Elsevier

Gene

Volume 372, 10 May 2006, Pages 92-102
Gene

NFATc1 regulation of the human β3 integrin promoter in osteoclast differentiation

https://doi.org/10.1016/j.gene.2005.12.012Get rights and content

Abstract

The transcription factor NFATc1 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 β3 integrin is required for normal osteoclast function. We therefore examined the role of NFATc1 in human β3 integrin expression in osteoclast differentiation. Analysis of the mouse and human β3 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 NFATc1. 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 β3 integrin promoter. Involvement of the NFATc1-calcineurin pathway in regulating the human β3 integrin promoter was further confirmed using the calcineurin pathway inhibitory peptide 11R-VIVIT. Together these results establish the β3 gene as a direct target of NFATc1 in RANKL-dependent osteoclast formation.

Introduction

Osteoclasts are multinucleated cells derived from hematopoietic progenitor cells of the monocyte/macrophage lineage that are unique in their capacity to resorb mineralized matrix (Baron, 1989). Studies have shown that receptor activator NFκB ligand (RANKL) (Kong et al., 1999), in the presence of M-CSF (Yoshida et al., 1990, Kodama et al., 1991, Tanaka et al., 1993), is the essential mediator of osteoclast differentiation. RANKL acts through its receptor RANK to initiate a signaling cascade that is crucial for osteoclast differentiation and activation. The transcription factor nuclear factor of activated T cells (NFATc1) is up-regulated by RANKL and has been identified as playing a crucial role in osteoclast differentiation and function (Ishida et al., 2002, Takayanagi et al., 2002, Hirotani et al., 2004).

NFATc1 is activated by the Ca2+/calmodulin-regulated phosphatase calcineurin (Macian et al., 2001). Over-expression of NFATc1 (Takayanagi et al., 2002) or ectopic expression of constitutively active NFATc1 (Hirotani et al., 2004) is able to bypass the requirement for RANKL in osteoclast differentiation. Selective inhibition of calcineurin-induced NFATc1 activation results in the impaired spreading of TRAP-positive cells and reduced bone-resorbing capacity (Hirotani et al., 2004). Pertinent to the current study are recent findings demonstrating the ability of NFATc1 to induce the expression of various osteoclast genes, including the β3 integrin (Hirotani et al., 2004).

NFATc1 is capable of inducing osteoclast precursors to differentiate into mature osteoclasts, however, the direct and key transcriptional target genes of NFATc1 have not been defined. Recent work has identified NFAT binding sites in the TRAP promoter, osteoclast-specific P3 promoter of the calcitonin receptor (CTR), the cathepsin K (cath K) promoter and the osteoclast associated receptor (OSCAR) promoter (Takayanagi et al., 2002, Matsumoto et al., 2004, Matsuo et al., 2004, Kim et al., 2005a) and demonstrated specific regulation of the promoters by NFATc1 (Anusaksathien et al., 2001, Matsuo et al., 2004, Kim et al., 2005b).

NFAT co-operatively binds with transcription factors of the AP-1 (Fos/Jun) family and AP-1 proteins to a number of functionally important sites in the promoters of numerous cytokine genes (Rao et al., 1997, Macian et al., 2001). For instance, the interaction between NFATc1 and c-Fos has been shown to be necessary in the regulation of the TRAP promoter in osteoclasts. In addition, in vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific TRAP and CTR promoters (Matsuo et al., 2004). It is possible that c-Fos or c-Jun interaction with NFATc1 may also be involved in the regulation of the human β3 promoter.

The integrin αvβ3 is expressed on bone resorbing osteoclasts (Shinar et al., 1993) and evidence suggests that it is involved in the attachment of osteoclasts to bone (Horton et al., 1991). Blocking experiments have identified the αvβ3 integrin as a major functional adhesion receptor in osteoclasts (Horton et al., 1991, Engleman et al., 1997), where it appears to be crucial for cell spreading (Grano et al., 1994). The αvβ3 integrin is also involved in cell migration and maintenance of the sealing zone (Nakamura et al., 1999). These observations suggest that αvβ3 plays a major role in the function of osteoclasts, mediating aspects of cellular organization and function (Horton et al., 1991, Grano et al., 1994, Engleman et al., 1997, Nakamura et al., 1999).

