ARTHRITIS & RHEUMATISM Vol. 56, No. 9, September 2007, pp 2854–2863 DOI 10.1002/art.22853 © 2007, American College of Rheumatology Regulation of Apoptosis in Fibroblast-like Synoviocytes by the Hypoxia-Induced Bcl-2 Family Member Bcl-2/Adenovirus E1B 19-kd Protein–Interacting Protein 3 Wafa Kammouni, 1 Keng Wong, 1 Guoping Ma, 1 Gary S. Firestein, 2 Spencer B. Gibson, 3 and Hani S. El-Gabalawy 1 Objective. Rheumatoid arthritis (RA) synovial hyperplasia is related in part to a resistance to apopto- sis exhibited by fibroblast-like synoviocytes (FLS). Since hypoxia is a regulator of apoptosis, and since RA synovium is hypoxic, we conducted this study to examine the effects of hypoxia on the Bcl-2 pathway and the role this may play in regulating apoptosis in FLS. Methods. Synovium samples from RA patients, osteoarthritis (OA) patients, and normal subjects were used for immunohistologic assessments and for gener- ating FLS lines in vitro. FLS were stimulated under conditions of hypoxia (1% O 2 ) and using 100 M CoCl 2 to simulate the effects of severe hypoxia. Changes in the gene expression profile of FLS were evaluated using microarrays and were confirmed by quantitative poly- merase chain reaction (PCR). Changes in protein ex- pression were detected by Western blotting. The effect of transient transfection with a BNIP3 plasmid on the apoptosis of FLS was evaluated in the presence and absence of cytokines. Results. Gene expression profiling demonstrated that BNIP3 was unique among the BCL2 family, in that it was induced by hypoxia in FLS. Quantitative PCR indicated a 2–3-fold induction of BNIP3 messenger RNA, and Western blotting showed a 3–5-fold increase in the 30-kd Bcl-2/adenovirus E1B 19-kd protein– interacting protein 3 (BNIP-3) monomer. BNIP-3 was widely expressed in RA synovium and was prominent in FLS from the lining layer. Overexpression of BNIP3 increased FLS apoptosis under hypoxic conditions, an effect that was inhibited by tumor necrosis factor  and interleukin-1. Conclusion. The proapoptotic protein BNIP-3 is induced in FLS by hypoxia and is widely expressed in RA synovium, but its proapoptotic effects may be inhib- ited in vivo by proinflammatory cytokines. Since over- expression of BNIP3 in FLS increases apoptosis, this may provide a novel approach for controlling synovial hyperplasia in RA. Rheumatoid arthritis (RA) is a chronic inflam- matory disease that leads to fundamental changes in the cellular composition and function of the synovial mem- brane (1). One aspect of this is that the resident fibroblast-like synoviocytes (FLS) that normally popu- late the thin synovial lining layer appear to increase in number and change their phenotype, becoming locally invasive to the adjacent cartilage and bone. This trans- formation of FLS, which resembles that seen in tumor cells, is evident in vitro as anchorage-independent growth, loss of contact inhibition, and in the expression of protooncogenes (2). Studies evaluating coimplanta- tion of RA FLS and articular cartilage into SCID mice have clearly demonstrated the invasive nature of these cells (3). Although in vivo indices of proliferation in RA synovium have generally been found to be low, the FLS derived from these tissues have a survival advantage when cultured in vitro. This has led to the hypothesis that the survival advantage may be related, at least in part, to an increase in the levels of several antiapoptotic Supported by the Arthritis Society of Canada (grant TAS 03-0093) and the Rheumatic Diseases Core Center at the University of California, San Diego. Dr. Firestein’s work was supported by NIH grant R01-AR-45347. 1 Wafa Kammouni, PhD, Keng Wong, BSc, Guoping Ma, MD, Hani S. El-Gabalawy, MD: University of Manitoba, Winnipeg, Mani- toba, Canada; 2 Gary S. Firestein, MD: University of California, San Diego, La Jolla; 3 Spencer B. Gibson, PhD: Manitoba Institute of Cell Biology, and University of Manitoba, Winnipeg, Manitoba, Canada. Address correspondence and reprint requests to Hani El- Gabalawy, MD, University of Manitoba Arthritis Centre, RR149, 800 Sherbrook Street, Winnipeg, Manitoba R3A 1M4, Canada. E-mail: elgabal@cc.umanitoba.ca. Submitted for publication August 14, 2006; accepted in revised form May 25, 2007. 2854