Abstract
IntroductionFibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.MethodsExpression of RasGRF1, MMP-1, MMP-3, and IL-6 was detected in synovial tissue by immunohistochemistry and stained sections were evaluated by digital image analysis. Expression of RasGRF1 in FLS and synovial tissue was also assessed by immunoblotting. Double staining was performed to detect proteins in specific cell populations, and cells producing MMP-1 and MMP-3. RasGRF1 expression was manipulated in RA FLS by cDNA transfection and gene silencing, and effects on MMP-1, TIMP-1, MMP-3, IL-6, and IL-8 production measured by ELISA.ResultsExpression of RasGRF1 was significantly enhanced in RA synovial tissue, and detected in FLS and synovial macrophages in situ. In cultured FLS and synovial biopsies, RasGRF1 was detected by immunoblotting as a truncated fragment lacking its negative regulatory domain. Production of MMP-1 and MMP-3 in RA but not non-RA synovial tissue positively correlated with expression of RasGRF1 and co-localized in cells expressing RasGRF1. RasGRF1 overexpression in FLS induced production of MMP-3, and RasGRF1 silencing inhibited spontaneous MMP-3 production.ConclusionsEnhanced expression and post-translational modification of RasGRF1 contributes to MMP-3 production in RA synovial tissue and the semi-transformed phenotype of RA FLS.
Highlights
Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs)
Given the similarities between FLS and transformed cancer cells, we examined the expression of Ras guanine nucleotide-releasing factor 1 (RasGRF1) in RA and non-RA synovial tissue and FLS, providing evidence that elevated RasGRF1 expression and post-translational modification of this protein in RA synovial tissue may contribute to joint destruction by stimulating MMP-3 production
Measurement of MMP-1, MMP-3, TIMP-1, IL-6 and IL-8 production by fibroblast-like synoviocytes Medium was removed from FLS 24 hours after introduction of cDNA or locked nucleic acid (LNA), and was replaced with starvation medium
Summary
Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). While transforming mutations in gene products involved in cellular transformation, such as Ras and PTEN, have not been detected in RA FLS [6,7], it is appreciated that signaling pathways regulated by proto-oncogene and tumor suppressor gene products are constitutively activated due to stimulation by inflammatory cytokines, chemokines, growth factors, and oxidative stress in RA synovial tissue [8]. The binding of GTP to Ras superfamily GTPases leads to a conformational change in the GTPase, allowing signaling to downstream effector proteins [10] Of these small GTPases, Ras family homologs (HRas, K-Ras, and N-Ras) are important in coupling extracellular stimuli to activation of a shared set of signaling pathways regulating cell proliferation and survival, including mitogen-activated protein kinase cascades, phosphoinositide 3-kinase and Ral GTPases [9,11]. GEF selectivity in activating different Ras homologs, as well as differential coupling of GEFs to specific types of cellular receptors – such as Son-of-sevenless coupling to tyrosine kinase-dependent receptors, and Ras guanine nucleotide-releasing factor 1 (RasGRF) coupling to G proteincoupled receptors – achieve specificity in Ras superfamily GTPase signaling
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