Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors

Abstract

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G 1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21 WAF1/Cip1 . Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes.

Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells

“…Although the function of TWIST is not fully established at the molecular level, recently TWIST has been indicated as a potential oncogene interfering with p53-related pathways leading to preventing myc-induced apoptosis in mouse embryonal fibroblasts (Maestro et al, 1999). TWIST is also found to be involved in the suppression of differentiation and protection of apoptosis through inhibition of p21 Waf1 via both p53-dependent and -independent pathways (Funato et al, 2001;Hjiantoniou et al, 2003). More recently, the antiapoptotic function of TWIST has been indicated through modulating NFkB pathways, and loss of TWIST expression leads to sensitization to TNFainduced apoptosis (Sosic et al, 2003).…”

“…As shown in Figure 3a, TWIST mRNA and protein expressions were 4.0-and 5.2-fold higher in HNE1-T3 cells as compared to HNE1 cells, respectively, which confirms the CGH results and indicates a TWIST gene amplification in HNE1-T3 cells. Since p53, p21 Waf1 , MDM2, Bax proteins have been reported to be downstream factors of the TWIST protein (Maestro et al, 1999;Funato et al, 2001;Yousfi et al, 2002), we next studied the expression of these proteins in HNE1-T3 and HNE1 cells. As shown in Figure 3b, p53, p21 Waf1 protein levels were low in HNE1-T3 cells, but MDM2 levels were high, which agrees with a previous study that TWIST may negatively interfere with the p53 tumour suppressor pathway (Maestro et al, 1999).…”

TWIST Family of Basic Helix-Loop-Helix Transcription Factors Mediate Human Mesenchymal Stem Cell Growth and Commitment

“…Previous reports have identified Twist-1 and Twist-2 expression in different murine osteoblastic cell lines, and have shown that both genes are downregulated during osteogenic differentiation in vitro [14,20,[23][24][25]. Glackin and colleagues reported that enforced expression of either Twist-1 or Twist-2 in human osteosarcoma cell lines inhibited their ability to undergo osteoblastic maturation, whereas overexpression of Twist-1 or Dermo-1 antisense increased bone cell maturation [11,13].…”

Twist and p53 reciprocally regulate target genes via direct interaction

“…Twist has been shown to be involved in several pathways that control tumor cell growth, apoptosis, differentiation and epithelial-mesenchymal transition (Maestro et al, 1999;Funato et al, 2001;Valsesia-Wittmann et al, 2004;Yang et al, 2004;Alexander et al, 2006). Moreover, Twist inhibits the p53-mediated response to cellular stress (Stasinopoulos et al, 2005).…”