Phosphate-Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra-1

Abstract

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra-1-deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra-1 transgenic osteoblasts, suggesting a role for Fra-1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi-dependent regulation of MGP. In MC3T3-E1 cells and primary calvaria-derived osteoblasts, Pi increased MGP and Fra-1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi-stimulated MGP and Fra-1 expression, indicating that ERK1/2 is required for Pi-dependent regulation of MGP and Fra-1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra-1 interacts with the MGP promoter in response to Pi in MC3T3-E1 cells. Finally, we found that in fra-1 knockdown MC3T3-E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra-1-deficient mice failed to exhibit a Pi-dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2-Fra-1 pathway.

The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals

“…These observations are in agreement with a study demonstrating an increase in ERK1/2 phosphorylation, but not p38 or JNK, in response to bisphosphonates that is further induced by the addition of 3 mM Pi [127]. In a recent study, we obtained similar results and confirmed that Pi effect induces the specific activation of the ERK1/2 signaling pathway in osteoblasts [83]. We have shown that this pathway is required for the effect of Pi on the expression of matrix gla protein (MGP), a known inhibitor of calcification.…”

“…Although more detailed studies are necessary, our observations support the idea that crystal formation more than Pi itself may be required for the increase in ERK phosphorylation and PiT1 and PiT2 expression [82]. In line with this work, we recently obtained similar data in mouse primary osteoblasts and MC3T3-E1 cells where the Pi stimulation of MGP expression via the ERK1/2 pathway [83] correlated with the presence of calcium in the medium and could be abolished by the use of crystal formation inhibitors (Khoshniat et al, unpublished observation).…”

“…Accordingly, previous studies have shown that phosphorylation of Fra-1 by the MAPK ERK1/2 resulted in Fra-1 protection from proteosomal degradation [130] and subsequent post-translational stabilization [131]. Interestingly, in a recent report, we have observed that the regulation of MGP expression by Pi through the ERK1/2 signaling pathway involves the stimulation of the DNA binding activity of Fra-1 to specific sequences in the MGP promoter [83].…”

“…Beck et al have found that Pi selectively activates the extracellular signal regulated kinase (ERK1/2) signaling pathway to mediate its effect on osteopontin expression in MC3T3 cells [83,126]. Increased extracellular Pi concentration caused a rapid and biphasic phosphorylation of ERK1/2 but did not activate the other mitogen-activated protein kinase (MAPK) signaling proteins, p38 or the c-jun N-terminal kinase (JNK).…”

Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and collagen production

“…In Fra1-deficient mice, the low bone mass phenotype was reported to be a result of defects in the late maturation of osteoblasts to bone matrix-producing cells accompanied by insufficient induction of the bone matrix components Mgp, Oc, and col12. Up-regulation of Oc and col12 expression was less pronounced in bones of Fra-1 tg mice compared with Mgp , which is directly regulated by Fra-1 (Julien et al, 2009). In this study, we did not detect any binding of Fra-1 to the Oc or col12 promoters, suggesting that Fra-2 might be the major AP-1 factor directly regulating this important collagen component.…”