Molecular cloning, characterization and expression of two hypoxia-inducible factor alpha subunits, HIF-1alpha and HIF-2alpha, in a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus).
Alteration of gene expression is a crucial component of adaptation by animals to hypoxic conditions and is mediated by specific transcription factors, hypoxia-inducible factors (HIFs), which are composed of alpha and beta subunits. In this study, we report the cloning and characterization of two HIF-alpha subunits, HIF-1alpha and HIF-2alpha, and their expression in various tissues of a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus). The full-length croaker HIF-1alpha (2805 bp) and HIF-2alpha (3205 bp) cDNAs contain open reading frames encoding proteins with 720 and 847 amino acids, respectively, which are highly homologous to the HIF-1alpha and HIF-2alpha proteins of other non-mammalian species. Croaker HIF-1 alpha shares only 43% sequence identity with the croaker HIF-2alpha subunit. However, the basic helix-loop-helix/Per-ARNT-Sim regions appear to be relatively well conserved between the two proteins, with identities of 75-83%. The core oxygen-dependent degradation domain regions in croaker HIFs are well conserved, suggesting a similar mechanism of HIF degradation to that in other vertebrate species. Northern blot analysis showed that croaker HIF-1alpha and HIF-2alpha mRNAs (transcript sizes 3.0-3.8 kb) are highly expressed in the brain, heart, liver, and gonads under hypoxic conditions, whereas muscle tissues show lower levels of expression. Short-term (1.7 mg/L dissolved oxygen, DO for 3 days to 1 week) and long-term (1.7, 2.7 and 3.7 mg/L DO for 3 weeks) hypoxia exposure caused significant increases in HIF-1alpha and HIF-2alpha mRNA expression in croaker ovaries compared to mRNA levels in fish held in normoxic conditions (DO: 6.5 mg/L). However, HIF transcript levels in hypoxia-exposed fish had returned to control values 24 h after the DO in the tanks was restored to normoxic levels. The results suggest that the upregulation of both HIF-1alpha and HIF-2alpha subunits at the transcriptional level is an important component of adaptation of croaker to chronic hypoxia and HIF-alphas are potentially useful molecular indicators of