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B Regulation by IB Kinase in Primary Fibroblast-Like Synoviocytes1
*
Division of Rheumatology, Allergy, and Immunology, University of California at San Diego School of Medicine, La Jolla, CA 92093; and
Signal Pharmaceuticals, 5555 Oberlin Drive, San Diego, CA 92121
NF-
B is a key regulator of inflammatory gene transcription and
is activated in the rheumatoid arthritis (RA) synovium. In resting
cells, NF-B is retained as an inactive cytoplasmic complex by its
inhibitor, IB. Phosphorylation of IB targets it for proteolytic
degradation, thereby releasing NF-B for nuclear translocation.
Recently, two related IB kinases (IKK-1 and IKK-2) were identified
in immortalized cell lines that regulate NF-B activation by
initiating IB degradation. To determine whether IKK regulates
NF-B in primary cells isolated from a site of human disease, we
characterized IKK in cultured fibroblast-like synoviocytes (FLS)
isolated from synovium of patients with RA or osteoarthritis.
Immunoreactive IKK protein was found to be abundant in both RA and
osteoarthritis FLS by Western blot analysis. Northern blot analysis
showed that IKK-1 and IKK-2 genes were constitutively expressed in all
FLS lines. IKK function in FLS extracts was determined by measuring
phosphorylation of recombinant IB in vitro. IKK activity in both RA
and osteoarthritis FLS was strongly induced by TNF-
and IL-1 in a
concentration-dependent manner. Activity was significantly increased
within 10 min of stimulation and declined to near basal levels within
80 min. Activation of IKK in FLS was accompanied by phosphorylation and
degradation of endogenous IB as determined by Western blot
analysis. Concomitant activation and nuclear translocation of NF-B
was documented by EMSA and immunohistochemistry. Transfection with a
dominant negative IKK-2 mutant prevented TNF--mediated NF-B
nuclear translocation, whereas a dominant negative IKK-1 mutant had no
effect. This is the first demonstration that IKK-2 is a pivotal
regulator of NF-B in primary human cells.
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