Identification of NFAT binding sites that mediate stimulation of cathepsin K promoter activity by RANK ligand.
The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL.
Balkan
Wayne
W
Geriatrics Research, Education and Clinical Center and Research Service, Miami Veterans Affairs Healthcare System, Miami, FL 33125, USA.
Martinez
Ariel F
AF
Fernandez
Isabel
I
Rodriguez
Maria A
MA
Pang
Manhui
M
Troen
Bruce R
BR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020013:Calcium Signaling; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000378:metabolism* / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D015398:Signal Transduction; D014162:Transfection
D017931:DNA Primers; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D053245:RANK Ligand; D034741:RNA, Small Interfering; C505689:Tnfsf11 protein, mouse; D002403:Cathepsins; D056657:Cathepsin K; C534132:Ctsk protein, mouse; C534133:Ctsk protein, rat