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

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.

TNF-α stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.

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.

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.