Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis.
The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells, we observed that thrombin markedly up-regulates growth-regulated oncogene-alpha (GRO-alpha) in several tumor cell lines as well as endothelial cells by mRNA and protein analysis. Thrombin enhanced the secretion of GRO-alpha from tumor cells 25- to 64-fold. GRO-alpha is a CXC chemokine with tumor-associated angiogenic as well as oncogenic activation following ligation of its CXCR2 receptor. GRO-alpha enhanced angiogenesis in the chick chorioallantoic membrane assay 2.2-fold, providing direct evidence for GRO-alpha as an angiogenic growth factor. Anti-GRO-alpha antibody completely inhibited the 2.7-fold thrombin-induced up-regulation of angiogenesis, as well as the 1.5-fold thrombin-induced up-regulation of both endothelial cell cord formation in Matrigel and growth in vitro. Thrombin as well as its PAR-1 receptor activation peptide [thrombin receptor activation peptide (TRAP)] as well as GRO-alpha all markedly increased vascular regulatory proteins and growth factors: matrix metalloproteinase (MMP)-1, MMP-2, vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), CD31, and receptors KDR and CXCR2 in human umbilical vein endothelial cells. All of the thrombin/TRAP gene up-regulations were completely inhibited by anti-GRO-alpha antibody and unaffected by irrelevant antibody. Similar inhibition of gene up-regulation as well as thrombin-induced chemotaxis was noted with small interfering RNA (shRNA) GRO-alpha KD 4T1 breast tumor and B16F10 melanoma cells. In vivo tumor growth studies in wild-type mice with shRNA GRO-alpha KD cells revealed 2- to 4-fold impaired tumor growth, metastasis, and angiogenesis, which was not affected by endogenous thrombin. Thus, thrombin-induced angiogenesis requires the up-regulation of GRO-alpha. Thrombin up-regulation of GRO-alpha in tumor cells as well as endothelial cells contributes to tumor angiogenesis.
Caunt
Maresa
M
Departments of Medicine, New York University School of Medicine, New York, New York 10016, USA.
Hu
Liang
L
Tang
Thomas
T
Brooks
Peter C
PC
Ibrahim
Sherif
S
Karpatkin
Simon
S
D000595:Amino Acid Sequence; D042702:Angiopoietin-2 / Q000096:biosynthesis; D000818:Animals; D048708:Cell Growth Processes / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D054360:Chemokine CXCL1; D019743:Chemokines, CXC / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002642:Chick Embryo; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
D042702:Angiopoietin-2; C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D013917:Thrombin; D020781:Matrix Metalloproteinase 1