Kruppel-like factor 4 is a novel mediator of Kallistatin in inhibiting endothelial inflammation via increased endothelial nitric-oxide synthase expression.
Kallistatin is a plasma protein that exhibits pleiotropic effects in vasodilation, anti-angiogenesis, and anti-inflammation. To isolate a kallistatin-binding protein that mediates the vascular actions of kallistatin, we screened and identified a positive clone from a human heart cDNA expression library by using an alkaline phosphatase-kallistatin fusion protein binding assay. Sequence analysis revealed that kallistatin-binding protein is human Kruppel-like factor 4 (KLF4). KLF4 was localized on the plasma membrane of HEK-293 cells and endothelial cells overexpressing KLF4. KLF4 and kallistatin complex formation was identified in endothelial cells by immunoprecipitation followed by immunoblotting. We showed that kallistatin inhibits tumor necrosis factor-alpha-induced NF-kappaB activation, as well as vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression in endothelial cells, whereas knockdown of KLF4 by small interfering RNA oligonucleotide abolished the effect of kallistatin. Kallistatin increased endothelial nitric-oxide synthase (eNOS) expression and nitric oxide levels, and these effects were also blocked by KLF4 small interfering RNA oligonucleotide. Moreover, inhibition of eNOS by RNA interference or by NOS inhibitor abolished the blocking effect of kallistatin on vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression. In summary, we identified KLF4 as a kallistatin-binding protein, which has a novel role in mediating the anti-inflammatory actions of kallistatin via increasing eNOS expression in endothelial cells. This study provides a new target for modulating endothelial function in vascular disease.
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Bo
B
Shen
Robert S
RS
Smith
Yi-Te
YT
Hsu
Lee
L
Chao
Julie
J
Chao
D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018932:Chemokine CCL2 / Q000378:metabolism / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D015723:Gene Library; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D014652:Vascular Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D016328:NF-kappa B; D015843:Serpins; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C051324:kallistatin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III