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S., Lacey D. GSK-3 in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling. (11, 12). Nuclear export of NFAT members is facilitated by phosphorylation, and several kinases have been suggested to regulate NFAT function, including GSK-3 (13), CK1 (14), p38 (15), and JNK1 (16). Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase originally identified for its role in the regulation of glycogen deposition. GSK-3 has two isoforms, GSK-3 and GSK-3 (17), both of which are implicated in many different biological processes including metabolism, transcription, translation, cell growth, and apoptosis (18). With respect to transcription, Enalapril maleate GSK-3 regulates a wide variety of transcription factors, including cyclin D1, c-Jun, NFATc, and -catenin (13, 19, 20). In resting cells, GSK-3 is constitutively active, and its activity is inhibited by various kinases via phosphorylation of a serine residue, Ser-21 in GSK-3 and Ser-9 in GSK-3 in response to different stimuli (21). Serine phosphorylation on GSK-3 blocks the access of substrate to the GSK-3 catalytic domain, thus inhibiting substrate phosphorylation (22). Of the two isoforms of GSK-3, GSK-3 is a more likely candidate for being an NFATc1 kinase, influencing NFATc1 subcellular localization through phosphorylation (13). However, the significance of the ability of GSK-3 to regulate NFATc1 during osteoclastogenesis has not yet been demonstrated. In addition, because GSK-3-deficient mice die (23), the relevance of GSK-3 in osteoclast precursors has not been well characterized. Therefore we investigated the role of GSK-3 in RANKL-mediated osteoclast differentiation and also clarified the relevance of GSK-3 and NFATc1. In addition, to understand the physiological role of GSK-3 (cytosolic Ca2+ concentration), single cells were viewed with a laser-scanning confocal system (FluoView Enalapril maleate 500, Olympus, Tokyo, Japan) attached to an upright microscope (BX51WI, Olympus). An argon laser (488 nm) was used for excitation, a green emission filter (505C525 nm) was used for fluo-4, and a red emission filter ( 660 nm) was used for fura red to observe the fluorescent images. The ratio of Enalapril maleate the fluorescence intensity of fluo-4 to fura red was calculated. The maximum intensity of [Ca2+]was obtained with the addition of 10 m ionomycin at the end of each experiment. The ratio of increase from the basal level was expressed as the percentage of maximum ratio increase. Generation of Transgenic Mice The constitutively active GSK-3 Enalapril maleate (GSK3-S9A) mutant cDNA was fused to the mouse TRAP gene promoter as described previously (29, 30). For generating transgenic mice, we used the standard pronuclear injection method with C57BL/6 mice (The Jackson Laboratory). Genomic DNA isolated from the tail was analyzed by polymerase chain reaction (PCR) using the specific primers (GT-F, 5-TAGCCATCAACAGCCGTCAGT; GT-R, 5-CTTCTGCCCCAGAGAATAAAG; GP-F, 5-CAGGGTACAGTTTAGAATGGG; GP-R, 5-GTACTAGGCAGACTGTGTAAAG) to detect the transgene. All the mouse experiments were performed with 4C6-week-old mice under the animal protocol approved by the Animal Care Committee of the Ewha Laboratory Animal Genomics Center. Bone Histomorphometry and Microcomputed Tomography Analysis Bones were fixed in 10% formaldehyde, decalcified in 0.5 m EDTA, pH 7.4, embedded in paraffin, and then cut into 4-m sections. Hematoxylin and eosin (H&E) or TRAP staining was performed according to a standard protocol (24). The histomorphometric data were analyzed by Osteomeasure XP (OsteoMetrics Enalapril maleate Inc.). Quantitative microcomputed tomography was performed with Skyscan 1076 (Skyscan N.V.). The data from scanned slices were used for the three-dimensional analysis to calculate femoral morphometric parameters by CT-AN 1.10 (Skyscan N.V.). The nomenclature and units were according to the recommendation of the Nomenclature Committee of the American Society for Bone and Mineral Research (31). RANKL-induced Bone Loss Five-week-old female mice were administered with a local calvarial injection of RANKL at 2 mg/kg of body weight. After 5 days, osteoclast number per millimeter of trabecular bone surface and the percentage of bone surface covered by osteoclasts (eroded surface) were Lif measured as described (32). Statistics Data are expressed as mean S.D. from at least three independent experiments. Statistical analyses were performed using the two-tailed Student’s test to analyze differences among groups. 0.05 was considered statistically significant. RESULTS GSK-3 Is Inactivated upon RANKL Treatment To examine the role of GSK-3 in RANKL-mediated osteoclast differentiation, we.