Drs. Pap and Nawrath contributed equally to this work.
Cooperation of Ras- and c-Myc–dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis
Article first published online: 9 SEP 2004
Copyright © 2004 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 50, Issue 9, pages 2794–2802, September 2004
How to Cite
Pap, T., Nawrath, M., Heinrich, J., Bosse, M., Baier, A., Hummel, K. M., Petrow, P., Kuchen, S., Michel, B. A., Gay, R. E., Müller-Ladner, U., Moelling, K. and Gay, S. (2004), Cooperation of Ras- and c-Myc–dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis. Arthritis & Rheumatism, 50: 2794–2802. doi: 10.1002/art.20461
- Issue published online: 9 SEP 2004
- Article first published online: 9 SEP 2004
- Manuscript Accepted: 5 MAY 2004
- Manuscript Received: 6 AUG 2003
- Swiss National Science Foundation. Grant Number: SNSF 32-64142.00
- Deutsche Forschungsgemeinschaft. Grant Numbers: DFG Pa 698/1-1, Pa 698/2-1, Mu 1383/3-3
To study the specific contribution of MAP kinase activator c-Raf-1 and one of its downstream transcription factors, c-Myc, to the growth and invasive behavior of rheumatoid arthritis synovial fibroblasts (RASFs).
RASFs were transduced with retroviral constructs expressing dominant-negative mutants of c-Raf-1 or c-Myc (DN c-Raf-1 or DN c-Myc, respectively) or with the mock vector. The expression of wild-type and mutant proteins was confirmed by Western blotting. Growth curves of RASFs were recorded, and apoptosis was measured by flow cytometry. Invasiveness of RASFs was assessed in the SCID mouse model of RA. Immunohistochemistry was used to study the effects of DN c-Raf-1 on phosphorylated c-Jun and matrix metalloproteinase 1 (MMP-1) in RASFs implanted into SCID mice. The phosphorylation of ERK and JNK in DN c-Raf-1– and mock-transduced RASFs was determined in vitro by Western blotting. The levels of MMPs in these cells were measured by quantitative polymerase chain reaction (PCR).
Neither DN c-Raf-1 alone nor DN c-Myc alone significantly altered proliferation or apoptosis of RASFs, but both mutants together rapidly induced apoptosis. Inhibition of c-Raf-1 or c-Myc significantly reduced the invasiveness of RASFs in the SCID mouse model. DN c-Raf-1 decreased the phosphorylation of ERK and JNK in vitro and reduced the in vivo expression of phosphorylated c-Jun as well as the expression of disease-relevant MMPs. As determined by quantitative PCR, the inhibition was most pronounced for MMP-1 and MMP-3.
The data demonstrate that Ras- and c-Myc–dependent signaling events cooperate to regulate the growth and invasiveness of RASFs. Targeting of both c-Raf-1 and c-Myc may constitute an interesting therapeutic approach in RA.