TGF-beta induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways.
BACKGROUND
Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-beta1 (TGF-beta1), but the mechanism remains unclear.


METHODS
We examined the role of TGF-beta1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-beta1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-beta1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation.


RESULTS
Induction of VEGF-A and TSP-1 by TGF-beta1 in NRK52E cells was associated with activation of pathway-restricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-beta1. Conditioned media from NRK52E, which was stimulated by TGF-beta1 for 24 hours, did not induce endothelial cell proliferation. However, conditioned media from Smad2 knockout induced endothelial cell proliferation, whereas endothelial cell proliferation was inhibited by Smad3 knockout-derived conditioned media.


CONCLUSIONS
R-Smads have distinct roles in mediating the expression of pro- and antiangiogenic growth factors in response to TGF-beta1.
Nakagawa
Takahiko
T
Division of Nephrology-Medicine, Baylor College of Medicine, Houston, Texas, USA. nakagt@medicine.ufl.edu
Li
Jin H
JH
Garcia
Gabriela
G
Mu
Wei
W
Piek
Ester
E
Böttinger
Erwin P
EP
Chen
Yan
Y
Zhu
Hong J
HJ
Kang
Duk-Hee
DH
Schreiner
George F
GF
Lan
Hui Y
HY
Johnson
Richard J
RJ
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D051899:Smad2 Protein; D051900:Smad3 Protein; D051906:Smad7 Protein; D019700:Thrombospondin 1 / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051906:Smad7 Protein; C497808:Smad7 protein, rat; C508962:Tgfb1 protein, rat; D019700:Thrombospondin 1; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat