QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 21, 2007
doi: 10.1242/10.1242/jcs.03419

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Castañares, C. Articles by Rodríguez-Pascual, F. Search for Related Content PubMed PubMed Citation Articles by Castañares, C. Articles by Rodríguez-Pascual, F.

Signaling by ALK5 mediates TGF--induced ET-1 expression in endothelial cells: a role for migration and proliferation

¹ Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (C.S.I.C.), Instituto `Reina Sofía' de Investigaciones Nefrológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
² Department of Molecular Cell Biology, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands

View larger version (19K): [in this window] [in a new window]   Fig. 1. TGF--mediated induction of the ET-1 gene. BAEC were incubated with or without 5 ng/ml TGF- for different time periods as indicated, and changes in the expression of ET-1 at the level of mRNA, peptide and promoter activity were determined as described in the Materials and Methods. (A) ET-1 mRNA levels were detected and analyzed by RT-qPCR. Values are represented as fold induction (mean ± s.d., n=4, *P<0.05 versus zero time). (B) ET-1 peptide levels were detected by specific ELISA and expressed as fmol/well (mean ± s.d., n=3, *P<0.05 versus zero time, #P<0.05 versus treated cells). (C) Transcriptional activation of the human ET-1 promoter was estimated from cells transfected with a -650/+172-bp fragment of the human ET-1 promoter, compared with empty vector pGL3-Basic. Basal and TGF--induced luciferase activity was measured by luminometry and expressed as fold induction with respect to the activity of human ET-1 promoter under basal conditions (mean ± s.d., n=4, *P<0.05 versus zero time, #P<0.001 versus human ET-1 promoter for the indicated times).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Smads and AP-1 cooperate to mediate TGF- induction of the ET-1 gene through activation of the Smad signaling pathway. (A) Specific point-mutations were introduced in the human ET-1 promoter-luciferase construct (wild type) that alter the Smad-binding element and/or the AP-1 site. BAEC transfected with this set of constructs were incubated with TGF- for 18 hours or left under basal conditions. Luciferase activity was measured and expressed as normalized arbitrary units (mean ± s.d., n=4, *P<0.05 versus wild type in the presence of TGF-, #P<0.05 versus wild type in the absence of TGF-). The involvement of JNK/AP-1 in the induction of ET-1 gene expression by TGF- was analyzed in endothelial cells incubated with the JNK inhibitor SP600125 (25 µM). (B) ET-1 mRNA levels were detected and analyzed by RT-qPCR. Values are represented as fold induction. (C) ET-1 peptide levels were detected by specific ELISA and expressed as fmol/well. (D) Human ET-1 promoter was estimated from cells transfected with the ET-1 promoter-luciferase construct and expressed as arbitrary units normalized to total protein content (mean ± s.d., n=4, *P<0.05 versus basal without the inhibitor, #P<0.05 versus TGF- stimulation without the inhibitor).

View larger version (51K): [in this window] [in a new window]   Fig. 3. Overexpression of TGF- receptors and Smad isoforms in endothelial cells. Cells were transiently transfected with expression constructs for wild type (wt), constitutively active (ca) and kinase-deficient (kd) forms of type I receptors ALK1 and ALK5, and Smad3, Smad1 and Smad5 isoforms. (A) For western blot analyses, whole-cell extracts were fractionated by 10% SDS-PAGE and transferred to blots. The membranes were then incubated with specific antibodies against ALK5, ALK1 and HA (epitope tag for receptor expression plasmids) and Smad3, Smad1 and Smad5. Bands show the presence of endogenous (control) and corresponding overexpressed levels of these proteins. On some occasions, for example for ALK1 and Smad1/5, multiple specific bands were detected. The housekeeping gene GAPDH was detected with an anti-GAPDH antibody for loading control purposes. (B) Immunofluorescence microscopy was used to detect endogenous (control) and overexpressed levels of Smad3, Smad1 and Smad5 in BAEC. Primary anti-Smad3 or anti-Smad1/5 were coupled to FITC-labeled secondary antibodies (green signal). Nuclei were stained with Hoechst 33342 (blue signal).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effect of the overexpression of ALK5, ALK1 and Smad isoforms on ALK5- and ALK1-specific reporters and on human ET-1 promoter. The effect of the overexpression of constitutively active (ca) forms of ALK5 and ALK1 on (A) ALK5- and (B) ALK1-specific luciferase reporters was analyzed by cotransfection experiments in BAEC. Overexpression plasmids were cotransfected together with a -650/+172-bp fragment of the human ET-1 promoter linked to a luciferase gene. (C) Effect of ALK5 and ALK1 ca in the absence of TGF- (open bars), and of ALK5 and ALK1 wild type (wt) and kinase-deficient (kd) forms in the presence of 5 ng/ml TGF- (filled bars). (D) Effect of Smad3, Smad1 and Smad5 overexpression in the presence of TGF-. Luciferase activity was measured by luminometry and expressed as fold induction with respect to control pCMV5 empty vector (mean ± s.d., n=4, *P<0.05 versus control).

