Protein kinase Cδ and c-Abl kinase are required for transforming growth factor β induction of endothelial-mesenchymal transition in vitro

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and nonfunctional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, transforming growth factor-β induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, transforming growth factor-β treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

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Anti-Fibrosis Effect of Relaxin and Spironolactone Combined on Isoprenaline-Induced Myocardial Fibrosis in Rats via Inhibition of Endothelial–Mesenchymal Transition

Cellular Physiology and Biochemistry ◽

10.1159/000464125 ◽

2017 ◽

Vol 41 (3) ◽

pp. 1167-1178 ◽

Cited By ~ 13

Author(s):

Jiejie Cai ◽

Xiao Chen ◽

Xingxing Chen ◽

Lingzhi Chen ◽

Gaoshu Zheng ◽

...

Keyword(s):

Growth Factor ◽

Myocardial Fibrosis ◽

Transforming Growth Factor ◽

Umbilical Vein ◽

Combined Therapy ◽

Fibrous Tissue ◽

Type I ◽

Mesenchymal Transition ◽

Endothelial Mesenchymal Transition

Background: The effect of relaxin and spironolactone combined on myocardial fibrosis has not been reported. Thus, we investigated the effect of the combined therapy on isoprenaline-induced myocardial fibrosis and the mechanism. Methods: Rats were injected subcutaneously with isoprenaline to induce myocardial fibrosis and underwent subcutaneous injection with relaxin (2 µg·kg-1·d-1) and given a gavage of spironolactone (30 mg·kg-1·d-1) alone or combined for 14 days. In vitro, the endothelial–mesenchymal transition was induced with transforming growth factor β (TGF-β) in human umbilical vein endothelial cells (HUVECs) pretreated with relaxin, 200 ng/ml, and/or spironolactone, 1uM. Results: Relaxin and spironolactone used alone or combined improved cardiac function and decreased cardiac weight indices; reduced fibrous tissue proliferation; reduced levels of type I and III collagen; decreased the expression of α–smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1), and increased the expression of cluster of differentiation-31 (CD31) in rats with isoprenaline-induced myocardial fibrosis. In vitro, compared with TGF-β treatment, relaxin and spironolactone used alone or combined with TGF-β decreased cell mobility, α-SMA and vimentin levels but increased vascular endothelial cadherin (VE-cadherin) and endothelial CD31levels. Especially, combined therapy had more remarkable effect than relaxin and spironolactone used alone both in vitro and in vivo. Conclusion: Relaxin and spironolactone combined affected isoprenaline-induced myocardial fibrosis in rats that the mechanism might be inhibition of the cardiac endothelial–mesenchymal transition.

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The Endothelial-mesenchymal Transition in Systemic Sclerosis Is Induced by Endothelin-1 and Transforming Growth Factor-β and May Be Blocked by Macitentan, a Dual Endothelin-1 Receptor Antagonist

The Journal of Rheumatology ◽

10.3899/jrheum.150088 ◽

2015 ◽

Vol 42 (10) ◽

pp. 1808-1816 ◽

Cited By ~ 38

Author(s):

Paola Cipriani ◽

Paola Di Benedetto ◽

Piero Ruscitti ◽

Daria Capece ◽

Francesca Zazzeroni ◽

...

Keyword(s):

Systemic Sclerosis ◽

Growth Factor ◽

Receptor Antagonist ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Mesenchymal Transition ◽

Endothelin 1 ◽

Smad Phosphorylation ◽

Endothelial To Mesenchymal Transition

Objective.High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation.Methods.EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects.Results.We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects.Conclusion.Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.

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Abstract 32: Investigating the Autoactivation of p38α Mitogen-Activated Protein Kinase Mediated by Transforming Growth Factor-β-Activated Protein Kinase Binding Protein 1

Circulation Research ◽

10.1161/res.111.suppl_1.a32 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Gian Felice De Nicola ◽

E D Martin ◽

Rekha Bassi ◽

Sharwari Verma ◽

Maria Conte ◽

...

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Transforming Growth Factor ◽

