Elsevier

Gene

Volume 396, Issue 2, 15 July 2007, Pages 273-282
Gene

Molecular cloning, characterization and expression of two hypoxia-inducible factor alpha subunits, HIF-1α and HIF-2α, in a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus)

https://doi.org/10.1016/j.gene.2007.03.009Get rights and content

Abstract

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 α and β subunits. In this study, we report the cloning and characterization of two HIF-α subunits, HIF-1α and HIF-2α, and their expression in various tissues of a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus). The full-length croaker HIF-1α (2805 bp) and HIF-2α (3205 bp) cDNAs contain open reading frames encoding proteins with 720 and 847 amino acids, respectively, which are highly homologous to the HIF-1α and HIF-2α proteins of other non-mammalian species. Croaker HIF-1α shares only 43% sequence identity with the croaker HIF-2α 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-1α and HIF-2α 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-1α and HIF-2α 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-1α and HIF-2α subunits at the transcriptional level is an important component of adaptation of croaker to chronic hypoxia and HIF-αs are potentially useful molecular indicators of environmental hypoxia exposure.

Introduction

Hypoxia-inducible factors (HIFs) are dimeric transcription factors that mediate changes in gene expression during adaptation of animals to low oxygen conditions (Semenza, 1998, Wenger, 2002). HIFs are composed of two subunits: the hypoxia-regulated α subunit, HIF-1α (or its paralogs HIF-2α and HIF-3α), and the oxygen-insensitive HIF-1β subunit (also known as the aryl hydrocarbon receptor nuclear translocator, ARNT) (Wenger and Gassmann, 1997, Semenza, 1998, Semenza, 2001). Both subunits are members of the basic helix–loop–helix (bHLH)-containing Per–ARNT–Sim (PAS) domain protein family (Wenger, 2002).

The biological activity of HIF-1 is determined by the expression and activity of the α subunit (Wiener et al., 1996, Jiang et al., 1996a, Jiang et al., 1997, Pugh et al., 1997, Kallio, 1998, Yu et al., 1998, Bergeron et al., 1999). Regulation of HIF-1α expression and activity can occur through multiple mechanisms, including alterations of mRNA expression (Wiener et al., 1996, Yu et al., 1998, Bergeron et al., 1999), protein expression (Jiang et al., 1996a, Pugh et al., 1997, Yu et al., 1998), nuclear localization (Kallio et al., 1998), and transactivation (Jiang et al., 1996a, Jiang et al., 1997, Pugh et al., 1997). Among these, the most intensively studied mechanism has been the regulation of steady-state HIF-1α protein levels in higher vertebrates. In mammals, HIF-1α protein levels and DNA-binding activity are tightly regulated by the level of oxygen concentration and are increased as the oxygen level decreases (Jiang et al., 1996b). Under normoxic conditions, HIF-1α and HIF-2α proteins are targeted for proteosomal degradation through the oxygen-dependent hydroxylation of proline residues in the degradation domain (Huang et al., 1998). However, the proteins are stabilized under hypoxic conditions and HIF-1α and HIF-2α form separate heterodimers with the HIF-1β (ARNT) protein. The HIF-α/β heterodimers translocate to the nucleus and bind to hypoxia response elements (HREs) on hypoxia-sensitive genes resulting in alterations in their rates of transcription (Bracken et al., 2003). Functional comparisons between HIF-1α and HIF-2α reveal many similarities concerning genomic organization, modular protein structure, hypoxic protein stabilization, heterodimerization, DNA binding, and the transactivation function of reporter genes (Ema et al., 1997, Hogenesch, 1997, Wenger and Gassmann, 1997, Wiesener et al., 1998, Maxwell et al., 1999, O'Rourke et al., 1999). HIF-3α is an additional homolog, on the other hand, whose function and expression pattern are less well characterized in higher vertebrates (Gu et al., 1998).

Molecular responses to hypoxia have not been studied extensively in teleost fishes, even though many fish species are periodically exposed to hypoxia in their natural habitats. Three HIF-α cDNA variants have been cloned and characterized in teleost fishes; HIF-1α in rainbow trout, Oncorhynchus mykiss (Soitamo et al., 2001) and grass carp, Ctenopharyngodon idellus (Law et al., 2006), HIF-2α in killifish, Fundulus heteroclitus (Powell and Hahn 2002), and HIF-4α in grass carp (Law et al., 2006). Based on bioinformatic searches of several databases, HIF-3α has not been identified in any teleost fishes. Activated HIF-1α protein, capable of binding to human erythropoietin HRE sequences, has been detected in rainbow trout cells exposed to hypoxia (Soitamo et al., 2001). The HIF-2α cDNA in an estuarine species, killifish, and the HIF-1α and HIF-4α cDNAs in a freshwater species, grass carp, show relatively high homology with mammalian HIFs (Powell and Hahn, 2002, Law et al., 2006). Several differences in the tissue distribution of 1α and 4α HIFs in grass carp and their expression patterns in response to hypoxia stress have been reported (Law et al., 2006). However, equivalent information is lacking for any marine teleost species and the characteristics, regulation and function of HIF-α subunits in fish inhabiting different hypoxic environments remain poorly understood.

