Erythropoietin gene from a teleost fish, Fugu rubripes

Abstract

In this paper we report the cloning and characterization of the erythropoietin (Epo) gene from the pufferfish, Fugu rubripes. This is the first nonmammalian Epo gene to be cloned. The Fugu Epo comprises 5 exons and 4 introns similar to the human EPO, and encodes a 185-amino acid protein that is 32% to 34% identical to Epo from various mammals. The synteny of genes at the Epo locus is conserved between the Fugu and humans. Unlike in mammals in which adult kidney is the primary Epo-producing organ, the heart is the main Epo-producing organ in adult Fugu. In addition to the heart, Fugu Epo is also expressed in the liver and brain similar to the human EPO. Interestingly, the transcripts in the Fugu brain are generated from a distal promoter and include an alternatively spliced first coding exon. No such brain-specific alternative splicing of Epo has been reported in mammals so far. Transient transfection studies in a fish hepatoma cell line (PLHC-1) and a human hepatoma cell line (HepG2) suggest that although the Fugu Epo promoter many not be hypoxia inducible, the gene may be regulated by hypoxia. ( IntroductionErythropoietin (Epo) is a glycoprotein hormone that plays a crucial role in ensuring supply of adequate oxygen to tissues by regulating the production of red blood cells. In mammals, Epo stimulates differentiation and proliferation of erythroid precursor cells in the bone marrow, in response to decreased environmental oxygen concentration or systemic oxygen deficiency caused by anemia. 1 In humans, fetal liver and adult kidney are the primary sites of EPO production. 1 In addition to these tissues, EPO is also produced in the central nervous system, bone marrow, spleen, heart, lung, ovary, testis, and breast cancer cells. [2][3][4][5][6][7] The production of Epo in the kidney, liver, and the central nervous system is greatly induced by hypoxic conditions. 8,9 The gene encoding the human EPO was first cloned in 1985 by 2 groups independently. 10,11 Since then, the Epo gene has been cloned from several mammalian species including nonhuman primates, rodents, ruminants, and felines. [12][13][14][15] The human EPO gene is transcribed from several start sites in the kidney and liver, and is thus independently regulated in these tissues. 16 Expression of the human EPO gene in transgenic mice has indicated that the cis-elements directing expression to the kidney are located between 6 kb and 14 kb upstream of the basal promoter, whereas the liver-specific elements are located in the 3Ј flanking region. 16 A 43-bp cis-element, capable of mediating the hypoxia response in the liver, also resides within the 3Ј flanking region, 120 bp downstream of the polyadenylation (polyA) signal. [16][17][18] Transient transfection studies in human hepatoma cell lines have demonstrated that the 3Ј enhancer induces 15-to 50-fold higher expression of a reporter gene in response to hypoxia.Although an "immunoreactive erythropoietin" that competes with human EPO for reaction with human EPO antibodies has been demonstrated in some teleos...

The hypoxia signaling pathway and hypoxic adaptation in fishes

“…The first Epo gene fish cloned was that of the pufferfish (Fugu; Takifugu rubripes). In Fugu, Epo is induced by hypoxia and is mainly expressed in the heart [80][81][82]. However, in other teleost fishes, such as rainbow trout, carp, and eel, Epo was expressed in the kidney, the kidney is the major organ for erythropoiesis in fishes [83].…”

Multiple and highly divergent IL-11 genes in teleost fish

“…Very recently, an IL-11 peptide was reported in rainbow trout (Oncorhynchus mykiss) (Wang et al 2005). Fish orthologues of several other mammalian four-helix bundle cytokines, including IL-12p35 (Yoshiura et al 2003) and EPO (Chou et al 2004), are known, and these cytokines invariably exhibited low overall amino acid similarity to their mammalian orthologues. Furthermore, a four-helix bundle cytokine designated M17 that shares similarities with OSM, LIF, and CNTF was recently described in carp (Fujiki et al 2003).…”

Functional conservation of erythropoietin signaling in zebrafish

“…fugu Epo lacks all 3 N-linked glycosylation sites, yet contains the O-linked glycosylation site. 29 Addition of glycan moieties to Epo in mammals and teleosts underscores the importance of posttranslational modifications for in vivo function, yet the localization and number of glycosylation sites appear to have diverged.EpoR belongs to the type I superfamily of single-transmembrane cytokine receptors. 21 This family shares conserved extracellular ligand-binding regions and a cytoplasmic box 1 motif, which selectively binds Jaks, conferring kinase activity to the receptor.…”