Role of Hydrogen Sulfide and 3-Mercaptopyruvate Sulfurtransferase in the Regulation of the Endoplasmic Reticulum Stress Response in Hepatocytes

Abstract

It is estimated that over 1.5 billion people suffer from various forms of chronic liver disease worldwide. The emerging prevalence of metabolic syndromes and alcohol misuse, along with the lack of disease-modifying agents for the therapy of many severe liver conditions predicts that chronic liver disease will continue to be a major problem in the future. Better understanding of the underlying pathogenetic mechanisms and identification of potential therapeutic targets remains a priority. Herein, we explored the potential role of the 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide (H2S) system in the regulation of the endoplasmic reticulum (ER) stress and of its downstream processes in the immortalized hepatic cell line HepG2 in vitro. ER stress suppressed endogenous H2S levels and pharmacological supplementation of H2S with sodium hydrogen sulfide (NaHS) mitigated many aspects of ER stress, culminating in improved cellular bioenergetics and prevention of autophagic arrest, thereby switching cells’ fate towards survival. Genetic silencing of 3-MST or pharmacological inhibition of the key enzymes involved in hepatocyte H2S biosynthesis exacerbated many readouts related to ER-stress or its downstream functional responses. Our findings implicate the 3-MST/H2S system in the intracellular network that governs proteostasis and ER-stress adaptability in hepatocytes and reinforce the therapeutic potential of pharmacological H2S supplementation.

Overproduction of hydrogen sulfide, generated by cystathionine β-synthase, disrupts brain wave patterns and contributes to neurobehavioral dysfunction in a rat model of down syndrome

“…Recent studies demonstrate that oxidative phosphorylation is markedly dysregulated in the brain in a mouse model of DS and AD [ 65 ]. Indeed, mitochondrial bioenergetic dysfunction and associated defects in protein processing have been already implicated in many forms of neurodegeneration [ [66] , [67] , [68] , [69] , [70] , [71] , [72] ]. In this context, it is interesting to note that Kanaumi and colleagues have previously detected a marked increase in CBS-positive astrocytes around senile plaques in adults with DS [ 17 ].…”

Role of the cystathionine β-synthase / H2S pathway in the development of cellular metabolic dysfunction and pseudohypoxia in down syndrome

“…Cell Proliferation kit II (XTT), Cell Proliferation ELISA BrdU kit and Cytotoxicity Detection KitPLUS (LDH) were employed the quantification of fibroblast viability, proliferation, and necrosis, respectively. The assays were formatted in Corning® Costar® TC-Treated, flat-bottom, transparent 96-well microplates and performed as previously described [ 12 , 13 ].…”

“…Fluorescent signals were read with Infinite® 200 PRO microplate reader and imaged with the upright laser scanning confocal LEICA TCS SP5 BIO microscope. AzMC and P3 fluorophores were employed for the quantification of the intracellular H 2 S levels, as per our previously published methodology [ 12 ], while the cell-permeant reagent 2′,7′-dichlorofluorescein diacetate (DCFDA) was utilized for measuring measures hydroxyl, peroxyl, and other ROS species within the cell in accord to the manufacturer's protocol. Briefly, 24 h post the treatment with AOAA, fibroblasts were incubated with 20 μM DCFDA, 100 nM Invitrogen™ MitoTracker™ Deep Red FM and 1 μg/ml DAPI in assay buffer supplemented with 10% FBS for 1h at 37 °C in a humidified incubator with 5% CO 2 and 95% air.…”

“…Measurement of Mitochondrial Respiration. Extracellular Flux (XF) analysis was employed for real-time quantification of oxygen consumption rate (OCR), proton efflux rate (PER) in intact cells; measurement of Complex IV activity was performed in permeabilized cells, using ascorbate and tetramethyl- p -phenylene diamine as Complex IV electron donors, according to our previously described methodology [ 9 , 12 ]. The assays were formatted in Seahorse XF24 cell-culture microplates, where diploid and 21-trisomic fibroblasts were seeded at a density of 2 × 10 4 cells per well in total 200 μl for 2 h. Cells were subsequently treated with 0 or 3 μM AOAA for 24 h before the mitochondrial respiration being assessed.…”

“…After two freeze/thaw cycles, the whole-cell lysate was collected and sonicated for 1 min (5 s ON/5 s OFF-6 cycles). Whole-cell proteins were extracted, quantified, handled and processed for Western blotting as previously published [ 9 , 12 ].…”