Regulation of chemotaxis by the cytoplasmic domain of tissue factor.
We previously demonstrated that FVIIa bound to tissue factor (TF) induces a hyperchemotactic response towards PDGF-BB. The aim of the present study was to investigate the role of the cytoplasmic domain of TF in cell migration. Porcine aortic endothelial (PAE) cells expressing human PDGF beta-receptors (PAE/PDGFRbeta) were transfected for expression of human wildtype TF (PAE/PDGFRbeta,TF), a construct lacking the cytoplasmic domain (PAE/PDGFRbeta,TFDeltacyto), a construct with alanine replacement of serine 258 (PAE/PDGFRbeta,TFS258A), or a construct with alanine replacement of serine 253, 258 and 263 in the cytoplasmic domain (PAE/PDGFRbeta,TF3SA). All stably transfected cell lines expressed functional TF. Chemotaxis was analyzed in a modified Boyden chamber assay. PAE/PDGFRbeta,TF cells stimulated with FVIIa migrated towards a 100-fold lower concentration of PDGF-BB than in the absence of FVIIa, however, hyperchemotaxis was not seen in PAE/PDGFRbeta,TFDeltacyto cells. PAE/PDGFRbeta/TFS258A and PAE/PDGFRbeta,TF3SA cells responded to low levels of PDGF-BB, but migrated a significantly shorter distance than PAE/PDGFRbeta,TF cells. Thus, hyperchemotaxis towards PDGF-BB is likely to depend in part on phosphorylation of the cytoplasmic domain of TF. We conclude that the cytoplasmic domain of TF plays a pivotal role in modulating cellular migration response. Our results suggest that the FVIIa/TF complex mediates intracellular signaling by alternative signal transduction pathway(s).
Siegbahn
Agneta
A
Department of Medical Sciences, Clinical Chemistry, Akademiska Hospital, S-751 85 Uppsala, Sweden. agneta.siegbahn@akademiska.se
Johnell
Matilda
M
Sorensen
Brit B
BB
Petersen
Lars C
LC
Heldin
Carl-Henrik
CH
D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D002633:Chemotaxis; D003593:Cytoplasm; D004730:Endothelium, Vascular / Q000166:cytology*; D015942:Factor VIIa / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics / Q000502:physiology*; D013552:Swine; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D013925:Thromboplastin; D020797:Receptor, Platelet-Derived Growth Factor beta; D015942:Factor VIIa