IL-34 modulates rheumatoid synovial fibroblasts proliferation and migration via ERK/AKT signalling pathway

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.

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.

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.

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.