title: Kallistatin inhibits TGF-β-induced endothelial-mesenchymal transition by differential regulation of microRNA-21 and eNOS expression.
abstract: Kallistatin, an endogenous protein, consists of two structural elements: active site and heparin-binding domain. Kallistatin exerts beneficial effects on fibrosis by suppressing transforming growth factor (TGF)-β synthesis in animal models. TGF-β is the most potent inducer of endothelial-mesenchymal transition (EndMT), which contributes to fibrosis and cancer. MicroRNA (miR)-21 is an important player in organ fibrosis and tumor invasion. Here we investigated the potential role of kallistatin in EndMT via modulation of miR-21 in endothelial cells. Human kallistatin treatment blocked TGF-β-induced EndMT, as evidenced by morphological changes as well as increased endothelial and reduced mesenchymal marker expression. Kallistatin also inhibited TGF-β-mediated reactive oxygen species (ROS) formation and NADPH oxidase expression and activity. Moreover, kallistatin antagonized TGF-β-induced miR-21 and Snail1 synthesis, Akt phosphorylation, NF-κB activation, and matrix metalloproteinase 2 (MMP2) synthesis and activation. Kallistatin via its heparin-binding site blocked TGF-β-induced miR-21, Snail1 expression, and ROS formation, as wild-type kallistatin, but not heparin-binding site mutant kallistatin, exerted the effect. Conversely, kallistatin through its active site stimulated the synthesis of endothelial nitric oxide synthase (eNOS), sirtuin 1 (Sirt1) and forkhead box O1 (FoxO1); however, these effects were blocked by genistein, a tyrosine kinase inhibitor. This is the first study to demonstrate that kallistatin's heparin-binding site is crucial for preventing TGF-β-induced miR-21 and oxidative stress, while its active site is key for stimulating the expression of antioxidant genes via interaction with an endothelial surface tyrosine kinase. These findings reveal novel mechanisms of kallistatin in protection against fibrosis and cancer by suppressing EndMT.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States.
authors.forename: Youming
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States.
authors.forename: Grant
authors.initials: G
authors.lastname: Bledsoe
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States.
authors.forename: Zhi-Rong
authors.initials: ZR
authors.lastname: Yang
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States.
authors.forename: Lee
authors.initials: L
authors.lastname: Chao
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, United States. Electronic address: chaoj@musc.edu.
authors.forename: Julie
authors.initials: J
authors.lastname: Chao
mesh_terms: D020134:Catalytic Domain; D042783:Endothelial Cells / Q000502:physiology*; D058750:Epithelial-Mesenchymal Transition; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015843:Serpins / Q000502:physiology*; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D017382:Reactive Oxygen Species; C000604407:SNAI1 protein, human; D015843:Serpins; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C051324:kallistatin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases
keywords: EndMT; Kallistatin; Oxidative stress; TGF-β; eNOS; miR-21