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.

Keywords: EndMT; Kallistatin; Oxidative stress; TGF-β; eNOS; miR-21.

Fig. 1

Kallistatin (KS) inhibits TGF-β1-induced EndMT. Kallistatin suppresses (A) TGF-β1-promoted morphology changes as indicated by light microscopy. Kallistatin also prevented TGF-β1-induced reduction of expression of the endothelial markers (B) VE-cadherin and (C) CD31, and elevation of expression of the mesenchymal marker (D) α-SMA in HUVECs, as indicated by representative immunofluorescence. Hoechst 33342 stains the nucleus. (E) Protein levels were determined by western blot and quantified. *P<0.05 vs. other groups. Values are expressed as mean±SE; n=3 experiments.

Fig. 2

Kallistatin (KS) inhibits TGF-β1-induced ROS generation and NADPH oxidase (Nox) expression and activity. (A) 2′,7′-dichlorofluorescein was used to quantify ROS formation. (B) Nox4 mRNA levels were measured by real-time PCR. (C) NADPH oxidase activity was determined by lucigenin–chemiluminescence. *P<0.05 vs. other groups. Values are expressed as mean±SE; n=3 experiments.

Fig. 3

Kallistatin (KS) inhibits TGF-β1-induced miR-21 synthesis, Akt and NF-κB activation, Snail1 expression, and MMP2 synthesis and activity. (A) miR-21 expression was detected by real-time PCR, (B) Akt phosphorylation was detected by western blot, (C) NF-κB activation was detected by enzyme-linked immunosorbent assay kit, (D) Snail1 mRNA level was determined by real-time PCR, and (E) MMP2 activity was determined by zymography, and MMP2 mRNA expression was determined by real-time PCR. *P<0.05 vs. other groups. Values are expressed as mean±SE; n=3 experiments.

Fig. 4

The heparin-binding site of kallistatin (KS) is essential to inhibiting TGF-β1-induced miR-21 synthesis, ROS production and Snail1 expression. (A) miR-21 synthesis was detected by real-time PCR, (B) ROS production was quantified with 2′,7′-dichlorofluorescein, and (C) Snail1 mRNA levels was determined by real-time PCR. WT-KS indicates wild-type kallistatin; HM-KS indicates heparin-binding site mutant kallistatin. *P<0.05 vs. control; #P<0.05 vs. TGF-β. Values are expressed as mean±SE; n=3 experiments.

Fig. 5

The active site of kallistatin is essential for increasing the expression of eNOS, Sirt1 and FoxO1 via a tyrosine kinase, as genistein blocked kallistatin’s effect. (A) eNOS, (B) Sirt1 and (C) FoxO1 mRNA levels were determined by real-time PCR. WT-KS indicates wild-type kallistatin; HM-KS indicates heparin-binding site mutant kallistatin; AM-KS indicates active site mutant kallistatin; GEN indicates genistein. *P<0.05 vs. control. Values are expressed as mean±SE; n=3 experiments.

Fig. 6

Hypothetic scheme: Kallistatin’s heparin-binding site is responsible for suppressing TGF-β-induced miR-21-Akt signaling and ROS formation, while its active site is key for stimulating the expression of eNOS, Sirt1 and FoxO1. Kallistatin’s effect on the expression of antioxidant genes is blocked by genistein, a tyrosine kinase inhibitor, suggesting that kallistatin’s active site interacts with a tyrosine kinase. Our current data, together with previous studies, suggest that kallistatin’s structural elements play an important role in attenuating endothelial-mesenchymal transition (EndMT).