Background/Purpose: Fibroblast-like synoviocytes (FLS) are key mediators of inflammation and joint damage in rheumatoid arthritis (RA) through the production of cytokines and matrix metalloproteinases (MMPs) as well as invasion into extracellular matrix. The search for potential kinases that target FLS for RA led to hematopoietic cell kinase (HCK) as a candidate target. HCK is a member of the Src tyrosine kinases and is primarily expressed by myeloid cells. HCK deficiency reduces the migration of M-CSF- and RANKL-induced murine bone marrow mononuclear cells in vitro, indicating that it might play a role in cell migration. However, its expression in mesenchymal cells is not defined, and nothing is known about HCK function in synoviocytes. To determine if HCK is a potential therapeutic target in RA, we evaluated HCK expression and function in RA FLS. These studies show that HCK is an inducible gene in RA FLS that regulates key pathogenic functions, thus allowing an inhibitor to target its effects primarily at sites of inflammation.

Methods: FLS were obtained from RA and osteoarthritis (OA) patients undergoing joint replacement surgery and used from passage 3 through 9. Three separate cell lines were studied for each experiment. To study HCK expression in FLS, RA and OA FLS were serum starved and treated with medium, IL-1β (2ng/ml) or TNF (50ng/ml) for various times. mRNA levels were determined by qPCR. For functional studies, we used a novel selective small molecule HCK inhibitor, with an IC50 of approximately 7nM. IL-6 and MMP expression were measured by qPCR. MTT assays were performed to determine PDGF-induced proliferation. Apoptosis was induced by H2O2 stimulation and MTT assay. For cell migration, scratch assays were performed on PDGF-stimulated FLS monolayers. The data were analyzed using student’s t test and one way ANOVA. 

Results: HCK mRNA levels were very low under basal conditions in cultured FLS. IL-1 and TNF increased HCK expression, with maximal introduction at 24 hr by 734- and 65-fold, respectively. TNF-induced IL-6 expression was decreased by 39% in cells treated with 1 μM of the HCK inhibitor (p = 0.025). MMP3 expression was also inhibited by 50±15% (p = 0.03) and 41±12% (p = 0.026) after the stimulation with IL-1β and TNF, respectively. The small molecule HCK inhibitor (1 μM) significantly reduced PDGF-induced FLS growth by 93±32% on day 3 (n=3 RA FLS, p = 0.001), and 81±19% on day 7 (p = 0.001). The compound had no effect on H2O2-induced apoptosis, suggesting that the effect on cell growth was due to decreased proliferation rather than increased cell death. HCK inhibition impaired PDGF-induced migration by 83±31% (p = 0.04).

Conclusion: HCK expressed was induced by IL-1β and TNF in RA FLS. HCK blockade decreased cytokine production, MMP production, proliferation, and cell migration in FLS. Because the gene is minimally expressed in resting cells, its main effect would be in cells at the site of inflammation.  Therefore, HCK could be a promising therapeutic target for RA that can regulate pathogenic behavior in a site and event specific manner.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hematopoietic-cell-kinase-hck-as-a-novel-regulator-of-fibroblast-like-synoviocyte-function-in-ra/