Background/Purpose: RA fibroblast-like synoviocytes (FLS) possess a unique aggressive phenotype characterized by increased cell growth, cytokine production and invasion. Previous unsuccessful attempts to target the downstream kinases in the mitogen-activated protein kinase (MAPK) pathway in RA have led to increased interest in the MAPK upstream kinases. These interconnected signal transduction pathways can involve the highest level of the MAPKs, namely the MAP3Ks. One of these MAP3Ks, apoptosis signal regulating kinase-1 (ASK1), is upstream of p38 and JNK and could potentially overcome the pro-inflammatory effects of selective p38 inhibitors. To address this question, we examined the expression, regulation and function of ASK1 in RA FLS.

Methods: FLS were obtained from RA and osteoarthritis (OA) synovial tissues at arthroplasty. ASK1 expression was determined by immunohistochemistry (IHC), confocal microscopy and qPCR. ASK1 promoter activity was measured using a minimal promoter luciferase reporter construct (pGL4.23-luciferase) that included a 994 kb region subcloned from the ASK1 promoter. ASK1 was blocked with the selective inhibitor ASKI-1 (IC50=4.7 nM; used at 0.02-2 uM). Migration was assessed using a scratch-wound assay, cell growth using an MTT assay and invasion using a Martigel matrix invasion assay.

Results: IHC demonstrated ASK1 expression in RA synovium, particularly in the synovial intimal lining. Because FLS reside in this region, we then studied its expression in these cells. ASK1 is constitutively expressed in FLS with similar levels in RA and OA as determined by qPCR (p>0.1, n= 6 each). Unexpectedly, IL-1β (2 ng/ml) or TNF (5 ng/ml) for 1 – 48 hours increased ASK1 mRNA levels. Levels peaked at 6 hours, with 9±4-fold (p<0.05, n=3) and 9±2-fold (p<0.05, n=3) increases, respectively. Dose responses for IL-1β and TNF showed a maximal effect with 0.02 ng/ml for IL-1β and 0.5 ng/ml for TNF. Confocal microscopy showed that ASK1 protein localizes to the nucleus and cytosol before and after treatment with IL-1β (2 ng/ml for 6 or 24 hr) with a 68±16% increase in protein levels compared to medium (p<0.01 at 24 hr, n=3). To explore the mechanism of increased gene expression, we used a luciferase reporter construct with the a portion of the ASK1 promoter. After 1 hr of IL-1β stimulation, luciferase expression increased 2±0.5-fold (p<0.03, n=3) confirming that ASK1 is induced at the transcriptional level. We then evaluated the effect of a selective inhibitor on FLS functions relevant to RA. ASK1 inhibition in RA FLS reduced invasion into Matrigel by 34±8% (24 hr, p<0.05, n=3), migration by 29±9% (24 hr, p<0.04, n =3) and cell growth by 29±8% (48 hr, p<0.03, n=3).

Conclusion:  ASK1 gene transcription is induced by inflammatory cytokines implicated in RA FLS. Because it is perhaps the only inducible MAPK family member, inhibition could have a degree of site and event selectivity for inflamed tissues. ASK1 inhibition also significantly reduced FLS functions related to joint damage. Therefore, targeting ASK1 is a novel therapeutic strategy for FLS-mediated damage in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ask1-is-regulated-by-il-1%ce%b2-and-tnf-and-modulates-key-rheumatoid-arthritis-ra-fibroblast-like-synoviocyte-functions-fls/