zhang et al., 2021) was >90%. FLS at passages 3-6 were used for subsequent experiments.
The DNA overexpression plasmids oe-RUNX1 and oe-CBF-β based on pEXP-RB-Mam vector and a control plasmid were purchased from Guangzhou RiboBio (China). Short hairpin RNA (shRNA) targeting
CCK8 kits (GlpBio, USA) were used to assess the proliferative capacity of RA-FLS. The cells were plated in 96-well plates at 1 × 103 cells/well, and the plates were pre-incubated in a humidified incubator for designated times (0, 24, 48, and 72 h). Next, 10 μl of CCK8 solution was added to each well of the plate, and the plates were incubated for 2 h. Cell proliferation was determined by measuring the optical density at 450 nm using a microplate reader.
The BeyoClickTM EdU-594 Cell Proliferation Assay Kit (Beyotime Biotechnology, China) was used to detect DNA synthesis by RA-FLS. The cells were plated in 96-well plates at 1 × 105 cells/well and incubated for 120 min at room temperature with a 20 μM EdU working solution. The cells were fixed with 4% neutral paraformaldehyde and treated with 0.5% Triton X-100 and Click-iT reaction mixture for 0.5 h at room temperature in the dark. The cells were then stained with 5 μg/ml Hoechst 33342 for 15 min at room temperature in the dark, and staining was detected by confocal microscopy (Carl Zeiss, Germany).
HTS Transwell®-96 Permeable Support with an 8.0 μm Pore Polyester Membrane (Corning Glass Works, USA) was used to evaluate the migration and invasion of RA-FLS. When used for invasion assays, the membranes were precoated with CeturegelTM Matrix LDEV-Free Matrigel (Yeasen Biotechnology, China). RA-FLS (1 × 105) were resuspended in serum-free medium and placed in the apical chamber of the Transwell, and the basolateral chamber was supplemented with medium containing 10% FBS. After 24 h of normal culture, cells on the upper surface of the membrane were wiped off, and the cells that migrated or invaded the lower surface of the membrane were fixed with 4% neutral paraformaldehyde and stained with crystal violet. The number of cells was observed under a microscope (Olympus Optical, Japan) to measure their ability to migrate and invade.
HUVECs (1 × 104 cells/well; ATCC, USA) were seeded in 96-well plates coated with CeturegelTM Matrix LDEV-free Matrigel and incubated at 37°C for 1 h. The supernatant of RA-FLS after different treatments was added to the culture wells of HUVECs as conditioned medium. The 96-well plates were incubated for 6 h and photographed under an inverted microscope for tube formation visualization. Tube-forming capability was quantified by counting the total number of closed polygons formed.
Pro-inflammatory cytokine concentrations in RA-FLS were measured using human interleukin (IL)-6 (K4143) and IL-1β (E4818) ELISA kits (BioVision, USA), according to the manufacturer’s protocol. Concentrations of pro-inflammatory cytokines in mouse synovial tissues were measured using mouse IL-1β (ab197742; Abcam) and IL-6 (ab222503; Abcam) kits.
Thirty-five 8-week-old male DBA/1J mice were purchased from Beijing HFK Bioscience (China). The mice were fed a normal rodent diet and provided water
Thirty mice were induced with CIA, and the remaining five mice were injected with an equal volume of PBS as a control. In CIA mice, bovine type II collagen (CII; Chondrex, USA) was emulsified in an equal volume of complete Freund’s adjuvant (Chondrex). Each mouse was intradermally injected at the tail root with 100 μl of an emulsion containing 100 μg of CII. Twenty-one days after the initial immunization, the mice received a booster injection of 100 μg CII emulsified with an equal volume of incomplete Freund’s adjuvant (Chondrex). After the second immunization of CIA mice (day 21), 30 mice were divided into the following six groups (n = 5 each): CIA, adeno-associated viruses (AAV)-NC, AAV-shLRRC15, AAV-RUNX1, AAV-RUNX1 + AAV-LRRC15, and AAV-RUNX1 + RO5-3335 groups.
The AAV used in animal experiments was purchased from VectorBuilder and had a virus titer of 2 × 1011 GC/ml. After the second immunization of CIA mice (day 21), each mouse subjected to AAV treatment was administered 50 μl of each AAV through the tail vein. Mice undergoing RO5-3335 treatment were administered 300 mg/kg/day RO5-3335 for 30 days starting on day 21.
