Osteopontin (OPN), a member of the SIBLING family of extracellular matrix proteins, plays an important role in regulating mineralization and modulating osteoclast-mediated resorption of bone. Previous studies by our laboratory (Foster et al., 2006
; Rutherford et al., 2006
) and others (Beck and Knecht, 2003
; Conrads et al., 2005
) have demonstrated that Pi regulates the expression of many genes in cementoblasts or osteoblasts, including OPN and other SIBLING proteins, as well as signaling molecules and transcription factors. In this study, we sought to determine if Pi affects OPN transcription directly or operates through a post-transcriptional mechanism. Our results demonstrate that Pi affects OPN expression directly by inducing OPN transcription, and further, that this response requires GR.
It is well-established that glucocorticoids play numerous key roles in bone development. Clinically, the use of glucocorticoids to treat inflammatory diseases often leads to osteoporosis, due to the inhibition of osteoclast-mediated bone resorption and arrested bone formation (Kim et al., 2006
). In osteoblasts and cementoblasts, glucocorticoids regulate the expression of many genes, including OPN, BSP, osteocalcin, and DMP-1 (Hakki et al., 2005
; Leclerc et al., 2005
; Ogata et al., 1995
; Mikami et al., 2008
). The mechanisms are not well-understood; however, recent evidence suggests that glucocorticoids function in part by regulating the phosphorylation state and activity of the transcription factor Runx2/Cbfa1 (Phillips et al., 2006
). The down-regulation of osteocalcin transcription by dexamethasone has been shown to involve inhibition of the activity of an Egr2/Krox20 enhancer located upstream of a Runx2 site (Leclerc et al., 2005
In this report, we demonstrated by EMSA that a nuclear protein was able to bind to the OPN GRE. However, we were unable to verify, via
supershift experiments with GR antibodies, that this DNA-binding protein was GR. Dexamethasone caused a slight increase in OPN gene transcription, though the increase was not significant. This finding is in accordance with previous studies in rat osteoblastic cell lines which demonstrated that dexamethasone caused only a slight increase in OPN gene transcription, measured by nuclear run-on assay (Ogata et al., 1995
). In contrast, BSP is up-regulated by glucocorticoids, and osteocalcin decreases in expression. Therefore, while the OPN GRE can be classified as a GRE based on sequence considerations, it does not function as a classic glucocorticoid-inducible motif. Rather, we propose that it functions in a Pi signaling pathway, probably in association with other transcription factors or co-activators (Ogata et al., 1995
; So et al., 2007
). The human OPN promoter also contains a potential GRE (5′ TTAATAaccTGTTCT 3′) at nucleotide positions -946 to -932. A potential GRE is present in the OPN promoter of other species, including dog, cow, gorilla, and macaque. Further studies will be needed to determine if this evolutionarily conserved GRE-like motif functions in other species to regulate Pi-mediated expression.
Phosphate is a potent regulator of bone mineralization; however, little is known about the molecular mechanisms involved (reviewed in Beck, 2003
; Foster et al., 2007
). In this study, we have shown that GR signaling is required for Pi-regulated transcription. It was previously reported that Pi regulation of OPN expression requires the activity of ERK1/2 and protein kinase C, as well as the proteosomal/ubiquination pathway (Beck and Knecht, 2003
). The well-established role of the proteasome pathway in the regulation of nuclear receptors, including GR (Dennis and O’Malley, 2005
), is consistent with a model in which GR turnover is an important component of Pi signaling and the induction of gene expression.
In conclusion, we have characterized a region of the murine OPN promoter that is required for Pi-mediated transcription. This region contains a GRE which is required for the Pi response. Further, the Pi-induced transcription was inhibited with a GR antagonist, implying that GR plays a direct role in the Pi signaling pathway. This work should facilitate further studies on OPN and other Pi-inducible genes to determine the precise role of GR signaling in regulating gene expression during the mineralization of bone.