View larger version (47K): [in this window] [in a new window]   Fig. 5. Effect of selective downregulation of ALK1 and ALK5 receptors and Smad1 and Smad3 isoforms by siRNA on TGF--induced ET-1 expression. (A) Efficiency of endogenous protein knockdowns by siRNA was confirmed by western blot assays. Functional validation of ALK1, ALK5, Smad1 and Smad3 siRNA was done by cotransfection with ALK1- and ALK5-specific reporters under overexpression of ALK1 constitutively active (ca) (B) and ALK5 ca (C) forms. Results are compared with the effect of an siRNA control. TGF--induced ET-1 expression under specific downregulation of ALK1 and ALK5 receptors and Smad1 and Smad3 isoforms was analyzed at the level of ET-1 promoter activity (D) and peptide secretion (E), as previously described. Values are expressed as fold induction with respect to siRNA control with pCMV5 empty vector (B,C) or without TGF- (D,E) (mean ± s.d., n=3, *P<0.05 versus control without activation, #P<0.05 versus control with activation).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Pharmacological inhibition of ALK5 with SB-431542 impaired TGF--induction of the ET-1 gene. Validation of compound SB-431542 (5 nM-10 µM) was done in BAEC transfected with (A) ALK1- and (B) ALK5-specific luciferase reporters together with ALK1 constitutively active (ca) and ALK5 ca forms, respectively. ET-1 mRNA levels, peptide accumulation and promoter activity were also analyzed in cells treated with SB-431542. (C) ET-1 mRNA expression was investigated using RT-qPCR to detect and quantify the level of transcripts. Values are represented as fold induction with respect to untreated cells. (D) Accumulation of ET-1 peptide in the extracellular medium was analyzed by specific ELISA and expressed as fmol/well. (E) ET-1 promoter activity was estimated by luminometry and values expressed as fold induction with respect to the corresponding control in the absence of TGF- (mean ± s.d., n=3).

View larger version (48K): [in this window] [in a new window]   Fig. 7. Effect of TGF--induced ET-1 on endothelial cell migration. Transwell assays: BAEC were seeded on Transwell inserts and cell-migration capacity analyzed as described in the Materials and Methods. (A) Schematic representation of filter device and representative pictures taken at corresponding sides of the filter membrane showing non-migrating cells (upper surface) and migrating cells (lower surface) for the indicated experimental conditions (basal, TGF- alone, TGF- plus SB-431542 and TGF- plus bosentan). (B) Cell-migration capacity is shown in the bar graph as the ratio of migrating versus non-migrating cells expressed as a percentage of control (untreated cells) (mean ± s.d., n=3, *P<0.05 versus basal, #P<0.05 versus TGF- alone). The potential effect of bosentan on TGF- signaling was analyzed by transfection of ALK5- and ALK1-specific reporters under overexpression of ALK5 constitutively active (ca) (C) and ALK1 ca (D) forms in cells treated with medium alone or with 10 µM bosentan. Promoter activity was estimated by luminometry and values expressed as fold induction with respect to the corresponding control without activation (mean ± s.d., n=3, *P<0.05 versus control without activation).

View larger version (63K): [in this window] [in a new window]   Fig. 8. Effect of TGF--induced ET-1 on endothelial cell migration. Wound healing assays: BAEC seeded on 24-well plates after confluency were wounded with a yellow pipette tip and incubated during reendothelization with medium alone (basal) or medium containing 5 ng/ml TGF-, TGF- plus 10 µM SB-431542 or TGF- plus 10 µM bosentan. (A) Representative images of the monolayers at 10x magnification showing initial lesions (zero time) and after 24 hours for the selected experimental conditions. (B) Estimation of cell-migration capacity was obtained by measurement of the wounded area expressed as the percentage of recovery (see Materials and Methods for details) (mean ± s.d., n=3, *P<0.05 versus basal, #P<0.05 versus TGF- alone).

View larger version (15K): [in this window] [in a new window]   Fig. 9. Role of ET-1 in TGF--mediated endothelial cell migration and proliferation. (A) Effect of exogenous ET-1 on endothelial cell migration. BAEC were seeded on Transwell inserts and cell-migration capacity analyzed for the indicated experimental conditions (basal, TGF- alone, TGF- plus SB-431542 and TGF- plus SB-431542 in the presence of 100 nM ET-1). A control experiment with ET-1 alone is also shown. Cell-migration capacity is shown as the ratio of migrating versus non-migrating cells expressed as a percentage of basal (mean ± s.d., n=3, *P<0.05 versus basal, #P<0.05 versus TGF- alone). (B) Effect of TGF--induced ET-1 on endothelial cell proliferation. BrdU incorporation: BAEC seeded on six-well plates were treated with TGF-, TGF- plus SB431542 or TGF- plus bosentan in the presence of 10 µM BrdU for 24 hours. They were then fixed, treated with pepsin/HCl, washed with PBS and incubated with anti-BrdU FITC-conjugated antibody. Estimation of cell proliferation was obtained by measurement of the mean fluorescence intensity from the FITC signal expressed as a percentage of basal (mean ± s.d., n=3, *P<0.05 versus basal, #P<0.05 versus TGF- alone).