Binding Protein ◽

Transforming Growth Factor Β ◽

Mitogen Activated Protein Kinase ◽

Scaffolding Protein ◽

Titration Calorimetry ◽

Activation Loop

Activation of p38α MAPK (p38α), by phosphorylation of two residues in the TGY motif of the activation loop, can occur independently of upstream kinases. One such mechanism involves the scaffolding protein Transforming Growth Factor-β-activated protein kinase binding protein 1 (TAB1). Under certain circumstances, such as myocardial ischemia, this activation can aggravate lethal injury. It is one of a few examples of activating autophosphorylation and poses a conundrum. How does an inactive kinase, and therefore with low affinity for ATP, phosphorylate its own activation loop when ATP binding is a prerequisite step for phosphotransfer? The aim of this study was to characterize the TAB1 binding of p38α. The binding characteristics of p38α and TAB1 were determined by Isothermal Titration Calorimetry, followed by the binding of p38α and ATPγS, a slowly hydrolysable form of ATP, in the presence and absence of TAB1. The binding of TAB1 to p38α increased significantly the affinity of p38α for ATP. Following the identification of a key region in TAB1 responsible for p38α binding, a synthetic peptide encompassing this region was used to analyze the biophysical and biological consequences of TAB1 binding. In vitro kinase assays were used to test the biochemical characteristics using a combination of wildtype kinase, kinase dead (K53M) or both in the absence or presence of TAB1(371-416). Using an antibody specific to the dual phosphorylation of the TGY motif as a readout, TAB1 binding to p38α increased p38α autophosphorylation in cis . NMR was employed to map the interaction surfaces between of p38α and TAB1 and to analyze the effect of TAB1-binding on p38α. The residues identified as important for the interaction between TAB1 and p38α were mutated and tested in cell free and biological systems to confirm their role as critical determinants for binding. In conclusion, we have further elucidated a mechanism whereby TAB1 binding to p38α alters the conformation of p38α, increasing its affinity for ATP and thereby facilitating autophosphorylation. We have identified the binding contacts of TAB1 and p38α that may be important in the design of therapeutics enabling selective and circumstance-specific inhibition of p38α activation.

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Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study

European Journal of Histochemistry ◽

10.4081/ejh.2018.2956 ◽

2018 ◽

Cited By ~ 1

Author(s):

Roberta Sferra ◽

Simona Pompili ◽

Luca Ventura ◽

Caroline Dubuquoy ◽

Silvia Speca ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Sphingosine Kinase ◽

Transforming Growth Factor Β ◽

Chronic Colitis ◽

Mesenchymal Transition ◽

Intestinal Fibrosis ◽

Sphingosine 1 Phosphate

A concomitant action of multiple profibrotic mediators appears crucial in the development and progression of fibrosis. Sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways are both involved in pathogenesis of fibrosis in several organs by controlling differentiation of fibroblasts to myofibroblasts and the epithelial to-mesenchymal transition. However, their direct involvement in chronic colitis-associated fibrosis it is not yet known. In this study we evaluated the immunohistochemical expression of some proteins implicated in sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in Dextrane Sodium Sulphate (DSS)-induced colorectal fibrosis in mice. Compared to control mice, DSS-induced chronic colitis mice developed a marked intestinal fibrosis associated with a concomitant overexpression of TGF-β, p-Smad3, α-SMA, collagen I-III, SPHK1, RhoA, PI3K, Akt, p-Akt, p-mTOR. This study highlights the relationship between the two pathways and the possible role of SPHK1 in the intestinal fibrosis. These results, if confirmed by in vitro studies, may have important clinical implications in the development of new therapeutical approaches in inflammatory bowel disease.

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Targeting the TGFβ pathway in uterine carcinosarcoma

Cell Stress ◽

10.15698/cst2020.11.234 ◽

2020 ◽

Vol 4 (11) ◽

pp. 252-260

Author(s):

Shailendra Kumar Dhar Dwivedi ◽

Geeta Rao ◽

Anindya Dey ◽

Megan Buechel ◽

Yushan Zhang ◽

...

Keyword(s):

Growth Factor ◽

Epithelial To Mesenchymal Transition ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

3D Culture ◽

Transforming Growth Factor Β ◽

Uterine Carcinosarcoma ◽

Mesenchymal Transition

Uterine carcinosarcoma (UCS) is a relatively infrequent, but extremely aggressive endometrial malignancy. Although surgery and chemotherapy have improved outcomes, overall survival (OS) remains dismal due to the lack of targeted therapy and biphasic (epithelial and mesenchymal) nature that renders the tumor aggressive and difficult to manage. Here we report a role of transforming growth factor-β (TGFβ) in maintaining epithelial to mesenchymal transition (EMT) phenotype and aggressiveness in UCS. Using a 3D-culture system, we evaluated the efficacy of the transforming growth factor-β receptor-I (TGFβR1) kinase inhibitor Galunisertib (GLT), alone and in combination with standard chemotherapeutic drugs used for the management of UCS. We demonstrate that GLT by inhibiting canonical and non-canonical signaling emanating from transforming growth factor-β1 (TGFβ1) reduces cellular viability, invasion, clonal growth and differentiation. Interestingly, GLT sensitizes UCS cells to chemotherapy both in vitro and in in vivo preclinical tumor model. Hence, targeting TGFβ signaling, in combination with standard chemotherapy, may be exploited as an important strategy to manage the clinically challenging UCS.

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