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.