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Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade.

ABSTRACT: Cellular damage and accumulation of extracellular matrix (ECM) protein in the glomerulo-interstitial space are the signatures of chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy), a high level of homocysteine (Hcy) is associated with CKD and further contributes to kidney damage. Despite a large number of studies, the signalling mechanism of Hcy-mediated cellular damage and ECM remodelling in kidney remains inconclusive. Hcy metabolizes to produce hydrogen sulphide (H2S), and a number of studies have shown that H2S mitigates the adverse effect of HHcy in a variety of diseases involving several signalling molecules, including forkhead box O (FOXO) protein. FOXO is a group of transcription factor that includes FOXO1, which plays important roles in cell growth and proliferation. On the other hand, a cell survival factor, Akt regulates FOXO under normal condition. However, the involvement of Akt/FOXO1 pathway in Hcy-induced mesangial cell damage remains elusive, and whether H2S plays any protective roles has yet to be clearly defined. We treated mouse mesangial cells with or without H2S donor, GYY4137 and FOXO1 inhibitor, AS1842856 in HHcy condition and determined the involvement of Akt/FOXO1 signalling cascades. Our results indicated that Hcy inactivated Akt and activated FOXO1 by dephosphorylating both the signalling molecules and induced FOXO1 nuclear translocation followed by activation of the FOXO1 transcription factor. These led to the induction of cellular apoptosis and synthesis of excessive ECM protein, in part, due to increased ROS production, loss of mitochondrial membrane potential (??m), reduction in intracellular ATP concentration, increased MMP-2, -9, -14 mRNA and protein expression, and Col I, IV and fibronectin protein expression. Interestingly, GYY4137 or AS1842856 treatment prevented these changes by modulating Akt/FOXO1 axis in HHcy. We conclude that GYY4137 and/or AS1842856 mitigates HHcy induced mesangial cell damage and ECM remodelling by regulating Akt/FOXO1 pathway.

SUBMITTER: Majumder S

PROVIDER: S-EPMC6561819 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade.

Majumder Suravi S Ren Lu L Pushpakumar Sathnur S Sen Utpal U

Cellular signalling 20190511

Cellular damage and accumulation of extracellular matrix (ECM) protein in the glomerulo-interstitial space are the signatures of chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy), a high level of homocysteine (Hcy) is associated with CKD and further contributes to kidney damage. Despite a large number of studies, the signalling mechanism of Hcy-mediated cellular damage and ECM remodelling in kidney remains inconclusive. Hcy metabolizes to produce hydrogen sulphide (H<sub>2</sub>S), and a ...[more]

PMID: 31085234

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Project description:IntroductionHydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer.ObjectivesThis study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs.MethodsEighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively.ResultsThe H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells.ConclusionThese findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Project description:The dysfunction of mesangial cells (MCs) in high-glucose (HG) conditions plays pivotal role in inducing glomerular sclerosis by causing the imbalance between generation and degradation of extracellular matrix (ECM) proteins, which ultimately leads to diabetic nephropathy. This study was designed to determine the function of forkhead box protein O1 (FoxO1), an important transcription factors in regulating cell metabolism and oxidative stress, in MCs in HG conditions. Up-regulation of fibronectin, collagen type IV, and plasminogen activator inhibitor (PAI-1) was observed under HG conditions in vivo and in vitro, accompanied with elevation of protein kinase B (Akt) phosphorylation and reduction of FoxO1 bioactivity. After overexpression of constitutively active (CA) FoxO1 in vivo and in vitro by using lentivirus vector, in vivo and in vitro, FoxO1 expression and activity was increased, in accordance with up-regulation of antioxidative genes (catalase and superoxide dismutase, leading to alleviated oxidative stress as well as attenuated Akt activity, whereas overexpression of wild type-FoxO1 only expressed partial effect. Moreover, CA-FoxO1 decreased the expression of fibronectin, collagen type IV, and PAI-1, causing amelioration of renal pathological changes and decrease of ECM protein deposition in glomerulus. Overexpression of CA-FoxO1 in renal cortex also decreased activin type-I receptor-like kinase-5 levels and increased signaling mothers against decapentaplegic (Smad) 7 levels, and simultaneously inhibited Smad3 phosphorylation. Results from in vitro study indicated that increased combination of FoxO1 and Smad3 may interfere with the function of Smad3, including Smad3 phosphorylation and translocation, interaction with cAMP response element binding protein (CREB)-binding protein, and binding with PAI-1 promoter. Together, our findings shed light on the novel function of FoxO1 in inhibiting ECM deposition, which is beneficial to ameliorate MC dysfunction.

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Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade.

Publications

Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade.

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