Smad7 and protein phosphatase 1alpha are critical determinants in the duration of TGF-beta/ALK1 signaling in endothelial cells.
BACKGROUND
In endothelial cells (EC), transforming growth factor-beta (TGF-beta) can bind to and transduce signals through ALK1 and ALK5. The TGF-beta/ALK5 and TGF-beta/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-beta signaling is an important specificity determinant for signaling responses. TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently.


RESULTS
The temporal activation of TGF-beta-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-beta/ALK1 signaling pathway. Smad7 and protein phosphatase 1alpha (PP1alpha) mRNA expression levels were found to be specifically upregulated by TGF-beta/ALK1. Ectopic expression of Smad7 or PP1alpha potently inhibited TGF-beta/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1alpha enhanced TGF-beta/ALK1-induced signaling responses. PP1alpha interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor.


CONCLUSIONS
Our results suggest that upon its induction by the TGF-beta/ALK1 pathway, Smad7 may recruit PP1alpha to ALK1, and thereby control TGF-beta/ALK1-induced Smad1/5 phosphorylation.
Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Vatnsmyrarvegur 16, 101 Reykjavik, Iceland. gudrunva@hi.is
Gudrun
G
Valdimarsdottir
Marie-José
MJ
Goumans
Fumiko
F
Itoh
Susumu
S
Itoh
Carl-Henrik
CH
Heldin
Peter
P
ten Dijke
D029404:Activin Receptors / Q000032:analysis / Q000502:physiology; D030301:Activin Receptors, Type II / Q000032:analysis / Q000502:physiology*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000502:physiology* / Q000821:virology; D005786:Gene Expression Regulation / Q000502:physiology; D047468:Immunoprecipitation; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051898:Smad1 Protein / Q000378:metabolism; D051906:Smad7 Protein / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000502:physiology*
D012333:RNA, Messenger; D051898:Smad1 Protein; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta; D029404:Activin Receptors; D030301:Activin Receptors, Type II; D010749:Phosphoprotein Phosphatases