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

Abstract

Background: 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.

Methods: The 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.

Results: Endogenous 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.

Conclusions: The 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.

Figure 1

Expression of TRPM3 in FLSs of synovium in rheumatoid arthritis. (a) PCR products from analysis of mRNA isolated from primary cultured FLSs from patients with (+) and without (-) prior reaction with reverse transcriptase (RT). On the left is the DNA marker ladder (M). The expected TRPM3 mRNA product size is indicated with an arrow. (b) Serial sections of RA synovial biopsy labeled with the primary (1°) anti-TRPM3 antiserum present (left image) or absent (right image). Positive labeling is green. The scale bar is 50 μm. (c) A section of RA synovial biopsy labeled with anti-TRPM3 antiserum (brown) and counter stained with haematoxylin to label cell nuclei (purple). The lining of the synovium (i) and a blood vessel (ii) are indicated by arrows.

Figure 2

Sphingosine-evoked Ca²⁺ signals in HIG-82 cells. Data were generated by intracellular Ca²⁺ measurement. Responses elicited by (a) sphingosine (SPH, 20 μM), (b) dimethylsphingosine (DMS, 20 μM) or (c) adenosine triphosphate (ATP, 100 μM) were tested in either 1.5 mM Ca²⁺-containing (+Ca²⁺) or Ca²⁺-free extracellular solution. Responses are each representative of 3 (n) independent experiments.

Figure 3

Inhibition of sustained sphingosine responses by anti-TRPM3 antibody in HIG-82 cells. Data were generated by intracellular Ca²⁺ measurement. (a) Cells were pre-treated with 1:4000 dilution of pre-immune serum (pre-imm.) or anti-TRPM3 antiserum before observing responses to 20 μM sphingosine (SPH). (b) Summary normalised data from 8 independent experiments showing inhibitory effect of TM3E3 on the sustained response to SPH. (c) Lack of effect of TM3E3 on Ca²⁺ entry in cells store-depleted by 1 μM thapsigargin (TG) for 30 min in Ca²⁺-free extracellular solution followed by re-addition of 1.5 mM Ca (+Ca²⁺); representative of N/n = 24/3. (d-f) Investigation of the transient response to SPH. (d) Effect of 10 μM ruthenium red (+RR.) on the SPH response in HIG-82 cells; representative of N/n = 12/3. (e) Responses to 10 μM 4α-phorbol 12,13-didecanoate (4α-PDD) in HIG-82 cells in the presence (+Ca²⁺) and absence of extracellular Ca²⁺; representative of N/n = 16/2. (f) Representative of 3 independent experiments, responses to sphingosine (SPH, 20 μM) in CHO cells stably expressing TRPV4.

Figure 4

Ca²⁺-entry evoked by sphingosine (SPH) in primary cultures of FLSs from patients with RA. (a) Intracellular Ca²⁺ measurement showing the effect of external application of 20 μM SPH in the presence and absence of extracellular Ca²⁺. (b-d) Intracellular Ca²⁺ measurements showing responses to externally applied substances: (b) 20 μM SPH, (c) 100 μM adenosine triphosphate (ATP), and (d) 30 μM lysophosphatidylcholine (LPC). In paired experiments on the same 96-well plate, responses were compared after pre-treatment with anti-TRPM3 antibody (TM3E3) or TM3E3 preadsorbed to its antigenic peptide ("+pep."; control group). (a-d) All data points are N = 8 and representative of 3 (n) independent experiments.

Figure 5

Ca²⁺-entry evoked by pregnenolone sulphate (PregS) in primary cultures of FLSs from patients with RA. (a) Intracellular Ca²⁺ measurement showing the effect of external application of 100 μM PregS in the presence and absence of extracellular Ca²⁺. (b) As for (a) but the effect of PregS (100 μM) and then addition of 20 μM SPH. (c) As for (a) but paired experiments on the same 96-well plate where responses were compared after pre-treatment with anti-TRPM3 antibody (TM3E3) or TM3E3 preadsorbed to its antigenic peptide ("+pep."; control group). (a-c) All data points are N = 8 and representative of 3 (n) independent experiments. (d) Concentration-dependence of Ca²⁺-entry evoked by PregS. Mean data are shown for measurement of the peak and sustained responses to PregS (e.g. see (c)); 4 (n) independent experiments. The fitted curves are Hill equations with mid-points at 35.7 μM (peak) and 39.5 μM (sustained). The shaded area on the left (i) indicates the highest concentrations reported for endogenous PregS [22,23], while the shaded area on the right (ii) indicates the higher concentrations of plasma PregS predicted on oral administration of 100 mg daily to adult humans.

Figure 6

Negative coupling to hyaluronan secretion in primary cultures of FLSs from patients with RA. Data are from measurements of hyaluronan derived from cells of 6 (a-c) or 3 (d) patient samples. (a) Mean effects of 100 μM pregnenolone sulphate (PregS), 10 ng/ml tumor necrosis factor (TNF) and 1 μg/ml dexamethasone; 'control' was the solvent. (b) Mean data showing no effect of anti-TRPM3 antibody (TM3E3) on basal hyaluronan secretion; one control was without TM3E3 (no Ab) and another was from cells pretreated with TM3E3 preadsorbed to its antigenic peptide (TM3E3 + pep.). (c) As for (b) but showing the effect 100 μM PregS under the different antibody (Ab) conditions; the control was cells without PregS. (d) Failure of TM3E3 to protect against the inhibitory effect of 1 μg/ml dexamethasone.