Inductive Effects of Dexamethasone on the Gene Expression of Cbfa1, Osterix and Bone Matrix Proteins During Differentiation of Cultured Primary Rat Osteoblasts

Abstract

Runx2/core binding factor alpha 1 (Cbfa1) and Osterix (Osx) are osteoblast-specific transcription factors essential for the development of a mature osteoblast phenotype and are thought to activate osteoblast marker genes in vivo to produce a bone-specific matrix. Dexamethasone (Dex) is known to be a potent stimulator of osteoblastic differentiation in vitro, however, the exact role is still unclear. To investigate the mechanisms of the stimulation of osteoblastic differentiation by Dex, we evaluated the effects of Dex on proliferation and mineralization as well as on mRNA expression of Cbfa1, Osx and osteoblast marker genes, osteocalcin (OC) and bone sialoprotein (BSP) mRNAs in differentiating foetal rat calvarial cells (FRCC), which were cultured for 35 days in the presence or absence of 10(-7) M Dex. Treatment of FRCC with Dex resulted in the stimulation of cell proliferation and increased the number of cells, which are able to produce bone-like nodules with a mineralized matrix when compared to untreated controls. Northern blot analysis revealed that, in the absence of Dex, Cbfa1 mRNA expressed at day 8, while Osx mRNA expressed at day 15. Subsequently expression of these mRNAs increased up to day 21, followed by constant expression during the culture period. The expression of OC and BSP mRNAs appeared to be synchronous with that of Osx mRNA and was detectable at day 15 with an increase thereafter. The presence of Dex resulted in an induction in Cbfa1 and Osx mRNA expression. The former appeared at day 5 and the latter appeared at day 11. Subsequently expression of Cbfa1 and Osx mRNAs increased up to day 15 with a decrease thereafter. Expression of OC and BSP mRNAs appeared to be coincident with that of Osx mRNA and was detectable at day 11 and reached a maximum at day 15 followed by constant expression. These observations indicate that induction of Cbfal and Osx mRNAs by Dex may be followed by activation of osteoblast marker genes such as OC and BSP mRNAs to produce a bone-specific matrix that subsequently becomes mineralized. Thus, it is likely that Dex may promote osteoblastic differentiation and mineralization of FRCC by inducing the expression of Cbfa1 and Osx genes in vitro.

Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

“…On the other hand, dexamethasone-treated osteoblasts underwent a concurrent down-regulation of E4BP4 and increased expression of both Runx2 and Osterix. Although E4BP4 is induced by dexamethasone in osteoblasts (21), this up-regulation in response to dexamethasone is in line with other studies (42), and correlates with its concomitant inhibition.…”

Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

“…However, overexpression of Osx in ES cells was unexpectedly found to upregulate Runx2/Cbfa1 expression [15] as a result of a possible feedback mechanism of Osx to Cbfa1. However, the overexpression of Osx in the latter study was performed in the presence of dexamethasone, which is a potent stimulator of osteogenic commitment and differentiation in vitro [26,27], and increases the expression of Runx2/Cbfa1 and Osx in differentiating fetal rat calvarial cells [28]. In our studies, osteogenic media that included 10 −8 M dexamethasone also increased levels of Cbfa1 and Osx in murine BMSC.…”

“…However, our study shows for the first time that overexpression of Osx in adult BMSC leads to the increased expression of osteogenic differentiation markers (OC, AP, BSP, and OPN) under experimental conditions that do not affect Runx2/Cbfa1 expression. The finding that RCAS-Osx transduced BMSC did not exhibit an upregulated expression of Runx2/Cbfa1 was not surprising, based on a previous report However, the overexpression of Osx in the latter study was performed in the presence of dexamethasone, which is a potent stimulator of osteogenic commitment and differentiation in vitro [26,27], and increases the expression of Runx2/Cbfa1 and Osx in differentiating fetal rat calvarial cells [28]. In our studies, osteogenic media that included 10 −8 M dexamethasone also increased levels of Cbfa1 and Osx in murine BMSC.…”

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

“…It has been reported that overexpression of BMP-2 inhibited the growth of a cell line derived from rat osteosarcoma, though it promoted their osteogenic differentiation (Huang et al, 2002). Other reports also indicated that proliferative abilities of osteoblasts and MSCs are influenced by osteogenic induction (Lee et al, 2004;Igarashi et al, 2004;Cho et al, 2005). It is generally believed that osteogenic induction on pluripotent cells with gene therapy approaches or chemical inducement decreases their proliferation rate.…”