Transcriptional activation of endothelial cell integrin alpha v by protein kinase C activator 12(S)-HETE.
Previous work demonstrated that 12(S)-HETE [12(S)-hydroxyeicosatetraenic acid], a lipoxygenase metabolite of arachidonic acid, stimulates the surface expression of integrin alpha v beta 3 on mouse lung vascular endothelial cells (CD clone 3) in a post-transcriptional and protein kinase C (PKC)-dependent fashion. In this study we examined the effect of 12(S)-HETE on the expression of integrin receptors alpha v beta 3 and alpha 5 beta 1 in a different clone of a mouse endothelial cell population derived from lung microvasculature (designated CD clone 4). The results indicated that 12(S)-HETE transcriptionally activates the gene expression of integrin alpha v as assessed by quantitative reverse transcription/polymerase chain reaction/Southern hybridization, RNase protection assay, solution hybridization, and northern blotting. The induction of alpha v mRNA occurred within 1 hour, peaked at approximately 4 hours (2- to 4-fold increase), persisted for up to 16 hours, and thereafter gradually declined. The PKC activator phorbol 12-myristate 13-acetate (PMA) induced the alpha v mRNA, in a similar way. 12(S)-HETE treatment did not, in contrast, alter the mRNA levels of integrin subunit alpha 5 or beta 1. The induction of alpha v mRNA appeared to be protein synthesis-independent, since cycloheximide did not alter the 12(S)-HETE effect. 12(S)-HETE also did not appear to alter the mRNA half-life of alpha v. On the other hand, 12(S)-HETE-induced increase in alpha v mRNA levels was PKC-dependent, since pretreatment of CD clone 4 cells with calphostin C significantly inhibited 12(S)-HETE-increased alpha v mRNA. Nuclear runoff experiments revealed that the increase in alpha v mRNA results from an enhanced gene transcription. Facilitated alpha v gene transcription resulted in an increased surface expression of alpha v beta 3 protein, which resulted in an increased cell adhesion to vitronectin. The above observations, in conjunction with our previous experimental data, suggest that 12(S)-HETE may employ diverse mechanisms to stimulate the integrin alpha v beta 3 expression in vascular endothelial cells, which could play important roles in tumor cell adhesion, angiogenesis, hemostasis, and many other vascular events.
Tang
D G
DG
Department of Radiation Oncology, Wayne State University, Detroit, MI 48202, USA.
Diglio
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Bazaz
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Honn
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KV
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D000906:Antibodies; D015703:Antigens, CD / Q000032:analysis / Q000096:biosynthesis*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D039564:Integrin alphaV; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D011652:Pulmonary Circulation; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects*
D000906:Antibodies; D015703:Antigens, CD; D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D039564:Integrin alphaV; D012333:RNA, Messenger; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate