FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation

Abstract

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1−/−; ERK2 flox/flox; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1−/−; ERK2 flox/flox; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.

Disruption of the ERK/MAPK pathway in neural crest cells as a potential cause of Pierre Robin sequence

“…This model is based on the activation pattern of the ERK pathway, in which P-ERK is detectable in mesenchymal cells that are committed to the osteogenic lineage but disappears at later stages in differentiated Col1a1-positive osteoblasts. In contrast to our data, late effects of the ERK pathway on osteoblast differentiation have been reported in limbs and calvarial bone (Ge et al, 2007;Kyono et al, 2012). Further analyses of downstream molecular events in Wnt1-Cre; Erk2 fl/fl mice are needed to understand the functional mechanism of the ERK pathway during mandible bone development.…”

“…Mandibular bone formation occurs via intramembranous ossification, in which mesenchymal cells differentiate directly into osteoblasts (Orliaguet et al, 1993). Previous in vitro and in vivo studies have shown that the ERK pathway is involved in the regulation of both endochondral and intramembranous ossification processes (Chen et al, 2014;Kyono et al, 2012). For example, Prx1-Cre;Erk1 −/− ;Erk2 fl/fl mice exhibit defective bone formation in their limbs and calvaria, which is mainly caused by a disruption of the osteoblast differentiation program after Runx2, Sp7 and Atf4 expression and before osteocalcin (Bglap) expression.…”

Fibroblast growth factor signaling in skeletal development and disease

“…Finally, in an osteocyte cell line, FGF2-stimulated ERK1/2 activation was found to increase the expression of Dmp1, a marker of osteocytes. In vivo, Dmp1 expression is reduced in limbs of mice lacking ERK1 and conditionally lacking ERK2 (Prx1-Cre), which show no osteocytes, indicating that FGF signaling coordinately regulates Dmp1 expression and osteocyte differentiation (Kyono et al 2012). Overall, these studies establish that multiple pathways activated by FGF/FGFR signaling control all steps of osteoblastogenesis (Fig.…”

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

“…In that study, the authors report abundant expression of DMP1 in murine, canine and human OS and evidence of osteoblastic/osteolytic lesions in mice injected with MLO-Y4 mouse osteocyte-like cell line [30]. Another study reported that FGF23 up regulates DMP1 mRNA in MLOY4 cells [31]. Neither the source and status of FGF23 nor its impact on oncogenic osteomalacia in OS is clear.…”