The severity of arthritis was scored every seven days from day 21 to day 56. Arthritis was monitored using the Arthritis Index scoring system described as follows (Bevaart et al., 2010): 0, normal; 1, swelling of one joint (wrist/ankle or digit); 2, swelling of > two joints; 3, swelling of all joints; and 4, swelling of all joints and ankylosis. A maximum score of 4 was reached per paw, resulting in a maximum score of 16 per mouse. At the end of the experiment (day 56), all mice were euthanized by sodium pentobarbital overdose (150 mg/kg, intraperitoneally), and bone, joint, and synovial tissues were harvested.
The tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Then, 5-μm sections were cut using a microtome (Leica Microsystems, Germany), dewaxed, dehydrated, and subjected to antigen retrieval. Subsequently, endogenous peroxidase was removed using 3% H2O2, and the tissue sections were incubated overnight at 4°C with primary antibodies against LRRC15 (1:200, M041540; Abmart Shanghai, China), CBF-β (1:200, ab231345; Abcam), and RUNX1 (1:1,000, PA5-85543; Thermo Fisher Scientific). The sections were incubated with HRP-coupled secondary IgG antibody (1:2,000, ab6721; Abcam) for 60 min at room temperature and stained with DAB reagent. After counterstaining with hematoxylin, the sections were photographed under a microscope and the tan-positive stained areas of each section were analyzed using ImageJ.
For H&E and Safranin O-fast green staining, paraffin-embedded sections were dewaxed, sequentially dehydrated, and stained using standard protocols (Beijing Solarbio Life Sciences, China). Finally, photographs were obtained under a light microscope to analyze pathological changes involving the mouse tissues.
The ability of RUNX1 to enrich the
The
Total protein in the cells was extracted using RIPA Lysis Buffer (Phygene) and centrifuged at 14,000 ×
Statistical analyses were performed using Prism 8.0 (Graphpad Software, USA). All results are presented as mean ± SD of at least three experiments. Statistical significance was determined using unpaired Student’s
The dataset GSE77298, containing 16 microarrays of end-stage RA synovial biopsies and 7 microarrays of synovial biopsies from individuals without joint disease, was selected from the GEO database to screen for differentially expressed genes in RA (Fig. 1A). Among them,
We isolated FLS from synovial tissues of patients with RA and controls (named RA-FLS and Control-FLS therein). We detected a much higher expression of LRRC15 in RA-FLS than in Control-FLS using RT-qPCR and western blotting (Figs. 2A and 2B). Subsequently, we transfected three shRNAs of
Remarkably, we observed a reduction in the migratory and invasive abilities of RA-FLS in the presence of sh-
RA mouse models were induced by CIA, and our preliminary experiments confirmed that an AAV titer of 2 × 1011 GC/ml was able to successfully infect the lung, liver, and synovial tissues of mice after 30 days (Supplementary Fig. S1). CIA mice were treated with AAV-shLRRC15 or AAV-NC (Fig. 4A). We observed a significant increase in the arthritis score in CIA mice compared to that in control mice, while AAV-shLRRC15 treatment reduced the arthritis score in CIA mice (Fig. 4B). The synovial tissues of CIA mice exhibited significantly enhanced expression of LRRC15, whereas AAV-shLRRC15 treatment decreased LRRC15 expression in the synovial tissues of mice, as evidenced by immunohistochemistry (Fig. 4C). Elevated levels of IL-6 and IL-1β in the synovial tissues of CIA mice were detected by ELISA, whereas the levels of these pro-inflammatory cytokines were significantly reduced after inhibition of LRRC15 (Fig. 4D). H&E staining of CIA mice showed pathological changes, including synovial hyperplasia and pannus formation, which were alleviated by inhibition of LRRC15 (Fig. 4E). We found that CIA mice exhibited erosion and destruction of articular bone and cartilage by staining with Safranin O-fast green, and loss of LRRC15 ameliorated such pathological changes in tissues (Fig. 4F).
To investigate the causes of LRRC15 overexpression in RA synovial tissues, we screened for upstream transcription factors responsible for the regulation of LRRC15 in hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget/#!/) and performed pathway enrichment analysis (Fig. 5A). Among the top-three enriched pathways, there was only one intersection, RUNX1 (Fig. 5B).
