Background In rheumatoid arthritis (RA), fibroblast-like synoviocytes (RAFLS) develop an aggressive phenotype, promoting cartilage damage and bone destruction through the secretion of proteolytic enzymes and cytokines. We have recently shown that human RAFLS and RA synovial tissues express a range of acid sensing proteins including Accn3, Gpr4, Gpr68 and Trpv1. In the present study, we have tested the hypothesis that acidification alters the phenotype of synoviocytes by measuring the expression of acid sensing proteins and matrix metalloproteinases in cultured RAFLS. In addition, we have investigated the colocalization of acid sensing proteins in human RA arthroscopy tissues by Immunofluorescence (IF) using an RAFLS-specific antibody.
To determine the localisation of acid sensing proteins in synovium from patients with RA.
investigate acid induced intracellular signaling mechanisms to determine their contribution to the pathological processes in RA
Determine the effects of acid sensing proteins on functional responses to acid in synoviocytes.
Methods RAFLS were cultured in DMEM containing 10% fetal bovine serum, glutamine and penicillin/streptomycin (normal culture medium). At 80% confluence the normal culture medium was replaced with medium acidified with 1N HCl to pH 7.0, 6.5, 6.0 or 5.5. After 24 hours total RNA was extracted, quantified, reverse transcribed and assayed for the expression of MMP-1, MMP-3, MMP-9, MMP-13 RANKL and the acid sensing proteins including Accn3, Gpr4, Gpr68 and Trpv1. These acid sensing proteins were also colocalized in relation to an antigen expressed by RAFLS (Fibroblast Surface Protein 1B10) in arthroscopic biopsies from five RA patients by IF.
Results There was a significant increase (P<0.05) in the expression of mRNA with decreasing pH, with highest expression for MMP-1 (20±7-fold), MMP-3 (4.8±2.7-fold), MMP-13 (8.8±0.5-fold), Accn3 (3.3±0.18-fold) and Gpr4 (3.2±0.28-fold), occuring at pH 6.0. In addition, expression of MMP-9 and RANKL was also pH-dependent, with maximum expression at pH 6.5 (MMP-9: 2.6±0.7-fold, RANKL 2.6±0.5-fold). All four acid sensing proteins were expressed in cells expressing the RAFLS antigen as well as in other cells throughout the synovial tissues.
Conclusions Our data show that in RA, acid sensing proteins are expressed throughout the synovium and not exclusively in RAFLS. Trpv1 and Gpr4 were expressed more widely throughout synovium compared with Asic3 and Gpr68. Under standard cell culture oxygenation, acidification of the extracellular environment leads to a significant up-regulation of MMP expression in human RAFLS. Our results support the concept that MMPs are important mediators of bone and cartilage destruction in the altered cellular environment of RA. Our data suggests that the acidic environment of the inflamed joint in RA can modulate the expression of mediators of tissue damage in RAFLS independently of hypoxia. The widespread expression in RA tissue of specific proteins known to act as acid sensors may implicate them in the pathophysiological destruction of cartilage and bone. Our ongoing studies will test the effects on MMP activity of inhibiting specific acid-sensing proteins in RAFLS and explore the molecular mechanisms by which acidity up-regulates MMP expression in RAFLS.
Disclosure of Interest None declared