NFATc1 regulation of the human β3 integrin promoter in osteoclast differentiation

The transcription factor NFATc I plays an essential role in transducing signals from RANKL in osteoclast differentiation. To date, however, the specific transcriptional targets of NFATc1 are unknown. Expression of the 133 integrin is required for normal osteoclast function. We therefore examined the role of NFATc1 in human beta(3) integrin expression in osteoclast differentiation. Analysis of the mouse and human beta(3) gene promoters revealed considerable sequence homology across a 1.3 kb region upstream of the transcription start site (TSS), with conserved NFAT binding elements present. The region - 1242 to + 29 (relative to the TSS) was cloned as a luciferase reporter construct (pB3-1.3) and a deletion construct removing to -997 (pB3-1) made. The deletion of 245 bp 5'removed three conserved NFAT sites including a consensus NFAT:AP-1 site. The pB3-1.3 reporter construct was induced by treatment with RANKL in the range 2.5-40 ng/ml and dose-dependently induced by co-transfection with human NFATc1 in RAW264.7 cells. The pB3-1 deletion construct was minimally induced with RANKL treatment and unresponsive to co-transfected NFATc1. Direct NFAT binding to two of the consensus NFAT sites within this 245 bp 5' region was demonstrated by EMSA and supershift with anti-NFAT antibodies. Mutation of two of the conserved NFAT sites in the - 1242 to - 997 fragment was required to prevent binding. The double NFAT mutant, in the context of the full-length promoter was unresponsive to RANKL treatment or co-transfected NFAR I. We generated cell-permeable TAT-dominant-negative (dn)NFATc1 fusion proteins to assess the effect of blockade of NFAT signaling. Transduction with dnNFAT inhibited RANKL induction of the human 1 3 integrin promoter. Involvement of the NFARc1-calcineurin pathway in regulating the human M integrin promoter was further confirmed using the calcineurin pathway inhibitory peptide 11R-VIVIT. Together these results establish the M gene as a direct target of NFATc1 in RANKL-dependent osteoclast formation. (c) 2006 Elsevier B.V. All rights reserved.