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First published online 26 February 2008
doi: 10.1242/jcs.023283

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L- and S-endoglin differentially modulate TGFβ1 signaling mediated by ALK1 and ALK5 in L₆E₉ myoblasts

¹ Instituto `Reina Sofía' de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, and Red de Investigación en Enfermedades Renales (RedinRen), Salamanca, Spain
² Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
³ Centro de Investigaciones Biológicas (CSIC), and Center for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain

View larger version (50K): [in this window] [in a new window]   Fig. 1. Expression of TGFβ receptors and Smads in L6E9 cells. L6E9 cells were serum starved for 24 hours before TGFβ1 treatment. (A) The expression of ALK1, ALK5 and TβRII from control (C) or 500 pM TGFβ1-treated (T) cells (24 hours) was analyzed by RT-PCR and western blot. (B) Myoblasts were stimulated with TGFβ1 for the indicated time periods. Total proteins extracts were analyzed by western blot with anti-phospho-Smad1, anti-phospho-Smad2, anti-Smad2/3 and anti-Smad4 antibodies. Loading controls included GAPDH, β-actin and tubulin. A representative blot from three independent experiments is shown. (C) Immunofluorescence of Smad1/5, Smad2/3 and Smad4 in L6E9 cells untreated or treated with TGFβ1 for 24 hours.

View larger version (38K): [in this window] [in a new window]   Fig. 2. TGFβ1-ALK1 and TGFβ1-ALK5 signaling pathways in L6E9 cells. Cells were serum starved for 24 hours before TGFβ1 treatments (30 minutes for p-Smads, 1 hour for Id1 and 24 hours for PAI1). (A) Cells were treated with the ALK5 inhibitor SB431542 (SB, 5 µM) 1 hour before treatment with TGFβ1. Whole-cell extracts were analyzed by western blot with anti-pSmad1, anti-pSmad2 and collagen I. Total protein extracts from control (C) or TGFβ1-treated (T) myoblasts (1 hour) were analyzed by western blot with anti-Id1 (B) and anti-PAI1 (D). L6E9 were transiently transfected with (Bre)2-Luc (C), and (CAGA)12-Luc (E) reporters; cells were incubated or not with TGFβ1 for 24 hours, before measuring the luciferase activity. Results are represented as fold induction of the TGFβ1 treated over the untreated counterparts. The histogram represents the mean of three independent experiments. *P<0.05, Student's t-test.

View larger version (27K): [in this window] [in a new window]   Fig. 3. L- and S-endoglin expression and their effects on TGFβ receptors. (A) Immunofluorescence and (B) western blot of endoglin in L6E9 mock, L-endoglin (L-Endo) and S-endoglin (S-Endo). (C,D) Mock-, L-Endo- and S-Endo-transfected cells were serum starved for 24 hours before TGFβ1 treatment. The expression of ALK1, ALK5 and TβRII in control (C), or TGFβ1-treated (T) cells was analyzed by RT-PCR (C) and western blot (D). A representative blot from three independent experiments is shown.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Effects of L- and S-endoglin on Smad phosphorylation. Mock-, L-Endo- and S-Endo-transfected cells were serum starved for 24 hours before 30 minutes of TGFβ1 treatment. (A) Total protein extracts from control (C) or TGFβ1-treated (T) myoblasts were analyzed by western blot with anti-phospho-Smad1/3 or anti-phospho-Smad2 antibodies; anti-tubulin was used as a loading control. (B) Mock-, L-Endo- and S-Endo-transfected cells were treated with the ALK5 inhibitor SB431542 (SB, 5 µM) 1 hour before treatment with TGFβ1. Total protein extracts from control (C) or TGFβ1-treated (T) myoblasts were analyzed by western blot with anti-phospho-Smad1/3, anti-phospho-Smad2 and anti-tubulin antibodies. A representative blot from three independent experiments is shown.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Effects of L- and S-endoglin on TGFβ1-ALK1 and TGFβ1-ALK5 signaling pathways. Cells were serum starved for 24 hours before TGFβ1 treatment. Total protein extracts from control (C) or TGFβ1-treated (T) myoblasts analyzed by western blot with anti-Id1 (A) and anti-PAI1 (C). Measures of densitometry of each band were performed and relative values are represented. Id1 and PAI1 histogram represents the mean of three different extracts. (B,D) Western blots of endoglin in L6E9 mock, L-endoglin (L-Endo) and S-endoglin (S-Endo). L6E9 cells were transiently transfected with (Bre)2-Luc reporter (*P<0.05 compared with mock and S-Endo) (B), and (CAGA)12-Luc reporter (*P<0.05 TGFβ1 compared with control; **P<0.05 compared with mock and S-Endo; ***P< 0.05 compared with mock and L-Endo). (D) Cells were incubated or not with TGFβ1 for 24 hours before measuring the luciferase activity.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Effect of L- and S-endoglin on collagen I and CTGF expression. (A,B) Mock, L- and S-endoglin cells were treated (T) or not (C) with TGFβ1 for 24 hours in serum-free medium. Total protein extracts were analyzed by western blot using a specific antibody for collagen I (A) or CTGF (B). Measures of densitometry of each band were performed and relative values are represented. Collagen I and CTGF histogram represents the mean of three different extracts. (C) L6E9 cells were transfected with a vector expressing either caALK1 or caALK5, and collagen expression analyzed by western blot. (D) Mock, L-Endo and S-Endo-transfected cells were treated with the ALK5 inhibitor SB431542 (SB, 5 µM) 1 hour before treatment with TGFβ1. Total protein extracts from control (C) or TGFβ1-treated (T) myoblasts were analyzed by western blot with anti-collagen I and anti-tubulin antibodies. A representative blot from three independent experiments is shown. The blot of the S-endoglin samples is under-exposed in order to visualize the differences caused by the SB431542 treatment.

View larger version (15K): [in this window] [in a new window]   Fig. 7. L- and S-endoglin modify cell proliferation. (A) Proliferation was assessed by the number of cells determined by MTT assay. Cell proliferation was analyzed at day 0 (day of treatment, data not shown) and 3 days after treatment. A representative experiment of three independent experiments using quadruplicate samples is shown. (B) Representative flow cytometry graph showing a higher proportion of L-endoglin cells in S and G2-M phases of the cell cycle.

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