Fractalkine stimulates cell growth and increases its expression via NF-κB pathway in RA-FLS

Abstract

Background: After the onset of rheumatoid arthritis (RA), fibroblast-like synoviocytes (RA-FLS) which are specialized types of fibroblasts, become tumor-like, keeping their ability to increase proliferation and invasion. The mechanism of their tumor-like growth is unclear. Fractalkine (FKN), also called CX3CL1, plays an important role in the proliferation of cells. FKN may stimulate the proliferation of RA-FLS and the by nuclear factor κB (NF-κB) pathway may be one of the steps in this process.

Objective: To investigate whether FKN can stimulate cell growth and increase its expression in RA-FLS, and the relationship between the NF-κB pathway and the function of FKN.

Methods: FLS were isolated from primary synovial tissue obtained from three patients with RA who had undergone total joint replacement surgery or synovectomy in the Third Hospital Affiliated to Sun Yat-sen University from February 2009 to January 2010. FKN was used in different concentrations to stimulate RA-FLS with or without NF-κB pathway blocker (PDTC), and to test the proliferation of FLS after 24 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RA-FLS was treated with 100 ng/mL FKN or 100 μM PDTC for different periods, and messenger RNA (mRNA) expression of FKN and CX3CR1 in RA-FLS was detected by reverse transcription - polymerase chain reaction. We then tested the protein expression of NF-κBp65 in the cytoplasm and nucleus, respectively by Western blotting after treating the RA-FLS with 100 ng/mL FKN for different time periods.

Results: FKN stimulated cell growth in RA-FLS at the concentration of 50 or 100 ng/mL (P = 0.005 and P = 0.022, respectively). NF-κB pathway blocker inhibited FKN, promoting proliferation of RA-FLS. RA-FLS could express FKN and CX3CR1 mRNA in vitro. FKN up-regulated FKN expression after 18-h treatment (P = 0.012). PDTC disturbed the expression of FKN mRNA after 16-18 h treatment (P = 0.001 and P < 0.001, respectively). After stimulation with FKN for 1 h, the expression of NF-κBp65 in cytoplasm began to decrease (P = 0.010), and the expression of NF-κBp65 in the nucleus began to increase after 2 h (P = 0.011).

Conclusion: These results suggest that FKN stimulates cells growth in RA-FLS and NF-κB pathway blocker inhibits FKN, promoting proliferation of RA-FLS. FKN induced activation of NF-κB activity. FKN up-regulates FKN mRNA expression in RA-FLS via the NF-κB pathway.