TRPM3 channel stimulated by pregnenolone sulphate in synovial fibroblasts and negatively coupled to hyaluronan

Paul Emery,Coziana Ciurtin,Yasser Majeed,Anne A English,David J Beech,Piruthivi Sukumar,Jacqueline Naylor

doi:10.1186/1471-2474-11-111

Abstract

BackgroundCalcium-permeable channels are known to have roles in many mammalian cell types but the expression and contribution of such ion channels in synovial cells is mostly unknown. The objective of this study was to investigate the potential relevance of Transient Receptor Potential Melastatin 3 (TRPM3) channel to fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis.MethodsThe study used RT-PCR and immunofluorescence to detect mRNA and protein. Intracellular calcium measurement detected channel activity in a FLS cell-line and primary cultures of FLSs from patients with rheumatoid arthritis. Enzyme-linked immunosorbent assays measured hyaluronan.ResultsEndogenous expression of TRPM3 was detected. Previously reported stimulators of TRPM3 sphingosine and pregnenolone sulphate evoked sustained elevation of intracellular calcium in FLSs. The FLS cell-line showed an initial transient response to sphingosine which may be explained by TRPV4 channels but was not observed in FLSs from patients. Blocking antibody targeted to TRPM3 inhibited sustained sphingosine and pregnenolone sulphate responses. Secretion of hyaluronan, which contributes adversely in rheumatoid arthritis, was suppressed by pregnenolone sulphate in FLSs from patients and the effect was blocked by anti-TRPM3 antibody.ConclusionsThe data suggest that FLSs of patients with rheumatoid arthritis express TRPM3-containing ion channels that couple negatively to hyaluronan secretion and can be stimulated by pharmacological concentrations of pregnenolone sulphate.

Highlights

Calcium-permeable channels are known to have roles in many mammalian cell types but the expression and contribution of such ion channels in synovial cells is mostly unknown
Endogenous expression of Transient Receptor Potential Melastatin 3 (TRPM3) Messenger RNA encoding TRPM3 was detected in fibroblast-like synoviocyte (FLS) from patients with Rheumatoid arthritis (RA) (Fig 1a)
Ca2+-entry evoked by sphingosine in HIG-82 cells Because of limited supply of synovial biopsies we first sought to investigate the functional relevance of TRPM3 expression through experiments on a FLS cell-line (HIG82 cells), a proposed model system of proliferating FLSs in joints

Summary

Introduction

Calcium-permeable channels are known to have roles in many mammalian cell types but the expression and contribution of such ion channels in synovial cells is mostly unknown. The objective of this study was to investigate the potential relevance of Transient Receptor Potential Melastatin 3 (TRPM3) channel to fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis. The mammalian TRP channels are divided into subgroups according to amino acid sequence similarities; one subgroup is the M (melastatin) subgroup of which there are eight members, while other subgroups include the V (vanilloid) and C (canonical) subgroups. We previously reported on androgens, oestrogen and progesterone It is sulphated in vivo and the sulphated form (pregnenolone sulphate) is suggested to have biological actions, including through glutamate and γ-aminobutyric acid receptors of neurones [12,13]. Hyaluronan from other cell types may have general adverse consequences in inflammatory conditions, including in atherosclerosis [18,19]

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