TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways
Abstract
Huey-ming LO1, 2, Tsung-hsuan LAI1, 3, 4, Chih-hung LI1, Wen-bin WU1, *
1School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan, China; 2Section of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, China; 3Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan, China; 4Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taoyuan, Taiwan, China
Aim: Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-α has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro.
Methods: Human umbilical vein endothelial cells (HUVECs) were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-α-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTT assay. Monocyte migration was measured with transwell migration assay.
Results: Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α (2, 5 ng/mL) induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α (5 ng/mL) caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor (SP600125), p38 MAPK inhibitor (SB202190) or PI-3K inhibitor (LY294002) significantly suppressed TNF-α-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-α-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone (up to 500 nmol/L) failed to affect TNF-α-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-α-induced CXCL1 from HUVECs attracted human monocyte migration.
Conclusion: TNF-α stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.
Keywords: chemokine; CXCL1; GRO-α; TNF; vascular endothelial cell; JNK; p38 MAPK; PI3K; monocyte; dexamethasone
The work was supported by a research grant(SKH-8302-99-DR-05) from Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, China.
* To whom correspondence should be addressed.
E-mail wenbin@mail.fju.edu.tw
Received 2013-06-20 Accepted 2013-10-10
Keywords:
1School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan, China; 2Section of Cardiology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, China; 3Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan, China; 4Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taoyuan, Taiwan, China
Aim: Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-α has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro.
Methods: Human umbilical vein endothelial cells (HUVECs) were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-α-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTT assay. Monocyte migration was measured with transwell migration assay.
Results: Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α (2, 5 ng/mL) induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α (5 ng/mL) caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor (SP600125), p38 MAPK inhibitor (SB202190) or PI-3K inhibitor (LY294002) significantly suppressed TNF-α-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-α-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone (up to 500 nmol/L) failed to affect TNF-α-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-α-induced CXCL1 from HUVECs attracted human monocyte migration.
Conclusion: TNF-α stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.
Keywords: chemokine; CXCL1; GRO-α; TNF; vascular endothelial cell; JNK; p38 MAPK; PI3K; monocyte; dexamethasone
The work was supported by a research grant(SKH-8302-99-DR-05) from Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, China.
* To whom correspondence should be addressed.
E-mail wenbin@mail.fju.edu.tw
Received 2013-06-20 Accepted 2013-10-10