ABSTRACT
BACKGROUND & AIMS: The organic solute transporters alpha and beta (OSTα-OSTß) form a heterodimeric transporter located at the basolateral membrane of intestinal epithelial cells and hepatocytes. Liver injury caused by ischaemia-reperfusion, cancer, inflammation or cholestasis can induce a state of hypoxia in hepatocytes. Here, we studied the effect of hypoxia on the expression of OSTα-OSTß. METHODS: OSTα-OSTß expression was measured in Huh7 cells and primary human hepatocytes (PHH) exposed to chenodeoxycholic acid (CDCA), hypoxia or both. OSTα-OSTß promoter activity was analysed in luciferase reporter gene assays. Binding of hypoxia-inducible factor-1 alpha (HIF-1α) to the OSTα-OSTß gene promoters was studied in electrophoretic mobility shift assays (EMSA). RESULTS: Expression of OSTα and OSTß increased in PHH under conditions of hypoxia. Exposure of Huh7 cells or PHH to CDCA (50 µM) enhanced the effect of hypoxia on OSTα mRNA levels. In luciferase assays and EMSA, the inducing effect of low oxygen could be assigned to HIF-1α, which binds to hypoxia responsive elements (HRE) in the OSTα and OSTß gene promoters. Site-directed mutagenesis of either the predicted HRE or the bile acid responsive FXR binding site abolished inducibility of the OSTα promoter, indicating that both elements need to be intact for induction by hypoxia and CDCA. In a rat model of chronic renal failure, the known increase in hepatic OSTα expression was associated with an increase in HIF-1α protein levels. CONCLUSION: OSTα-OSTß expression is induced by hypoxia. FXR and HIF-1α bind in close proximity to the OSTα gene promoter and produce synergistic effects on OSTα expression.
Subject(s)
Hepatocytes/metabolism , Liver/metabolism , Membrane Transport Proteins/metabolism , Animals , Binding Sites , Cell Hypoxia , Cell Line , Chenodeoxycholic Acid/pharmacology , Disease Models, Animal , Hepatocytes/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Failure, Chronic/metabolism , Liver/drug effects , Membrane Transport Proteins/drug effects , Membrane Transport Proteins/genetics , RNA Interference , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Response Elements , Transfection , Up-RegulationABSTRACT
Azaspiracid-1 is a novel algal toxin, which causes an instantaneous rise of intracellular messengers, and an irreversible disarrangement of the actin cytoskeleton. Little is known regarding the molecular mechanisms that are involved in azaspiracid-1 toxicity. This study investigated global changes in protein expression by stable-isotope labelling with amino acids in culture and mass spectrometry, following exposure of human neuroblastoma cells to azaspiracid-1. The most highly upregulated proteins were involved in cellular energy metabolism, followed by cytoskeleton regulating proteins. The majority of downregulated proteins were involved in transcription, translation and protein modification. In addition, two proteins, component of oligomeric Golgi complex 5 and ras-related protein RAB1, which are involved in the maintenance of the Golgi complex and vesicle transport, respectively, were downregulated. Electron microscopy revealed a disruption of the Golgi complex by azaspiracid-1, and an accumulation of vesicles. In this study, the differential protein expression was examined prior to changes of the cytoskeleton structure in order to capture the primary effects of azaspiracid-1, however the observed changes were of unexpected complexity. Azaspiracid-1 caused a pronounced, but temporary depletion of ATP, which may be the reason for the observed complexity of cellular changes.
