Cell Biology – Immunology – Pathology

Kidney International (2004) 66, 605–613; doi:10.1111/j.1523-1755.2004.00780.x

TGF-bold beta induces proangiogenic and antiangiogenic factorsvia parallel but distinct Smad pathways1

TAKAHIKO NAKAGAWA, JIN H LI, GABRIELA GARCIA, WEI MU, ESTER PIEK, ERWIN P BÖTTINGER, YAN CHEN, HONG J ZHU, DUK-HEE KANG, GEORGE F SCHREINER, HUI Y LAN and RICHARD J JOHNSON

Division of Nephrology-Medicine, Baylor College of Medicine, Houston, Texas; Department of Cell Biology and Applied Biology, University of Nijmegen, Nijmegen, Denmark; Division of Nephrology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York; Department of Medical and Molecular Genetics and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana; Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia; Division of Nephrology, Ewha Women's University Hospital, Seoul, Korea; and Scios, Inc., Sunnyvale, California

Correspondence: Takahiko Nakagawa, M.D., Ph.D., Division of Nephrology, Hypertension and Transplantation, University of Florida, PO Box 100224, Gainesville, FL 32610–0224. E-mail:nakagt@medicine.ufl.edu

1See Editorial by Woolf, p. 862.

Received 2 December 2003; Revised 29 January 2004; Accepted 20 February 2004.

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Abstract

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.

Conclusion

 

R-Smads have distinct roles in mediating theexpression of pro- and antiangiogenic growth factors in response to TGF-beta1.

Keywords:

VEGF, TSP-1, sFlt-1, Smad KO cell, endothelial cell proliferation

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