TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim: Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-alpha 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-alpha-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-alpha-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-alpha, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-alpha (2, 5 ng/mL) induced CXCL1 release and mRNA expression in the cells in concentration-and time-dependent manners. TNF-alpha (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-alpha-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-alpha-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone (up to 500 nmol/L) failed to affect TNF-alpha-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-alpha-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion: TNF-alpha stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.