Abdalkhalig Elkhider1, Jing Wei1, Mahmoud Al-Azab1, Yawei Tang1, Williams Walana1, Weiping Li1, Bo Yuan1, Yunshan Ye1, Guan Wang1, Yan Zhang2, Xia Li3. 1. Department of Immunology, College of Basic Medical Science, Dalian Medical University, Liaoning, China. 2. Department of Rheumatology and Immunology, The Second Affiliated Hospital of Dalian Medical University, Liaoning, China. zhangy1971@163.com. 3. Department of Immunology, College of Basic Medical Science, Dalian Medical University, Liaoning, China. lixia0416@dlmedu.edu.cn.
Abstract
OBJECTIVES: The interface between pro-inflammatory cytokines and rheumatoid synovial fibroblast (sFLS) has central effects on rheumatoid arthritis (RA). The present study aimed to explore the role of IL-34 expression as one of major cytokine implicated in RA. METHODS: We examined the expression of IL-34 after RA sFLS stimulated by IL-1β and TGF-β1 separately by reverse transcription polymerase chain reaction (RT-PCR). Transwell and wound closure techniques were used to detect whether IL-34 is involved in promoting cell migration. Cellular viability was determined via CCK-8 and cultural morphology assays between IL-34 downregulated group and non-transfected counterpart. We also tested the expression of VEGF gene with RT-PCR analysis and activation of the major signalling pathways by western blot in IL-34 down-regulated group, IL-1β or TGF-β1 treated groups. Propidium iodide (PI) staining and fluoresceine isothiocyanate (FITC) Annexin V and propidium iodide apoptosis assay were used to analyse cell cycle arrest and apoptosis separately in IL-34 down-regulated cells. RESULTS: We found that IL-1β significantly enhanced IL-34 expression, while contrarily, TGF-β1 restrained IL-34 gene expression. Transwell and wound closure techniques showed that IL-34 was involved considerably in promoting cell migration. However, IL-34 knock-down restricted sFLS migration possibly through the diminishing of MMP2 and MMP9 expression. Interestingly, IL-34 down-regulated cells exhibited significantly low cellular viability compared with the non-transfected counterpart via CCK-8 and cultural morphology assays. We found that IL-34 down-regulated cells have low VEGF gene expression compared with treated cells. PI staining showed a G0/G1 cell cycle arrest in IL-34 down-regulated cells. FITC Annexin V and propidium iodide apoptosis assay verified that IL-34 down-regulated cells induced massive apoptosis through apoptotic signalling caspase3, while IL-1β treated cells presented termination of cellular apoptosis signalled by BCL-2. Furthermore, we observed IL-34 induced activation of ERK1/2 and AKT pathways while IL-34 down-regulation significantly decreased the activation of these pathways. CONCLUSIONS: Our data add novel insights into the pathogenesis of RA and we suggest that IL-34 plays a dominant role in controlling migration and proliferation of sFLS. Consequently, therapeutic strategies targeting IL-34 could be a potent therapy for RA.
OBJECTIVES: The interface between pro-inflammatory cytokines and rheumatoid synovial fibroblast (sFLS) has central effects on rheumatoid arthritis (RA). The present study aimed to explore the role of IL-34 expression as one of major cytokine implicated in RA. METHODS: We examined the expression of IL-34 after RA sFLS stimulated by IL-1β and TGF-β1 separately by reverse transcription polymerase chain reaction (RT-PCR). Transwell and wound closure techniques were used to detect whether IL-34 is involved in promoting cell migration. Cellular viability was determined via CCK-8 and cultural morphology assays between IL-34 downregulated group and non-transfected counterpart. We also tested the expression of VEGF gene with RT-PCR analysis and activation of the major signalling pathways by western blot in IL-34 down-regulated group, IL-1β or TGF-β1 treated groups. Propidium iodide (PI) staining and fluoresceine isothiocyanate (FITC) Annexin V and propidium iodide apoptosis assay were used to analyse cell cycle arrest and apoptosis separately in IL-34 down-regulated cells. RESULTS: We found that IL-1β significantly enhanced IL-34 expression, while contrarily, TGF-β1 restrained IL-34 gene expression. Transwell and wound closure techniques showed that IL-34 was involved considerably in promoting cell migration. However, IL-34 knock-down restricted sFLS migration possibly through the diminishing of MMP2 and MMP9 expression. Interestingly, IL-34 down-regulated cells exhibited significantly low cellular viability compared with the non-transfected counterpart via CCK-8 and cultural morphology assays. We found that IL-34 down-regulated cells have low VEGF gene expression compared with treated cells. PI staining showed a G0/G1 cell cycle arrest in IL-34 down-regulated cells. FITCAnnexin V and propidium iodide apoptosis assay verified that IL-34 down-regulated cells induced massive apoptosis through apoptotic signalling caspase3, while IL-1β treated cells presented termination of cellular apoptosis signalled by BCL-2. Furthermore, we observed IL-34 induced activation of ERK1/2 and AKT pathways while IL-34 down-regulation significantly decreased the activation of these pathways. CONCLUSIONS: Our data add novel insights into the pathogenesis of RA and we suggest that IL-34 plays a dominant role in controlling migration and proliferation of sFLS. Consequently, therapeutic strategies targeting IL-34 could be a potent therapy for RA.