Isoform-specific effects of transforming growth factor β on endothelial-to-mesenchymal transition - PubMed (original) (raw)

. 2018 Nov;233(11):8418-8428.

doi: 10.1002/jcp.26801. Epub 2018 Jun 1.

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Isoform-specific effects of transforming growth factor β on endothelial-to-mesenchymal transition

Harika Sabbineni et al. J Cell Physiol. 2018 Nov.

Abstract

Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor β (TGFβ) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGFβ1, TGFβ2, and TGFβ3 on EndMT and characterize the underlying mechanisms. In our results, all three TGFβ isoforms induced EndMT in human microvascular endothelial cells after 72 hr, as evidenced by the increased expression of mesenchymal markers N-cadherin and α-smooth muscle actin as well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGFβ2 was the most pronounced. At 1 ng/ml, only TGFβ2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38-MAPK and increased expression of mesenchymal transcription factors Snail and FoxC2. Intriguingly, we observed that treatment with 1 ng/ml TGFβ1 and TGFβ3, but not TGFβ2, resulted in an increased expression of TGFβ2, thus indicating that EndMT with TGFβ1 and TGFβ3 treatments was due to the secondary effects through TGFβ2 secretion. Furthermore, silencing TGFβ2 using small interfering RNA blunted the expression of EndMT markers in TGFβ1- and TGFβ3-treated cells. Together, our results indicate that TGFβ2 is the most potent inducer of EndMT and that TGFβ1- and TGFβ3-induced EndMT necessitates a paracrine loop involving TGFβ2.

Keywords: EndMT; FoxC2; N-cadherin; Snail; TGFβ1; TGFβ2; TGFβ3.

© 2018 Wiley Periodicals, Inc.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that no conflict of interest exists.

Figures

FIGURE 1

FIGURE 1. TGFβ- induced EndMT in HMECs is a long-term process

(A–C) Representative Western blot images and the corresponding bar graph of band densitometry showing a gradual increase in the expression of mesenchymal marker N-Cadherin in HMECs treated with 1 ng/ml of TGFβ1, 2 and 3 for 0, 12, 24, 48 and 72 hours**. (D–E)** Representative Western blot images and the corresponding bar graph of band densitometry showing a gradual decrease in the expression of endothelial marker eNOS in HMECs treated with 1 ng/ml of TGFβ1, 2 and 3 for 0, 12, 24, 48 and 72 hours. Data are represented as mean ± SD. (n=3–5), *p<0.05; #p<0.0;, $p<0.001.

FIGURE 2

FIGURE 2. TGFβ2 is a more potent inducer of mesenchymal markers in HMECs compared to TGFβ1 and TGFβ3

(A) Representative Western blot images and the corresponding bar graph of band densitometry showing increased expression of mesenchymal marker N-cadherin in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing increased expression of the mesenchymal marker αSMA in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p<0.05.

FIGURE 3

FIGURE 3. TGFβ2 is a more potent suppressor of the endothelial marker expression in HMECs compared to TGFβ1 and TGFβ3

(A) Representative Western blot images and the corresponding bar graph of band densitometry showing reduced expression of endothelial marker eNOS in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing no significant change in the expression of endothelial receptor VE-cadherin in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p<0.05.

FIGURE 4

FIGURE 4. TGFβ2 exhibits higher potency in activating both canonical and non-canonical pathways in HMECs compared to TGFβ1 and TGFβ3

(A) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation and total expression of canonical transcription factor Smad2/3 in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing increased phosphorylation of non-canonical, stress-induced p38 MAPK in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p<0.05.

FIGURE 5

FIGURE 5. TGFβ2 is a more potent stimulator of mesenchymal transcription factor expression in HMECs compared to TGFβ1 and TGFβ3

(A) Representative Western blot images and the corresponding bar graph of band densitometry showing increased expression of mesenchymal transcription factor Snail in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Representative Western blot images and the corresponding bar graph of band densitometry showing increased expression of mesenchymal transcription factor FoxC2 in HMECs in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. Data are represented as mean ± SD. (n=3–5), *p<0.05.

FIGURE 6

FIGURE 6. TGFβ2 induces EndMT at lower doses

(A) Representative Western blot images showing a gradual increase in the expression of mesenchymal marker N-Cadherin associated with increased p-smad2/3 and decreased eNOS levels in HMECs treated with TGFβ1 for 72 hours with 0, 50, 100, 250, 500 and 1000 pg/ml doses**. (B–D)** Corresponding bar graph of band densitometry showing a gradual decrease a gradual increase in the expression of mesenchymal marker N-Cadherin associated with increased p-smad2/3 and decreased eNOS levels in HMECs treated with TGFβ1 for 72 hours with 0, 50, 100, 250, 500 and 1000 pg/ml doses. Data are represented as mean ± SD. (n=3–5), *p<0.05; $p<0.001.

FIGURE 7

FIGURE 7. TGFβ2 predominantly induce endothelial cell scattering compared to other isoforms

Representative images showing the predominant effect of TGFβ2 on cell scattering, a feature of the invasive mesenchymal cells compared to control, TGFβ1 and TGFβ2 treated cells after 72 hours of treatment. Scale bar: 20 μM.

FIGURE 8

FIGURE 8. TGFβ1- and TGFβ3-induced EndMT needs activation of a paracrine loop in HMECs involving TGFβ2

(A) Representative Western blot images showing increased expression of the most potent EndMT stimulating TGFβ isoform, TGFβ2, in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (B) Bar graph of Western blot band densitometry analysis showing increased expression of TGFβ2 in response to 1, 2.5 and 5 ng/ml of TGFβ1, TGFβ2, and TGFβ3 for 72 hours. (C) Representative Western blot images showing increased expression of N-Cadherin, αSMA, and TGFβ2 by both TGFβ1 and TGFβ3, both of which were blunted upon SiRNA-mediated knockdown of TGFβ2. (D–E) Bar graph of Western blot band densitometry analysis showing increased expression of N-Cadherin and αSMA by both TGFβ1 and TGFβ3, which were blunted upon SiRNA-mediated knockdown of TGFβ2. Data are represented as mean ± SD. (n=3–5), *p<0.05; #p<0.01.

FIGURE 9

FIGURE 9. Diagrammatic sketch of the working hypothesis

Both TGFβ1 and TGFβ3, but not TGFβ2 itself, induces the generation of TGFβ2 in HMECs, which in turn, promote EndMT pathways leading to Snail and FoxC2-induced transcriptional activation of mesenchymal markers and repression of endothelial markers.

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