ARTHRITIS & RHEUMATISM Vol. 50, No. 1, January 2004, pp 247–258 DOI 10.1002/art.11486 © 2004, American College of Rheumatology Proline-Rich Tyrosine Kinase 2 and Src Kinase Signaling Transduce Monosodium Urate Crystal–Induced Nitric Oxide Production and Matrix Metalloproteinase 3 Expression in Chondrocytes Ru Liu, 1 Fre ´de ´ric Liote ´, 2 David M. Rose, 1 Denise Merz, 1 and Robert Terkeltaub 1 Objective. Articular deposition of monosodium urate monohydrate (MSU) crystals may promote carti- lage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondro- cytes. Methods. Nitric oxide (NO) release, and expres- sion of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP-3) were assessed in cultured chondrocytes treated with MSU. MSU-induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family mem- bers proline-rich tyrosine kinase 2 [Pyk-2] and FAK) was also examined using selective pharmacologic inhib- itors and transfection of kinase mutants. Results. MSU induced MMP-3 and iNOS expres- sion and NO release in chondrocytes in a p38-dependent manner that did not require interleukin-1 (IL-1), as demonstrated by using IL-1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk-2 and FAK, their adaptor protein paxillin, and interacting kinase c-Src. Pyk-2 and c-Src signaling both mediated p38 MAPK activation in response to MSU. Pyk-2 and c-Src signaling played a major role in transducing MSU-induced NO production and MMP-3 expression. But, despite the observed FAK phosphorylation, a se- lective pharmacologic FAK inhibitor and a FAK dominant-negative mutant both failed to block MSU- induced NO release or MMP-3 expression in parallel experiments. Conclusion. In chondrocytes, MSU crystals acti- vate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk-2, Src, and p38 kinases transduce MSU-induced NO production and MMP-3 expression. Our results identify Pyk-2 and c-Src as novel sites for potential therapeutic intervention in cartilage degrada- tion in chronic gout. In gout, the deposition of monosodium urate monohydrate (MSU) crystals in synovium and cartilage can promote chronic inflammation that may lead to cartilage catabolism and bone erosion (1,2). MSU crys- tals stimulate both acute neutrophilic inflammation and chronic synovitis in large part via their capacity to directly activate both resident articular connective tissue cells and mononuclear phagocytes (3–5). Direct activa- tion of cells by MSU crystals can trigger the expression of a broad array of inflammatory mediators, including cyclooxygenase 2 and the cytokines tumor necrosis factor , interleukin-1 (IL-1), IL-6, and IL-8 (6–10). Also consistent with the remarkable intensity of the clinical and pathologic inflammatory response in gout (1,3,4) is the capacity of MSU crystals to activate phagocytes by triggering multiple signal transduction pathways (11–14). In leukocytes, MSU crystals nonspecifically en- gage plasma membrane proteins, such as the Fc receptor CD16 and the 2 integrin CD11b/CD18 (11). Concor- Supported by research grants from the Department of Vet- erans Affairs, the NIH (HL-61731, R03-AR-49416-10, P01-AG- 07996), the Arthritis Foundation, the University of California, San Diego, Medicine Education Research Foundation, the Stein Institute for Research on Aging, Assistance Publique Ho ˆpitaux de Paris, Paris 7 University, and the Philippe Foundation. 1 Ru Liu, PhD, David M. Rose, PhD, Denise Merz, BS, Robert Terkeltaub, MD: VA Medical Center and University of California, San Diego; 2 Fre ´de ´ric Liote ´, MD, PhD: Ho ˆpital Lariboi- sie `re, Paris 7 University, Paris, France. Drs. Liu and Liote ´ contributed equally to this work. Address correspondence and reprint requests to Robert Ter- keltaub, MD, VA Medical Center, MC111K, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail: rterkeltaub@ucsd.edu. Submitted for publication March 18, 2003; accepted in revised form October 13, 2003. 247