Original Article

Sphingosine-1-phosphate induces VEGF-C expression through a MMP-2/FGF-1/FGFR-1-dependent pathway in endothelial cells in vitro

Authors: Chi-hao Chang, Yuan-li Huang, Ming-kwang Shyu, Shee-uan Chen, Chih-hsin Lin, Tsai-kai Ju, JenHer Lu, Hsinyu Lee
DOI: 10.1038/aps.2012.186

Abstract

Chi-hao CHANG1, #, Yuan-li HUANG2, #, Ming-kwang SHYU3, #, Shee-uan CHEN3, Chih-hsin LIN1, Tsai-kai JU4, 5, JenHer LU6, *, Hsinyu LEE1, 7, 8, 9, 10, *
1Institute of Zoology, National Taiwan University, Taipei, Taiwan, China; 2Department of Biotechnology, Asia University, Taichung, Taiwan, China; 3Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan, China; 4Instrumentation Center, National Taiwan University, Taipei, Taiwan, China; 5Technology Commons, College of Life Science, National Taiwan University, Taipei, Taiwan, China; 6Department of Pediatrics and Pediatric Cardiology, Veterans General Hospital-Taipei, National Yang Ming University, Taipei, Taiwan, China; 7Department of Life Science, National Taiwan University, Taipei, Taiwan, China; 8Center for Biotechnology, National Taiwan University, Taipei, Taiwan, China; 9Angiogenesis Research Center, National Taiwan University, Taipei, Taiwan, China; 10Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan, China

Aim: To investigate whether sphingosine-1-phosphate (S1P), a potent angiogenic factor, induced vascular endothelial growth factor-C (VEGF-C) expression in endothelial cells in vitro and to examine its underlying mechanisms.

Methods: Human umbilical vein endothelial cells (HUVECs) were examined. VEGF-C mRNA expression in the cells was assessed using real-time PCR. VEGF-C protein and FGFR-1 phosphorylation in the cells were measured with ELISA. RNA interference was used to downregulate the expression of matrix metalloproteinase-2 (MMP-2), fibroblast growth factor-1 (FGF-1) and FGF receptor-1 (FGFR-1).

Results: Incubation of HUVECs with S1P (1, 5, and 10 μmol/L) significantly increased VEGF-C expression. The effect was blocked by pretreatment with the MMP inhibitor GM6001 or the FGFR inhibitor SU5402, but not the EGFR inhibitor AG1478. The effect was also blocked in HUVECs that were transfected with FGFR-1 or MMP-2 siRNA. Furthermore, incubation of HUVECs with S1P (5 μmol/L) significantly increased FGFR-1 phosphorylation, which was blocked by GM6001. Moreover, knockdown of FGF-1, not FGF-2, in HUVECs with siRNAs, blocked S1P-induced VEGF-C expression.

Conclusion: S1P induces VEGF-C expression through a MMP-2/ FGF-1/FGFR-1-dependent pathway in HUVECs.


Keywords: sphingosine-1-phosphate; VEGF-C; matrix metalloproteinase-2; fibroblast growth factor-1; fibroblast growth factor receptor-1; GM6001; SU5402; transactivation; human umbilical vein endothelial cells; RNA interference

The research was supported by two grants, NSC100-2325-B-002-045- from the National Science Council and NHRI-EX101-10130BI from the National Health Research Institutes of Taiwan, China.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail hsinyu@ntu.edu.tw (Hsinyu LEE); jenherlu@gmail.com (JenHer LU)
Received 2012-07-30 Accepted 2012-12-12
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