Effect of celecoxib on Ca 2þ movement and cell proliferation in human osteoblasts Jue-L. Wang a , Ko-L. Lin a , Jin-S. Chen b , Yih-C. Lu c , Bang-P. Jiann b , Hong-T. Chang b , Shu-S. Hsu b , Wei-C. Chen d , Jong-K. Huang b , Chin-M. Ho e , Chung-R. Jan e,* a Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan, ROC b Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan, ROC c Department of Orthopaedic Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan, ROC d Department of Surgery, Ping Tung Christian Hospital, Ping Tung 900, Taiwan, ROC e Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan, ROC Received 4 July 2003; accepted 5 November 2003 Abstract In human osteoblasts, the effect of the widely prescribed cyclooxygenase-2 inhibitor celecoxib on intracellular Ca 2þ concentrations ([Ca 2þ ] i ) and cell proliferation was explored by using fura-2 and the tetrazolium assay, respectively. Celecoxib at concentrations greater than 1 mM caused a rapid rise in [Ca 2þ ] i in a concentration-dependent manner (ec 50 ¼ 10 mM). Celecoxib-induced [Ca 2þ ] i rise was reduced by 90% by removal of extracellular Ca 2þ , and by 30% by L-type Ca 2þ channel blockers. Celecoxib-induced Mn 2þ -associated quench of intracellular fura-2 fluorescence also suggests that celecoxib-induced extracellular Ca 2þ influx. In Ca 2þ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2þ -ATPase, caused a monophasic [Ca 2þ ] i rise, after which the increasing effect of celecoxib on [Ca 2þ ] i was greatly inhibited. Conversely, pretreatment with celecoxib to deplete intracellular Ca 2þ stores totally prevented thapsigargin from releasing more Ca 2þ . U73122, an inhibitor of phoispholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca 2þ mobilizer)-induced, but not celecoxib-induced, [Ca 2þ ] i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, partly inhibited celecoxib-induced [Ca 2þ ] i rise in Ca 2þ -containing medium. Separately, overnight treatment with 1–100 mM celecoxib inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human osteoblasts, celecoxib increases [Ca 2þ ] i by stimulating extracellular Ca 2þ influx and also by causing intracellular Ca 2þ release from the endoplasmic reticulum via a phospholiase C-independent manner. Celecoxib may be cytotoxic at higher concentrations. # 2003 Published by Elsevier Inc. Keywords: Ca 2þ ; Ca 2þ stores; Celecoxib; Fura-2; MG63; Osteoblasts 1. Introduction Cyclooxygenase-2 (COX-2) is an important cellular target for both therapy and/or prevention of inflammatory disorders and cancer. The advent of selective COX-2 inhibitors now allows a more precise and safer treatment approach [1–3]. Because of their better gastrointestinal risk profile, the newly developed selective COX-2 inhibitors celecoxib and rofecoxib are discussed as cost-effective alternatives to common NSAIDs [1]. Due to the wide prescription of celecoxib in treating bone-related disorders, understanding the in vitro effect of celecoxib on osteoblasts is crucial. However, this issue has not been addressed before except that the prevention of prostaglandin synth- esis by inflammatory cytokines in bone cells was thought to contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis [3]. A regulated rise in cytosolic free Ca 2þ levels ([Ca 2þ ] i ) is a key signal in all cell types, and can trigger many physio- pathological events [4–6]; but an unregulated elevation in [Ca 2þ ] i is often cytotoxic [7]. Thus, it is important to examine the effect of an agent on cellular Ca 2þ signaling in order to understand its in vitro effect. The effect of celecoxib on [Ca 2þ ] i in osteoblasts is unclear. However, in prostate cancer cells (PC3), it was shown that exposure of PC3 cells to celecoxib stimulates an immediate [Ca 2þ ] i rise in a dose- and time-dependent manner. This activity is highly specific for celecoxib, and is not noted with other Biochemical Pharmacology 67 (2004) 1123–1130 0006-2952/$ – see front matter # 2003 Published by Elsevier Inc. doi:10.1016/j.bcp.2003.11.004 * Corresponding author. Tel.: þ886-7-3422121x1509; fax: þ886-7-3468056. E-mail address: crjan@isca.vghks.gov.tw (C.-R. Jan).