Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin‐D3, dexamethasone, and local growth factors in primary human osteoblasts

Abstract

Core binding factor alpha 1 (Cbfa1) is an osteoblast-specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic-differentiating agents is still unclear. In this study, we assessed the effects of 1,25-(OH)(2)-D3 (D3), ascorbic acid, bone morphogenetic protein-2 (BMP-2), dexamethasone (Dex), and transforming growth factor-beta (TGF-beta) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT-PCR) in an in vitro model of osteoblast differentiation. TGF-beta increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6-fold in a time-dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time-dependent fashion by up to 4.6-fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP-2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2-fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5-fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14-fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation.

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT

“…ALP is an important early marker of osteoblast differentiation (Kim et al 2004, ten Dijke et al 2003 which controls the precipitation of hydroxyapatite crystals in the early phase of mineralization in bone formation process (Anderson 2003, Manolagas 2000. Moreover, Chaudhary et al (2004) have reported that the ALP activity is closely associated with the formation of mineralized matrix while OCN, BGN and COL1A2 are major proteins commonly found in extracellular bone matrix and are believed to play an important role in bone formation (Chen et al 2003, 2004, Kuroki et al 1994, Parisuthiman et al 2005, Pischon et al 2004, Regazzoni et al 2001, Sierra et al 2003, Takeuchi et al 1996, Takuwa et al 1991, Viereck et al 2002, Young et al 2002.…”

Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Seeded on Melt Based Chitosan Scaffolds for Bone Tissue Engineering Applications

“…Runx2 is essential for the differentiation of MSCs into mature osteoblasts in the skeletal development of numerous mammalian organisms. 42,43 Osteocalcin, one of the few osteogenic specific genes, is a bone matrix protein and is known to play an important role in the differentiation of osteoblast progenitor cells, with significant up-regulation observed both in matrix synthesis and in the mineralization process. 43 BSP is secreted, bind cell surface integrin receptors, and regulate mineralization, and type I collagen represents the majority of the organic part of bone matrix.…”

Runx2 Integrates Estrogen Activity in Osteoblasts

“…In contrast, high levels of glucocorticoid, such as those commonly used therapeutically to suppress inflammatory disease or tissue rejection, cause generalized bone loss leading to fractures as well as gastrointestinal and immunological problems (17,18). Although in some instances glucocorticoid may enhance Runx2 mRNA expression, in vitro glucocorticoid in excess rapidly depletes the amount of functional Runx2 nuclear protein, with an accompanying inhibitory effect on Runx2-dependent gene expression (19,20). This seems to reprise the downstream events that occur with glucocorticoid therapy or with Runx2 gene deletion in vivo (7) and would essentially limit osteoblast activity in the adult remodeling skeleton.…”