Fig. 1: The Melanocortin (MC) system in synovial fibroblasts.
a Expression of several components of the MC pathway in RA SF fibroblasts as determined by end-point PCR using 1 μg of RNA (n = 4). b MCRs expression was further analyzed by real-time PCR using 1 μg of RNA. Cycle threshold (CT) values normalized against the reference control HPRT1 are shown (lower CT values denote higher expression). Numbers above bars indicate overexpression of MC1R compared to the other receptors calculated as 2−ΔΔCt. Data represent mean values, min to max range (n = 12, one-way ANOVA, Dunn’s correction; **p < 0.01; ***p = 0.001). c ACTH quantification in supernatants on SF cultured for 7 days without media replacement. Data are mean ± SD (n = 15). Dotted line represents values quantified in media without incubation with SF cells, serving as a negative control. d ERK-phosphorylation as analyzed by western blotting after 5 min stimulation of SF with MC agonists at the indicated concentrations. e Intracellular cAMP accumulation was quantified by EIA after 15 min SF cell stimulation with BMS or αMSH. The adenylyl cyclase activator forskolin (3 μM) was used as a positive control, yielding 0.58 pmol/ml. Data are mean ± SE (n = 3). f Ca2+ mobilization in SF upon addition of αMSH or BMS (from 1 μM then serially diluted up to 10 pM), and recorded for 86 s. Ionomycin (1 μM) was used as a positive control, yielding 0.15 units of absorbance ratio at 340/380 nm. Data are mean ± SE (n = 3). g In vitro wound healing assay conducted using ibidi® chambers on SF cells stimulated with 10 μM αMSH and 1 μM BMS. Representative images show gap closure. Scale bars indicate 100 μm. Data are mean ± SE (n = 6, two-way ANOVA). h Cytokines release from SF stimulated with SAA at 10 μg/ml and treated with αMSH (30 μM) or BMS (10 μM) for 24 h, determined by ELISA. Data are mean ± SE (n = 15, one-way ANOVA vs. control; *p < 0.05). i Transwell® inserts were used for SF migration (Mig) and invasion assays on Matrigel®-coated wells (Inv). Cells were treated overnight with 10 μM BMS. Data are mean ± SE (n = 6, Student’s t-test vs. control). Source data are provided as Source Data file.
