Figure 1.
Hypoxia inhibits the expression of RUNX2 and the mineralization capacity of MSCs and zebrafish.
MSCs form three individuals (D1, D2, and D3) were induced in OIM under normoxia (Nor) or hypoxia (Hyp) (A, B) or treated with indicated concentration of DFX for 3 days (C, D) (n = 3). Cells were analyzed by quantitative RT-PCR (A, C) and Western blotting (B, D). Results are shown as the mean ± SD values. Significance was determined by Student's t-test. (* p<0.05 and ** p<0.01 versus Nor or without DFX). E, MSCs without (CTR) or with induction in OIM under normoxia or hypoxia for 14 or 21 days were stained by Alizarin Red S (ARS) and quantification of staining was performed by optic density (O.D.) measurement at O.D.550 nm (n = 3).
Figure 2.
Expression of Type1 and Type2 RUNX2 in MSCs induced for osteogenic differentiation.
Quantitative RT-PCR for mRNA levels of indicated genes. A, MSCs were induced in OIM for indicated time period (n = 3). B, MSCs were induced in OIM without or with noggin treatment for 24 h (n = 3). C, MSCs were induced in OIM under normoxia (Nor) or hypoxia (Hyp) for 3day (n = 3). D, E, MSCs were induced in OIM or with indicated concentration of DFX for indicated time period (n = 3). F, MSCs were induced in OIM in the presence of DFX without or with rhBMP2 for 24 h (n = 3). Results are shown as the relative expression to GAPDH (mean ± SD), and significance was determined by Student's t-test. (* p<0.05 and ** p<0.01 versus black bar).
Figure 3.
Hypoxia inhibits osteogenesis by MSCs through the HIF-1α-TWIST pathway.
A, B, MSCs form three individuals (D1, D2, and D3) were induced in OIM under normoxia (Nor) or hypoxia (Hyp) or treated with indicated concentration of DFX for 3 days. Cells were analyzed by Western blotting. C, MSCs were transfected with control pFLAG-CMV1 or pFLAG-TWIST vector followed by induction in OIM without DFX treatment (Nor) for 2 days (n = 3). D, MSCs were transfected with control pSuper-Scramble or pSuper-TWIST-si vector followed by induction in OIM in the presence of 100 µM DFX (Hyp) for 2 days (n = 3). Cells were assayed by Western blotting and quantitative RT-PCR. Results are shown as the mean ± SD values, and significance was determined by Student's t-test. (* p<0.05 and ** p<0.01 versus control vector).
Figure 4.
TWIST inhibits Type 1 RUNX2 transcription via binding to its promoter.
A, Genomic organization of the region flanking the promoter region of human RUNX2 P2 (upper panel) and the schematic representation of the pGL3-RUNX2 P2 reporter construct. Transcription start site, TSS. (B, C) Reporter assays showing, in a MSC cell line (B) or 293T (C), DFX, TWIST and exposure to hypoxia repress the RUNX2 P2 promoter activity in a dose dependent manner (n = 3). β-galactosidase was used as a control of transfection efficiency. D, Mutational analysis of E1-box and E5-box sites in the RUNX2 P2 promoter in 293T cells. Reporter constructs containing wild-type RUNX2 P2 (WT), E1-box (E1M) or E5-box (E5M) mutations, or double mutations (E1E5M) were generated and used to analyze the importance of these sites in mediating repression by TWIST (n = 3). E, Truncation of the bHLH (basic and helix loop helix) domain (tb TWIST) inhibits TWIST-mediated repression of RUNX2 P2 promoter activity. (n = 3). Each ratio was normalized to the control (pGL3 basic vector), and significance was determined by Student's t-test. (* p<0.05 and ** p<0.01 versus control without DFX, TWIST or exposure to hypoxia). EMSA and ChIP assays demonstrate TWIST binds to the E-box motif in the RUNX2 P2 promoter (−1324 to −1144). F, Oligonucleotides for EMSA were the 28 bp probe from the RUNX2 P2 promoter, which contained a TWIST E1-box sequence (TEB). Nuclear extracts prepared from 293T cells, transiently transfected with pFLAG-TWIST, were incubated with [α-32P]-CTP-labelled probe before electrophoresis. No protein extracts: (lane 1, 7 and 13). Competition assays were performed in the presence of excess wild type (WT) (lane 3, 4, 9–12) or mutation type (MT) (lane 5 and 6) of unlabelled oligonucleotides containing the TBE. The supershift assay was performed in the presence of an isotype (lane 14) or anti-TWIST antibody (lane 15), and the position of supershifted bands is indicated (arrow). G, ChIP analysis of MSCs and 293T after transfection of pFLAG-TWIST. The two chromatins were incubated either without antibodies, with an anti-TWIST antibody or with isotype IgG antibody. The two samples with 180 bp fragment of the E1-box in the RUNX2 P2 promoter were amplified by PCR (left panel) and were also quantified with quantitative RT-PCR (right panel). Input, 2% of total input lysate. Results are shown as the mean ± SD values.