In the GSE77298 dataset, RUNX1 (GPL570 annotation platform No. 1563591_at) expression was downregulated in RA synovial tissues, but the difference was not statistically significant (Fig. 5C). We speculated that individual differences in patients with RA resulted in the generation of two extreme points that deviated far from the mean. Poor expression of RUNX1 was also determined in RA-FLS by RT-qPCR and western blotting (Figs. 5D and 5E). Using the ChIP-seq database Cistrome Data Browser (http://cistrome.org/db/#/), we found that there was a binding peak between RUNX1 and the
An overexpression DNA plasmid of
Further immunohistochemical staining and western blotting showed that CBF-β was slightly upregulated in RA synovial tissues and RA-FLS (Supplementary Figs. S2A and S2B). We upregulated CBF-β in RA-FLS by transfection with DNA overexpression plasmids, and we observed a decrease in LRRC15 expression upon overexpression of CBF-β using RT-qPCR and western blotting, indicating that CBF-β regulation can also affect LRRC15 expression (Supplementary Figs. S2C and S2D). Decreased protein content in immunoprecipitation experiments was detected using a Co-IP assay, indicating diminished interactions between RUNX1 and CBF-β in RA-FLS relative to Control-FLS (Supplementary Fig. S2E). It is worth noting that, considering the basal expression of RUNX1 and CBF-β in RA (only RUNX1 expression was significantly reduced in RA), the elevated LRRC15 expression and reduced interaction between RUNX1 and CBF-β in RA were mainly affected by the downregulation of RUNX1. The direct binding site of RUNX1 on the
We transfected the overexpression DNA plasmid of
CIA mice were further treated with AAV-RUNX1 in combination with RO5-3335 or AAV-LRRC15 (Fig. 7A). We observed a significant decrease in arthritis scores in AAV-RUNX1 mice compared to that in AAC-NC mice, whereas the administration of RO5-3335 or AAV-LRRC15 resulted in increased arthritis scores in mice (Fig. 7B). The expression of RUNX1 and LRRC15 was detected using immunohistochemistry (Fig. 7C). A significant increase in RUNX1 expression and a significant decline in LRRC15 expression were observed in the synovial tissues of AAV-RUNX1-administrated mice. The application of RO5-3335 or AAV-LRRC15 did not affect RUNX1 expression but significantly increased LRRC15 expression in mouse synovial tissues. Overexpression of RUNX1 also decreased IL-6 and IL-1β levels in mouse synovial tissues as detected by ELISA, while RO5-3335 or AAV-LRRC15 treatment restored the levels of pro-inflammatory cytokines (Fig. 7D). H&E staining showed that the inhibitory effect of RUNX1 on synovial hyperplasia and pannus formation was significantly reversed by RO5-3335 or AAV-LRRC15 treatment (Fig. 7E). By staining with Safranin O-fast green, we found that RUNX1 ameliorated the erosion and destruction of articular bone and cartilage in mice, whereas RO5-3335 and AAV-LRRC15 reversed this protective effect of RUNX1 on bone tissues (Fig. 7F).
LRRC15 is involved in cell-cell and cell-matrix interactions and has been recognized as a promising anti-cancer target because of its overexpression in mesenchymal-derived tumors and cancer-associated fibroblasts in breast, head and neck, lung, and pancreatic cancers (Ray et al., 2022). To elucidate potential regulators participating in the pathogenesis of RA, we screened the GEO database and found that LRRC15 levels were much higher in synovial tissues from RA patients than in normal controls. We demonstrated that LRRC15 deficiency suppressed RA-FLS proliferation and inflammation
Classically, epigenetic mechanisms describe chemical modifications of the histone tails or directly of DNA, thereby determining which genes in a cell are expressed or silenced (Ospelt et al., 2017). Dysregulation of transcription factors and co-factors may lead to bone deformities and bone mass disorders, and understanding the mechanisms involved in the modulation of these factors may provide solutions for treating bone diseases (Chan et al., 2021). To dissect the upstream mechanism of LRRC15 overexpression in RA, we conducted a series of bioinformatic predictions. It was thus identified that RUNX1 directly binds to the
In summary, our study convincingly demonstrated that LRRC15 loss, mediated by the RUNX1/CBF-β complex, can ameliorate RA by reducing the aggressiveness of FLS, inducing pro-inflammatory cytokine secretion, synovial hyperplasia, angiogenesis, cartilage erosion, and bone destruction (Fig. 8). Further investigations are required to determine the downstream effectors of LRRC15 in RA.
H.D., X.M., and J.Z. conceived and performed experiments, wrote the manuscript, and secured funding. P.F. and L.L. performed experiments. T.G. provided expertise and feedback.
The authors have no potential conflicts of interest to disclose.
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