title: PU.1 and NFATc1 mediate osteoclastic induction of the mouse beta3 integrin promoter.
abstract: Expression of the alpha(v)beta(3) integrin is required for normal osteoclast function. We previously showed that an evolutionary conserved NFATc1 binding site is required for RANKL induction and NFATc1 transactivation of the human beta(3) promoter. The mechanism conferring specificity for RANKL induction and NFATc1 transduction of the beta(3) gene in osteoclast differentiation is unclear since NFATc1 is expressed and activated in numerous cell types that do not express the beta(3) gene. PU.1 is an ETS family transcription factor in myeloid cells associated with expression of various osteoclast genes. The present study investigates the role of NFATc1 in concert with PU.1 in osteoclast-specific transcription of the mouse beta(3) integrin gene. The mouse beta(3) promoter was transactivated by NFATc1 in RAW264.7 cells and deletion or mutation of either of the conserved NFAT and PU.1 binding sites abrogated transactivation. NFATc1 transactivation of the mouse beta(3) promoter was specifically dependent on co-transfected PU.1 in HEK293 cells, to the exclusion of other ETS family members. Direct binding of NFATc1 and PU.1 to their cognate sequences was demonstrated by EMSA and NFATc1 and PU.1 occupy their cognate sites in RANKL-treated mouse marrow precursors in chromatin immuno-precipitation (ChIP) assays. TAT-mediated transduction with dominant-negative NFATc1 dose-dependently blocked endogenous expression of the mouse beta(3) integrin and the formation of TRAP positive multinucleated cells in RANKL-treated mouse macrophages. These data provide evidence that NFATc1, in concert with PU.1, are involved in regulation of beta(3) integrin expression during osteoclast differentiation and suggest that PU.1 confers specificity to the NFATc1 response to macrophage lineage cells.
authors.affiliation: The New England Baptist Bone and Joint Institute, Department of Rheumatology, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02115, USA. tcrottie@bidmc.harvard.edu
authors.forename: Tania N
authors.initials: TN
authors.lastname: Crotti
authors.forename: Sudarshana M
authors.initials: SM
authors.lastname: Sharma
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Fleming
authors.forename: Merrilee R
authors.initials: MR
authors.lastname: Flannery
authors.forename: Michael C
authors.initials: MC
authors.lastname: Ostrowski
authors.forename: Steven R
authors.initials: SR
authors.lastname: Goldring
authors.forename: Kevin P
authors.initials: KP
authors.lastname: McHugh
mesh_terms: D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D039661:Integrin beta3; D050778:NFATC Transcription Factors; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1