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First published online 14 November 2006
doi: 10.1242/jcs.03281

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Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-ß-dependent mechanism

¹ Medical Research Center for Ischemic Tissue Regeneration of Pusan National University and the Medical Research Institute, College of Medicine, Pusan National University, Busan 602-739, Republic of Korea
² Department of Plastic Surgery, Medical Research Institute, College of Medicine, Pusan National University, Busan 602-739, Republic of Korea
³ Department of Physiology, Medical Research Institute, College of Medicine, Pusan National University, Busan 602-739, Republic of Korea

View larger version (49K): [in this window] [in a new window]   Fig. 1. Dose- and time-dependent effects of SPC on the expression levels of -SMA. Serum-starved hATSCs were treated with the indicated concentrations of D-erythro-SPC (A) or L-threo-SPC (B) for 4 days. (C) Serum-starved hATSCs were treated with vehicle or 2 µM D-erythro-SPC for the indicated time periods. (D) Serum-starved hATSCs were treated with 0.1% DMSO (control), 5 µM LPC, 1 µM S1P or 5 µM D-erythro-SPC for 4 days. The expression levels of -SMA and actin were determined by western blotting with anti--SMA and anti-actin antibodies, respectively (upper panels). The densities of -SMA and actin were quantified from three independent experiments, and the expression levels of -SMA were normalized to total actin levels in the samples (lower panels). The data are presented as a percentage of control. *Significantly different from control value (P<0.05).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Effects of SPC and TGF-ß on the differentiation of hATSCs to SMCs. (A) Serum-starved hATSCs were treated with indicated concentrations of TGF-ß1 or TGF-ß3 for 4 days, and the expression levels of -SMA and actin were determined by western blotting with anti--SMA and anti-actin antibodies, respectively. Representative data from three independent experiments are shown. (B) The quantities of -SMA and actin were determined from the three experiments in A, and the expression levels of -SMA were normalized to total actin levels in the samples. (C) Serum-starved hATSCs were challenged with 2 µM D-erythro-SPC and/or 2 ng/ml TGF-ß3 for 4 days. The expression levels of -SMA, h1-calponin, and actin were probed with anti--SMA, anti-h1-calponin and anti-actin antibodies, respectively. Representative data from three independent experiments are shown. (D) The quantities of -SMA, h1-calponin and actin were determined from the three experiments in C, and the expression levels of -SMA and h1-calponin were normalized to total actin levels in the samples. The data are presented as a percentage of control (mock-treated cells). These data were analysed using Student's t-test and considered to be significantly different from the control when P<0.05 (*).

View larger version (41K): [in this window] [in a new window]   Fig. 3. Effects of SPC and TGF-ß3 on the transcriptional levels of smooth-muscle-specific markers. (A) Serum-starved hATSCs were treated with vehicles, 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for the indicated time, and -SMA mRNA was quantified by real time RT-PCR analysis. Data represent average values ± s.e.m. of triplicate determinants. *Significantly different from control value (P<0.05). (B) Serum-starved hATSCs were exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 24 hours, and the levels of -SMA, h1-calponin, SM22 and GAPDH mRNA were determined by semi-quantitative RT-PCR using gene-specific primers. Representative data from three independent experiments are shown. (C) hATSCs were treated with vehicles, 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 24 hours, and the expression levels of h1-calponin and SM22 were quantified by real time RT-PCR. Data represent average values ± s.e.m. of triplicate determinants. *Significantly different from control value (P<0.05).

View larger version (117K): [in this window] [in a new window]   Fig. 4. Effects of SPC and TGF-ß3 on the formation of actin stress fibers and the localization of -SMA in hATSCs. Serum-starved hATSCs were treated with vehicles, 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days, and immunostaining performed. -SMA and F-actin were double-stained with anti--SMA and Alexa Fluor 568 phalloidin, and analysed using a Leica TCS-SP2 laser scanning confocal microscope (Leica Microsystems, Germany) with a magnification of 400x. The overlaid images of the double staining are shown. Representatives of three independent experiments are shown.

View larger version (49K): [in this window] [in a new window]   Fig. 5. The role of PTX-sensitive G proteins in the SPC-induced differentiation of hATSCs to SMCs. (A) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for the indicated time periods, and the phosphorylation level of Smad2 was determined by western blotting with anti-phospho-Smad2 antibody. Anti-actin antibody was used to confirm equal loading. (B) The densities of p-Smad2 and actin were quantified in duplicate, and the phosphorylation levels of Smad2 were normalized to total actin levels in the samples. Relative densities of p-Smad2 are shown as mean ± s.e.m. Asterisks indicate significant difference from control values (*P<0.05, #P<0.01). (C) hATSCs were pretreated with 100 ng/ml PTX for 24 hours. The cells were then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 10 minutes or 4 days, and the phosphorylation level of Smad2 was determined by western blotting with an anti-phospho-Smad2 antibody. Anti-actin antibody was used to confirm equal loading. Representative data from three independent experiments are shown. (D) Serum-starved hATSCs were pretreated with 100 ng/ml PTX for 24 hours. The cells were then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days, and the expression levels of -SMA and actin were determined by western blot analysis. Representative data from three independent experiments are shown. (E) The densities of -SMA and actin were quantified from the duplicate determinations, and the expression levels of -SMA were normalized to total actin levels in the samples. The data are presented as a percentage of control (mock-treated cells). *Significantly different from control value (P<0.05).