The requirement for αvβ3 in normal osteoclast function is clearly demonstrated by genetic ablation of the β3 gene in mice (McHugh et al., 2000). Osteoclasts from mutant mice fail to spread in vitro (McHugh et al., 2000), supporting the results from blocking experiments using β3 antibodies (Horton et al., 1991). In vivo, osteoclasts in β3 knockout mice fail to form actin rings and do not form ruffled membranes, impeding them from resorbing bone effectively and resulting in an osteosclerotic phenotype (McHugh et al., 2000).

Expression of the αvβ3 integrin is regulated by expression of the β3 subunit during osteoclast differentiation (Kitazawa et al., 1995, Li et al., 1995, Zhu et al., 1996). We have previously shown that IL-4-dependent expression of the αvβ3 integrin, in osteoclast precursors is mediated by expression of the β3 gene (Kitazawa et al., 1995) and, using β3 gene promoter-reporter constructs, we have demonstrated that IL-4 dependent up-regulation is directly mediated by the transcription factor STAT-6 (McHugh et al., 2001). Thus far, little is known regarding the transcriptional mechanisms mediating β3 integrin expression during osteoclast differentiation. Therefore, we sought to understand the molecular mechanisms governing expression of the human β3 gene. In this study, we focused on the role of the calcineurin/NFATc1 pathway in the regulation of the human β3 integrin promoter during osteoclast differentiation. Using reporter constructs containing the promoter region the human β3 promoter, we demonstrated induction by RANKL and provide evidence that induction is mediated directly by NFATc1.

Section snippets

Phylogenic foot printing

The mouse and human genomic sequences encompassing the human β3 gene were identified by basic local alignment search tool (BLAST) (NCBI;www.ncbi.nlm.nih.gov/BLAST). VISTA (www.gsd.lbl.gov/vista/index.shtml) was used to identify regions of high identity and conserved regulatory elements (comparing the human promoter to the mouse genome). Putative transcription factor binding sites in the − 1242 to + 29 bp region of the human β3 gene, were identified using MATCH based on the TRANSFAC4.0 matrices (//transfac.gbf.de/TRANSFAC/

Homologous regions in the proximal promoters of mouse and human β3 integrin genes

Alignment of the 5′ end of the mouse and human β3 integrin gene sequences revealed regions of greater than 80% sequence homology across a 1.3 kb region upstream of the TSS (Fig. 1). Conservation between mouse and humans suggests that this region contains key regulatory elements (Fig. 1B). Furthermore, multiple conserved transcription factor binding elements are present within the region, as determined using the TRANSFAC transcription factor data base (Wingender et al., 2001) and confirmed with

Discussion

RANKL has been shown to act downstream of its receptor, RANK, by signaling through TRAF6 and c-Fos pathways (Takayanagi et al., 2002). RANKL selectively induces NFATc1 expression via these two pathways and triggers a sustained NFATc1-dependent transcriptional program during osteoclast differentiation (Takayanagi et al., 2002). In these studies we have shown that 1.3 kb of genomic DNA upstream of the human β3 gene, with > 80% homology to the mouse gene, is sufficient to act as a promoter in

Acknowledgements

The author of this paper holds a National Health and Medical Research Council (Aust) CJ Martin Fellowship (I.D. 200078). This work was supported by National Institute of Health Grants NIAMS R01 AR45472 (to SRG), NIAMS R01 AR47229 (to KPM) and aided by a grant from the Orthopaedic Research and Education Foundation (# 00-020, to KPM). We thank Dr P. Bray for the human β3 construct and Dr G.R. Crabtree for the NFATc1 expression plasmid.

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