Hypoxia is a natural seasonal phenomenon in estuaries and coastal regions, the incidence of which has greatly increased over the past 25 years due to eutrophication caused by several-fold increases in anthropogenic inputs of nitrogen (Smith, 2003). For example, seasonal hypoxia now affects a 12–16 × 103 km2 coastal region in the northern Gulf of Mexico and 30% of the total estuarine area along the Gulf of Mexico coastline of the United States (Engle et al., 1999, Rabalais et al., 2002). There is mounting concern over the impacts of these changes on marine ecosystems (Diaz and Rosenberg, 1995). However, the long-term effects of chronic seasonal hypoxia on the sustainability of fishery resources cannot be predicted accurately at present because of the current lack of knowledge of the molecular, physiological and population responses of marine fish to this environmental stressor.

The Atlantic croaker (Micropogonias undulatus) is a hypoxia-tolerant species (Bell and Eggleston, 2005, Eby and Crowder, 2002) that is abundant in estuaries along the United States Atlantic and Gulf of Mexico coasts and also inhabits the extensive hypoxic region in the northern Gulf of Mexico. Thus, Atlantic croaker is an excellent marine teleost model for investigating the molecular and physiological responses to hypoxia. In the present study, we cloned and characterized two distinct HIF-α isoforms, HIF-1α and HIF-2α, from croaker ovarian tissue. In addition, we examined their mRNA expression patterns in response to short- and long-term hypoxia exposure.

Section snippets

Experimental fish

Young-of-the year Atlantic croaker (length 14–16 cm, 30–38 g body weight, BW), M. undulatus, were collected by otter trawl in the vicinity of Port Aransas, Texas, by local fisherman in August, 2004. The fish were transported to the University of Texas Marine Science Institute, Port Aransas, and transferred to large indoor tanks (4727 L) equipped with a recirculating seawater system (salinity 30–32‰) and maintained under control photoperiod (photophase 12 h) and temperature (27 °C) conditions.

Cloning and characterization of croaker HIF-1α and -2α cDNAs

Partial coding regions of croaker HIF-1α (947 bp) and HIF-2α (997 bp) cDNAs were isolated by RT-PCR amplification. Following the design of the gene-specific primers, the 5′ and 3′ ends of these partial sequences were amplified by 5′- and 3′-RACE. In the overlapping portions, the sequences of the RACE products were found to be identical at the nucleotide level to the partial sequences. The full-length croaker HIF-1α cDNA consists of 2805 bp, which contains a 5′-untranslated region (5′-UTR,

Discussion

The present study is the first report of cloning and characterization two HIF-α cDNAs, HIF-1α and -2α, from a hypoxia-tolerant marine teleost and their mRNA expression levels during adaptation to chronic hypoxia. The predicted amino acid sequences of croaker HIF-1α and -2αs are very similar to those of other vertebrates and contain all of the characteristic motifs of HIF-α proteins, suggesting they have similar functions in adaptation to hypoxia as in other vertebrate species. There were

Acknowledgements

The authors thank to Susan Lawson for fish care and maintenance of the DO conditions in the experimental tanks. This study was supported by Environmental Protection Agency STAR Grant EPA R-82945801 (to P.T.).

First page preview

First page preview
Click to open first page preview

References (36)

  • T. Uchida

    Prolonged hypoxia differentially regulates hypoxia-inducible factor (HIF)-1α and HIF-2α expression in lung epithelial cells: implication of natural antisense HIF-1α

    J. Biol. Chem.

    (2004)
  • D.P. Wang

    Hypoxia-inducible factor 1α cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai–Tibetan plateau

    Biochem. Biophys. Res. Commun.

    (2006)
  • C.M. Wiener et al.

    In vivo expression of mRNAs encoding hypoxia-inducible factor 1

    Biochem. Biophys. Res. Commun.

    (1996)
  • M.S. Wiesener

    Induction of endothelial PAS domain protein-1 by hypoxia: characterization and composition with hypoxia-inducible factor-1α

    Blood

    (1998)
  • T.B. Zhao

    Cloning of hypoxia-inducible factor 1α cDNA from a high hypoxia tolerant mammal-plateau pike (Ochotona curzoniae)

    Biochem. Biophys. Res. Commun.

    (2004)
  • G.W. Bell et al.

    Species-specific avoidance responses by blue crabs and fish to chronic and episodic hypoxia

    Mar. Biol.

    (2005)
  • M. Bergeron et al.

    Induction of hypoxia-inducible factor 1 (HIF-1) and its target genes following focal ischemia in rat brain

    Eur. J. Neurosci.

    (1999)
  • C.P. Bracken et al.

    The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses

    Cell. Mol. Life Sci.

    (2003)
  • Cited by (127)

    • Role of HIF in fish inflammation

      2023, Fish and Shellfish Immunology
    View all citing articles on Scopus
    View full text