Heme oxygenase 1 (Hmox1), a ubiquitous enzyme degrading heme to carbon monoxide, iron, and biliverdin, is one of the cytoprotective enzymes induced in response to a variety of stimuli, including cellular oxidative stress. Gangliosides, sialic acid-containing glycosphingolipids expressed in all cells, are involved in cell recognition, signalling, and membrane stabilization. Their expression is often altered under many pathological and physiological conditions including cell death, proliferation, and differentiation. The aim of this study was to assess the possible role of Hmox1 in ganglioside metabolism in relation to oxidative stress. The content of liver and brain gangliosides, their cellular distribution, and mRNA as well as protein expression of key glycosyltransferases were determined in Hmox1 knockout mice as well as their wild-type littermates. To elucidate the possible underlying mechanisms between Hmox1 and ganglioside metabolism, hepatoblastoma HepG2 and neuroblastoma SH-SY5Y cell lines were used for in vitro experiments. Mice lacking Hmox1 exhibited a significant increase in concentrations of liver and brain gangliosides and in mRNA expression of the key enzymes of ganglioside metabolism. A marked shift of GM1 ganglioside from the subsinusoidal part of the intracellular compartment into sinusoidal membranes of hepatocytes was shown in Hmox1 knockout mice. Induction of oxidative stress by chenodeoxycholic acid in vitro resulted in a significant increase in GM3, GM2, and GD1a gangliosides in SH-SY5Y cells and GM3 and GM2 in the HepG2 cell line. These changes were abolished with administration of bilirubin, a potent antioxidant agent. These observations were closely related to oxidative stress-mediated changes in sialyltransferase expression regulated at least partially through the protein kinase C pathway. We conclude that oxidative stress is an important factor modulating synthesis and distribution of gangliosides in vivo and in vitro which might affect ganglioside signalling in higher organisms.

• MeSH
• Gangliosides metabolism   MeSH
• Heme Oxygenase-1 metabolism   MeSH
• Liver metabolism   MeSH
• Humans MeSH
• Brain metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Oxidative Stress physiology   MeSH
• Signal Transduction physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Gangliosides MeSH
• Heme Oxygenase-1 MeSH

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

• Keywords
• 17α- ethinylestradiol, bile acids, heme, multidrug resistance-associated protein 3, nuclear factor erythroid-2-related factor-2,
• MeSH
• ATP-Binding Cassette Transporters genetics   MeSH
• Alkaline Phosphatase blood   MeSH
• Bilirubin blood   pharmacology   MeSH
• Cholestasis blood   chemically induced   enzymology   MeSH
• Enzyme Induction drug effects   MeSH
• Ethinyl Estradiol MeSH
• Gene Expression drug effects   MeSH
• Heme pharmacology   MeSH
• Heme Oxygenase (Decyclizing) biosynthesis   genetics   MeSH
• Hepatocytes drug effects   enzymology   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Taurocholic Acid pharmacology   MeSH
• Protective Agents pharmacology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Rats, Wistar MeSH
• Primary Cell Culture MeSH
• Multidrug Resistance-Associated Proteins genetics   MeSH
• Bile Acids and Salts blood   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Abcc2 protein, rat MeSH Browser
• Abcc4 protein, rat MeSH Browser
• Alkaline Phosphatase MeSH
• Bilirubin MeSH
• Ethinyl Estradiol MeSH
• Heme MeSH
• Heme Oxygenase (Decyclizing) MeSH
• Taurocholic Acid MeSH
• Protective Agents MeSH
• Multidrug Resistance-Associated Proteins MeSH
• Bile Acids and Salts MeSH