View larger version (35K): [in this window] [in a new window]   Fig. 6. The role of ERK in SPC-induced expression of -SMA and delayed phosphorylation of Smad2. (A) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for the indicated time periods. (B) hATSCs were pretreated with 100 ng/ml PTX for 24 hours, and then exposed to 2 µM D-erythro-SPC for 10 minutes. (C) hATSCs were pretreated with 10 µM U0126 for 15 minutes, and then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days. (D) hATSCs were pretreated with 10 µM U0126 for 15 minutes, and then exposed to 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 10 minutes. The expression levels of -SMA, actin, and ERK were determined by western blotting with anti--SMA, anti-actin and anti-ERK antibodies, respectively. The phosphorylation levels of ERK and Smad2 were determined by western blotting with anti-p-ERK and anti-p-Smad2 antibodies, respectively. Representative data from three independent experiments are shown.

View larger version (43K): [in this window] [in a new window]   Fig. 7. The role of the TGF-ß-dependent pathway in SPC-induced SMC differentiation and the late activation of Smad2. (A) Serum-starved hATSCs were pretreated with 10 µM SB-431542 or vehicle, and then treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days. The expression levels of -SMA and actin, and the phosphorylation levels of ERK and Smad2 were determined by western blotting. Representative data from three independent experiments are shown. (B) hATSCs were exposed to 2 µM D-erythro-SPC for 2 days in the absence or presence of 10 µM U0126, and then the conditioned media were subjected to ELISA for quantification of TGF-ß1 protein. Data are shown as mean ± s.e.m. (n=4) and representatives of three independent experiments. *P<0.05. (C) Serum-starved hATSCs were exposed to 2 µM D-erythro-SPC for the indicated time periods, and TGF-ß1 and GAPDH mRNAs were quantified by semi-quantitative RT-PCR. Representative data from three independent experiments are shown. (D) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC in the absence or presence of 10 µM U0126 for 6 hours. The mRNA levels of TGF-ß1 and GAPDH were determined by semi-quantitative RT-PCR. (E) The relative level of TGF-ß1 was normalized to those of GAPDH. The data are shown as mean ± s.e.m. of triplicate determinations. *P<0.01. (F) Serum-starved hATSCs were treated with 2 µM D-erythro-SPC, 2 ng/ml TGF-ß1, or 2 ng/ml TGF-ß3 together with anti-TGF-ß1 neutralizing antibody (0.2 µg/ml) for 4 days. The expression levels of -SMA and actin, and the phosphorylation level of p-Smad2 were determined by western blotting. Representative data from three independent experiments are

View larger version (66K): [in this window] [in a new window]   Fig. 8. The role of Smad2 in the SPC-induced differentiation of hATSCs to SMCs. Serum-starved hATSCs were transfected with control siRNA (Control) or Smad2 siRNA (si-Smad2), and then treated with 2 µM D-erythro-SPC or 2 ng/ml TGF-ß3 for 4 days. The phosphorylation level of Smad2 and the expression level of Smad2 were determined by western blotting with anti-p-Smad2 and anti-Smad2 antibodies, respectively. The expression levels of -SMA, h1-calponin and actin were determined by western blotting. Representative data from three independent experiments are shown.

View larger version (33K): [in this window] [in a new window]   Fig. 9. The role of SRF and myocardin in the SPC-induced differentiation of hATSCs to SMCs. Serum-starved hATSCs were treated with vehicles, 2 µM D-erythro-SPC, or 2 ng/ml TGF-ß3 for 24 hours, and SRF and myocardin mRNA levels were determined by semi-quantitative RT-PCR (A) or real time RT-PCR (B). Representative data from three independent experiments are shown in A. The data in B are shown as mean ± s.e.m. (n=3). (C) hATSCs were transfected with control siRNA or siRNAs specific for SRF or myocardin, respectively. The levels of SRF and myocardin mRNAs were determined by semi-quantitative RT-PCR. Representative data from three independent experiments are shown. (D) The siRNA-transfected hATSCs were treated with vehicle, 2 µM D-erythro-SPC, or 2 ng/ml TGF-ß3 for 4 days, and the expression levels of -SMA and actin were determined by western blot analysis. (E) The quantity of -SMA and actin were determined in triplicate, and the expression levels of -SMA were normalized to total actin levels in the samples. The data are presented as a percentage of mock-treated control. *Significantly different from control value (P<0.05).

View larger version (35K): [in this window] [in a new window]   Fig. 10. Schematic illustration of the molecular mechanisms involved in the SPC-induced differentiation of hATSCs. SPC increases the expression of smooth-muscle-specific genes, including -SMA, through a Gi/o-ERK-dependent pathway. SPC-induced activation of ERK stimulates the expression and secretion of TGF-ß isoforms, which elicit late activation of Smad2 through TGF-ß type I receptor kinase. Activated Smad2 cooperates with SRF and myocardin to induce expression of smooth-muscle-specific genes.

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