Endocrinol Metab.  2020 Jun;35(2):384-395. 10.3803/EnM.2020.35.2.384.

Gemigliptin Inhibits Interleukin-1β–Induced Endothelial-Mesenchymal Transition via Canonical-Bone Morphogenetic Protein Pathway

• Hong OK
• Lee SS
• Yoo SJ
• Lee MK
• Kim MK
• Baek KH
• Song KH
• Kwon HS

• KMID: 2503269
• DOI: http://doi.org/10.3803/EnM.2020.35.2.384

Abstract

Background
Endothelial-to-mesenchymal transition (EndMT) contributes to inflammatory conditions inducing conversion of endothelial cells (ECs) into activated fibroblasts, promoting fibrotic diseases. Pro-inflammatory cytokine is the most potent inducer of EndMT. We investigated inhibition of interleukin-1β (IL-1β)-induced EndMT by gemigliptin, a dipeptidyl peptidase-IV inhibitor.
Methods
We exposed human umbilical vein endothelial cells (HUVECs) to 10 ng/mL IL-1β/20 μM gemigliptin and analyzed the expression of endothelial, smooth muscle, mesenchymal, and osteoblastic markers, bone morphogenetic protein (BMP), Smad, and non-Smad signaling pathway proteins.
Results
Morphological changes showed gemigliptin blocked IL-1β-induced EndMT, upregulated EC markers, and downregulated smooth muscle and mesenchymal markers. IL-1β activation of HUVECs is initiated by the BMP/Smad and non-smad BMP signaling pathways. Gemigliptin inhibited IL-1β induction of BMP2 and 7, activin receptor type IA, BMP receptor type IA, and BMP receptor type II. Reversal of IL-1β-mediated inhibition of BMP-induced Smad1/5/8, Smad2, and Smad3 phosphorylation by gemigliptin suggests involvement of the Smad pathway in gemigliptin action. In the non-Smad BMP pathway, gemigliptin treatment significantly increased the deactivation of extracellular regulated protein kinase (ERK), p38, and JNK by IL-1β. Gemigliptin treatment suppressed BMP-2-induced expression of key osteoblastic markers including osterix, runt-related transcription factor 2, and hepcidin during IL-1β-induced EndMT.
Conclusion
We demonstrated a novel protective mechanism of gemigliptin against fibrosis by suppressing IL-1β-induced EndMT.

Keyword

LC15-0444; Dipeptidyl-peptidase IV inhibitors; Interleukin-1beta; Bone morphogenetic proteins; Endothelial-to-mesenchymal transition

Figure

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  Fig. 1 Gemigliptin significantly but not completely suppressed the interleukin-1β (IL-1β)-induced smooth muscle cell (SMC) markers and mesenchymal stem cell (MSC) markers, reversed endothelial cell (EC) markers. Human umbilical vein endothelial cells (HUVECs) were treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 24 hours. (A) Representative Western blot image comparing changes in the expression of the endothelial cell marker endothelial nitric oxide synthase (eNOS), vascular endothelial cadherin (VE-cadherin), von Willebrand factor (vWF), the smooth muscle marker α-smooth muscle actin (α-SMA), transgelin (SM22), and mesenchymal cell marker fibroblast-specific protein 1 (FSP-1), fibronectin (FN), and collagen type I (Col I). (B) Quantification of protein expression by densitometry analysis of Western blots. Results were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) and expressed as fold-change relative to control without IL-1β. Values are mean±standard error of the mean (n=3). aP<0.05, control vs. IL-1β; bP<0.05, IL-1β vs. IL-1β+gemigliptin.

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  Fig. 2 Gemigliptin inhibits interleukin-1β (IL-1β)-induced the bone morphogenetic protein (BMP) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 24 hours. (A) Representative Western blot image comparing changes in the expression of the BMP2, BMP4, BMP7 and activin receptor type IA (Act-RIA), activin receptor type IIA (Act-RIIA), and activin receptor type IIB (Act-RIIB), and BMP receptor type IA (BMP-RIA) and IB (BMP-RIB), and type II (BMP-RII). (B, D) Quantification of protein expression by densitometry analysis of Western blots. (C) Representative Western blot image comparing changes in the expression of the transforming growth factor β1 (TGF-β1) and TGF-β receptor type I (TGF-β-RI), and type II (TGF-β-RII). Results were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) and expressed as fold-change relative to control without IL-1β. Values are mean±standard error of the mean (n=3). aP<0.05, Control vs. IL-1β; bP<0.05, IL-1β vs. IL-1β+gemigliptin.

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  Fig. 3 Gemigliptin inhibits interleukin-1β (IL-1β)-induced the Smad-dependent pathway. Human umbilical vein endothelial cells (HUVECs) were treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 24 hours. (A) Representative Western blot image comparing changes in the expression of the Smad1, Smad2, Smad3 and the phosphorylation of Smad1 (pSmad1), Smad2, Smad3. (B) Quantification of protein expression by densitometry analysis of Western blots. Results were expressed as fold-change relative to control without IL-1β. Values are mean±standard error of the mean (n=3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05, Control vs. IL-1β; bP<0.05, IL-1β vs. IL-1β+gemigliptin.

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  Fig. 4 Gemigliptin inhibits interleukin-1β (IL-1β)-induced the non-Smad bone morphogenetic protein pathway. Human umbilical vein endothelial cells were treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 15 minutes. (A) Representative Western blot image comparing changes in the expression of the extracellular regulated protein kinase (Erk), P38, JNK and the phosphorylation of Erk (pErk), P38 (pP38), JNK (pJNK) after exposure to treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 15 minutes. (B) Quantification of protein expression by densitometry analysis of Western blots. Values are mean±standard error of the mean (n=3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05, Control vs. IL-1β; bP<0.05, IL-1β vs. IL-1β+gemigliptin.

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  Fig. 5 Gemigliptin inhibits interleukin-1β (IL-1β)-induced osteogenic-specific transcription factor. Human umbilical vein endothelial cells were treated IL-1β (10 ng/mL) in the presence of absence of 20 μM gemigliptin for 15 minutes or 24 hours. (A) Representative Western blot image comparing changes in the expression of the osterix, runt-related transcription factor 2 (RUNX2), and hepcidin. (B) Quantification of protein expression by densitometry analysis of Western blots. Results were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control) and expressed as fold-change relative to control without IL-1β. Values are mean±standard error of the mean (n=3). aP<0.05, Control vs. IL-1β; bP<0.05, IL-1β vs. IL-1β+gemigliptin.

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