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First published online 20 March 2007
doi: 10.1242/jcs.000067

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Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling

¹ Division of Gene Therapy Science, Graduate School of Medicine, Osaka University 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
² Department of Orthopedic Surgery, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
³ Department of Orthopedic Surgery, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan

View larger version (29K): [in this window] [in a new window]   Fig. 1. Twist-1 overexpression suppresses BMP2-induced osteoblast differentiation. (A) Immunoprecipitation of overexpressed Flag-tagged Twist-1 in the nuclear extracts of MC3T3-E1-Tw1 and MC3T3-E1-WT cells. (B) MC3T3-E1-Tw1 and MC3T3-E1-WT cells were grown in the presence or absence of rhBMP2 (300 ng/ml) for 6 days. ALP activity was measured as described in the Materials and Methods (*P<0.01). (C) MC3T3-E1-Tw1 and MC3T3-E1-WT cells were grown in the presence or absence of rhBMP-2 (300 ng/ml) and total RNA was isolated on day 6. Northern blot analysis was performed using osteopontin and osteocalcin cDNA probes.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Downregulation of endogenous Twist-1 using RNAi methods enhances BMP-induced transcriptional activity mediated by Smads. (A) C3H10T1/2 cells were transfected with either Twist-1-specific siRNA (siTwist-604, siTwist-691, siTwist-645, siTwist-481) or control siRNA (Scrambled) as described in the Materials and Methods. Total RNA was extracted 24 hours later, and northern blot analysis was performed using a Twist-1 cDNA probe. (B) C3H10T1/2 cells were transfected with either siTwist-691 or control siRNA (Scrambled). Cells were transfected with 3GC2-Lux and TK-Renilla luciferase 24 hours after siRNA transfection. At 12 hours after second transfection, cells were treated, with BMP (300 ng/ml) or left untreated, for 12 hours. Cells were lysed and luciferase activity was assayed (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in the scrambled sample without BMP was approximately 2,000 and 34,000 RLU (relative light unit), respectively. (C) C3H10T1/2 cells were transiently transfected with either 90 pmol of siTwist-691 or Scrambled in combination with 100 ng of 3GC2-Lux luciferase construct, Smad1, Smad4 and BMPR-IB(QD). Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in the scrambled sample without BMPR-1B(QD) was approximately 411,000 and 30,000 RLU, respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Twist-1 inhibits BMP-induced Smad transcriptional activity with E-protein. (A) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1, Smad4, E47 and increasing doses (12.5 or 50 ng) of Twist-1 expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in cells transfected with luciferase expression plasmids was approximately 109,000 and 681,000 RLU, respectively. (B) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1, Smad4, E47, Twist-1 and Id1 construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in cells transfected with luciferase expression plasmids was approximately 51,000 and 199,000 RLU, respectively. (C) Flag-tagged full-length (FL) Twist-1 or deletion Twist-1 mutant (Twist-NBCT) and Myc-tagged E47 constructs were transfected into COS-7 cells. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Myc antibody. (D) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1 and Smad4, E47, full-length Twist-1 and deletion Twist-1 mutant (Twist-NBCT) expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (**P<0.01, *P<0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Twist-1 can interact with HDAC1 and Smad4. (A) MC3T3-E1-WT and MC3T3-E1-Tw1 cells were treated with BMP2 (300 ng/ml) for 24 hours. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Smad4 and HDAC1 antibody. 10% In, 10% of input; IP, immunoprecipitated fraction. (B) MC3T3-E1-Tw1 cells were treated with BMP alone [TSA (–)] or the mixture of BMP and TSA (82.5 µM) [TSA (+)]. After 24 hours, total RNA was extracted and the expression of ALP, Runx2 and osteopontin was quantified by real-time PCR. The expression levels were normalized by GAPDH, and the ratio was shown in each sample. Data are presented as mean ± s.d. of triplicate samples (*P<0.05).

View larger version (42K): [in this window] [in a new window]   Fig. 5. Twist-1 protein stability is increased by the formation of a heterodimer complex with E47. (A) Myc-tagged Twist-1 and E47 constructs and GFP expression vector were transfected into COS-7 cells. Western blot analysis was performed with anti-Myc antibody. To show transfection efficiency, GFP protein was also detected by western blot. (B) COS-7 cells were transfected with the Flag-tagged Twist-1, Myc-tagged E47 and/or Myc-tagged Id1. At 24 hours after transfection, cells were pulsed with [35S]methionine and cysteine for 3 hours, and chased with unlabeled medium for the indicated times. Labeled cell lysates were immunoprecipitated using anti-Flag antibody. Flag-tagged Twist-1 was visualized using SDS-PAGE.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Id1 inhibits Twist-1 function by sequestering E47 from Twist-1. (A) COS-7 cells were transiently transfected with Flag-Twist-1, Myc-tagged E47 and increasing doses (0.5, 1 or 2 µg) of Myc-tagged Id1 construct. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Flag and anti-Myc-antibody. (B) COS-7 cells were transiently transfected with Flag-Id1, Myc-tagged E47 and increasing doses (0.5, 1 or 2 µg) of Myc-tagged Twist-1 construct. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Flag and anti-Myc-antibody. (C) Endogenous E47 in C3H10T1/2 cells was precipitated using anti-E47 antibody. Then, Id1 was detected in the immunoprecipitates by western blot. (D) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1 and Smad4, 25 ng of Twist-1 and E47, and increasing doses (25 or 100 ng) of Id1 expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). (E) MC3T3-E1-Tw1 cells were transiently transfected with either Id1 or GFP expression construct by electroporation (Amaxa). The cells were grown in the presence or absence of rhBMP2 (300 ng/ml) for 6 days. ALP activity was measured as described in the Materials and Methods. Data are presented as mean ± s.d. of triplicate samples (*P<0.05).

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