Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells

Abstract

Periodontal ligament (PDL) is a thin fibrous connective tissue located between two mineralized tissues, alveolar bone and cementum, which maintains a constant width physiologically. The mechanisms by which PDL resists mineralization are not well understood. Twist is a basic helix loop helix protein that plays a central role in regulation of early osteogenesis. We investigated the localization of Twist in PDL and compared the expression of Twist and osteoblast-related genes in PDL cells with those in osteoblast-like cells in the presence or absence of recombinant human bone morphogenetic protein (BMP)-2. Histochemical analysis showed that Twist was expressed along alveolar bone surface in PDL. PDL cells constitutively expressed Twist gene and the expression level was higher than that in osteoblast-like cells. In osteoblast-like cell culture, BMP-2 enhanced osteoblast-related gene expression, while Twist expression was slightly decreased. In contrast, BMP-2 increased runt-related transcription factor (Runx)-2, but failed to enhance alkaline phosphatase (ALP) and osteocalcin (OCN) gene expression in PDL cells. Interestingly, unlike in osteoblast-like cells, Twist expression was upregulated by BMP-2 in PDL cells. We transiently knocked down Twist gene in PDL cells using a short interference RNA expression vector (siTwist) and found that ALP, osteopontin (OPN), bone sialoprotein (BSP) genes expression and basal level of ALP activity were slightly increased, whereas Runx2 and OCN genes were not affected. Collectively, these results suggest that Twist may act as a negative regulator of osteoblastic differentiation in PDL cells.

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

“…Our functional studies showed that enforced expression of either Twist-1 or Dermo-1 caused a marked reduction in the capacity of human MSC to undergo osteogenic differentiation in vitro and in vivo, similar to the findings of Komaki and colleagues who showed that Twist-1 negatively regulated osteo/cementoblast differentiation in human periodontal ligament cells [44]. Potential mechanisms may involve heightened expression of Runx2 in our TWIST-1 and DERMO-1 overexpressing MSC lines, whereas previous studies reported that high levels of Runx2 caused a reduction in the expression of target genes such as OCN, resulting in an inhibition of bone cell differentiation [47][48][49].…”

“…cells, including BSP, OPN, and OCN. Previous findings have demonstrated an increase in ALP, OPN, and BSP gene expression following siRNA knockdown of Twist in human periodontal ligament cells [44], and Twist antisense knockdown in osteosarcoma cell lines [11]. Other studies have also reported a reduction in Runx2 expression and activity in TWIST haploinsufficiency mutant osteoblasts [45], and in cancer cell lines, following shRNA knockdown of Twist-1 [46].…”

Scleraxis and osterix antagonistically regulate tensile force-responsive remodeling of the periodontal ligament and alveolar bone

“…The regions of the control (A-D,I-L) and experimental (E-H,M-P) sides correspond to the boxed regions in Fig. 4C and D that Mgp, asporin, msh homeobox 2 (Msx2) and twist-related protein 1 (Twist1) act as inhibitors of the mineralization of the PDL (Hashimoto et al, 2001;Kaipatur et al, 2008;Komaki et al, 2007;Murshed et al, 2004;Yamada et al, 2007;Yoshizawa et al, 2004). Scx is also a member of the twist subfamily of basic helix-loophelix transcription factors (Atchley and Fitch, 1997).…”

Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer

“…The explants were covered with sterilized glass cover slips in a 100-mm culture dish and incubated with Dulbecco's modified Eagle medium (DMEM) (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 50 mg ml -1 streptomycin and 50 U ml -1 penicillin (GIBCO BRL) at 37 1C in a humidified atmosphere of 5% CO 2 and 95% air. 43 Cells from passages 3-8 were used in the experiments. HEK 293 cells and Saos-2, a human osteogenic sarcoma cell line, were cultured under the same conditions.…”