Regulation of JNK by MKK-7 in fibroblast-like synoviocytes.
OBJECTIVE
JNK regulates matrix metalloproteinase (MMP) gene expression and joint destruction in rheumatoid arthritis (RA). Previous studies demonstrated that the 2 upstream MAPK kinases (MKK-4 and MKK-7) are phosphorylated in RA synovium and form a complex with JNK in fibroblast-like synoviocytes (FLS). However, the functional hierarchy of MKK-4 and MKK-7 in FLS has not been determined. We determined the relative contributions of these MKKs by evaluating the effect of MKK-4 and MKK-7 gene knockdown in cultured FLS.


METHODS
FLS were transfected with MKK-4 and/or MKK-7 small interfering RNA, and protein levels were determined by immunoblotting. After stimulation with interleukin-1/beta (IL-1beta), tumor necrosis factor alpha(TNFalpha, or anisomycin, kinase function was determined by in vitro kinase assay. Activator protein 1 (AP-1) binding and transcriptional activity were determined by electrophoretic mobility shift assay and AP-1-luciferase promoter assay, respectively. MMP-3 expression was determined by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction.


RESULTS
IL-1beta-induced JNK phosphorylation was dependent on MKK-7 but not on MKK-4; however, anisomycin-activated JNK required both kinases. In vitro kinase assay demonstrated that IL-1beta-or TNFalpha induced JNK activity was only MKK-7 dependent, while anisomycin-activated JNK was both MKK-4 and MKK-7 dependent. IL-1beta-induced AP-1 binding activity and AP-1-driven gene expression were strictly MKK-7 dependent. Finally, MMP-3 production only required MKK-7, and there was no effect of MKK-4 deficiency.


CONCLUSIONS
These data indicate that only MKK-7 is required for JNK activation in FLS after cytokine stimulation; however, other forms of cellular stress utilize MKK-4. Thus, JNK function might be modulated by targeting MKK-7 to suppress cytokine-mediated FLS activation while leaving other stress responses intact.
Inoue
Tomoyuki
T
Division of Rheumatology, Allergy and Immunology, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656, USA.
Hammaker
Deepa
D
Boyle
David L
DL
Firestein
Gary S
GS
D000841:Anisomycin / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D048688:MAP Kinase Kinase 7 / Q000235:genetics / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D007375:Interleukin-1; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D000841:Anisomycin; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human; D019278:Matrix Metalloproteinase 3