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


CONCLUSIONS
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.
Department of Rheumatology, Third Hospital Affiliated to Sun Yat-sen University, No. 600 Tianhe Road, Guangzhou, China.
Xinghua
X
Guo
Yunfeng
Y
Pan
Chuyin
C
Xiao
Yuqiong
Y
Wu
Daozhang
D
Cai
Jieruo
J
Gu
D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D015153:Blotting, Western; D000073883:CX3C Chemokine Receptor 1; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D054428:Chemokine CX3CL1 / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D006801:Humans; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013583:Synovial Membrane / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D015854:Up-Regulation
D000073883:CX3C Chemokine Receptor 1; C515538:CX3CL1 protein, human; C095235:CX3CR1 protein, human; D054428:Chemokine CX3CL1; D016328:NF-kappa B; D011759:Pyrrolidines; C498143:RELA protein, human; D012333:RNA, Messenger; D019707:Receptors, Chemokine; D013859:Thiocarbamates; D051996:Transcription Factor RelA; C020972:pyrrolidine dithiocarbamic acid