Changes in gene expression induced by hypoxia in cultured gas gland cells from Atlantic cod (Gadus morhua)

Abstract

Low level of oxygen, called hypoxia, triggers the transcription of regulatory genes that promote O2 delivery and anaerobic metabolism. The majority of this transcriptional response to hypoxia is mediated by the Hypoxia inducible factor (HIF-1), comprising the two subunits HIF-1α and HIF-1β. The HIF-1α is expressed ubiquitously in all cells and targeted for proteosomal degradation. When fish are exposed to hypoxia condition, HIF-1α degradation is inhibited and subsequently activates or inhibits the expression of specific genes. In hypoxia, prolylhydroxylase domain (PHD) is less active, so the degradation is inhibited and subsequently allows HIF-1α to make Aryl hydrocarbon nuclear translocator (ARNT) - HIF1α complex and translocates to the nucleus. Finally, this complex binds to the target genes and recruit transcriptional co-activators for full transcriptional activity and either activate or inhibit the expression of specific genes. In this study, I analyzed the expression of hypoxia inducible genes, Hypoxia inducible factor-1α (HIF-1α), Lactate dehydrogenase a (LDHa), Glucose transporter-1 (GLUT-1) and Carbonic anhydrase (CA-6) genes in cultured gas gland cells from Atlantic cod (Gadus morhua). Gas gland cells of the swim bladder were cultured in growth medium and treated with the PHD inhibitor FG-9245 diluted in three different concentrations (10, 50 and 90µM) using DMSO. Total duration of cell culture was 16 days. Total mRNA was extracted from the cells and a qPCR analysis was performed by using a Light cycler® 480 platform to quantify the mRNA levels of the four genes. Relative expression of all four genes had increased in the FG-9245 treated group than the control group. However, LDHa and GLUT-1 showed low relative gene expression in 90µM PHD inhibitor treated cells then 50µM inhibitor treatment. In 90µM inhibitor treated cells, there was a sharp increase of relative gene expression of CA-6. The HIF-1α protein was detected in the gas gland tissue of Atlantic cod at normal oxygen level by Western blotting using a specific HIF-1α antibody. Although expected molecular weight was 86kDa, but the molecular weight was observed 28 kDa probably due to degradation in environmental oxygen level. Relative gene expression pattern of HIF-1α, LDHa, GLUT-1 and CA-6 suggests the efficacy of PHD inhibitor on determining the degree of hypoxia in Atlantic cod in vitro.