Understanding Hypoxia-Induced Gene Expression in Early Development: In Vitro and In Vivo Analysis of Hypoxia-Inducible Factor 1-Regulated Zebra Fish Insulin-Like Growth Factor Binding Protein 1 Gene Expression

Abstract

Hypoxia triggers the transcription of regulatory genes that promote O 2 delivery and anaerobic metabolism, suppress major energy-requiring processes, and inhibit growth and development in animals ranging from invertebrates to mammals (14,15,40). Hypoxia also influences several human pathological processes, such as tumorigenesis and intrauterine growth restriction (IUGR). The majority of these transcriptional responses to hypoxia are mediated by the hypoxia-inducible factor 1 (HIF-1) complex. HIF-1 is a heterodimeric complex composed of 45). HIF-1␤, also known as aryl hydrocarbon receptor nuclear translocator, is constitutively expressed and insensitive to O 2 availability. When oxygen levels are high, HIF-1␣ is bound to the von Hippel-Lindau tumor suppressor (pVHL) and targeted for ubiquitination and proteosomal degradation (30, 34). Hypoxic conditions inhibit this degradation, which allows HIF-1␣ to accumulate in the cell (16,18,27). HIF-1␣ is then translocated to the nucleus, dimerizes with HIF-1␤, binds to DNA, and activates target gene expression (3). Hypoxia response elements (HREs) are cis-regulatory DNA sequences that specifically bind to HIF-1 and are required for transcriptional induction upon hypoxia exposure.Insulin-like growth factor binding protein 1 (IGFBP-1) is a hypoxia-inducible gene. Earlier studies have shown that circulating levels of IGFBP-1 are elevated in IUGR fetuses (7,12,42,43). In vitro studies using cultured human cells and in vivo studies using rodent and fish models suggest that IGFBP-1 gene expression is elevated under hypoxic conditions (13,29,31,35,41). Since IGFBP-1 binds IGFs and inhibits IGF action on cell growth in vitro (4, 9) and because IGFBP-1 overexpression reduced birth weights in mice (5,11,36), it was postulated that elevated IGFBP-1 plays a major role in hypoxia-induced IUGR by binding IGFs and inhibiting their growth-promoting activities (41). Using the transparent and free-living zebra fish embryo as a model system, we have recently shown that (i) hypoxia strongly up-regulates IGFBP-1 expression and delays growth and developmental rate in zebra fish embryos; (ii) IGFBP-1 knockdown partially abrogates these hypoxic effects, whereas IGFBP-1 overexpression decreases growth and developmental rates under normoxia; and (iii) reintroduction of IGFBP-1 to the knocked down embryos restores the hypoxic effects (19). These findings provide strong evidence arguing that up-regulation of IGFBP-1 by hypoxia plays a key role in coordinating embryonic growth rate and developmental timing in response to environmental oxygen availability. Although there is in vitro evidence that overexpression of HIF-1␣ in cultured human hepatoma (HepG2) cells increases human IGFBP-1 promoter activity (41), how hypoxia triggers IGFBP-1 gene expression in vivo is not clear, and the cis-regulatory elements responsible for hypoxia-induced IGFBP-1 transcription in vivo are not well defined.The objectives of this study are (i) to determine when the HIF-1 pathway becomes operational in early development and

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“…This difference in temporal expression pattern signifies that the species of the present study can better tolerate short-term hypoxia, up to 72 h without the aid of a high HIF expression. Also, HIF expression pattern suggests that HIF-1 α is involved in the adaptive response to hypoxia in C. mrigala and helps the fish to survive under short-term hypoxia which is in agreement with several earlier studies (Kajimura et al, 2006;Rahman & Thomas, 2007;Rytkonen et al, 2007). It was also observed that the response of HIF-1 α to low environmental oxygen tensions was quite rapid as the expression level increased even at one-hour exposure to 0.5±0.16 mg/L oxygen level.…”

“…HIF-1 is a heterodimeric transcription factor formed by the association of a constitutively expressed beta subunit and an inducible alpha subunit, which is rapidly degraded in the presence of oxygen level (Wang & Semenza, 1995). Furthermore, the metabolic and haematological responses during hypoxia are mediated by HIF, which is well studied in several fish species (Kajimura, Aida, & Duan, 2006;Rahman & Thomas, 2007;Rytkonen, Vuori, Primmer, & Nikinmaa, 2007;Terova et al, 2008). However, the time of exposure to hypoxia and its intensity determine the threshold and magnitude of these responses, which is critical to enable the survival of the fish in the hypoxic water.…”

Metabolic hormones regulate insulin-like growth factor binding protein-1 mRNA levels in primary cultured salmon hepatocytes; lack of inhibition by insulin

“…However, in mammalian hepatocyte culture, hypoxia directly increases hepatocyte IGFBP-1 mRNA levels (Popovici et al 2001, Scharf et al 2005. Further, the zebrafish IGFBP-1 promoter contains a functional hypoxia-response element, indicating that cellular pathways for direct hypoxia induction of IGFBP-1 exist in fishes (Kajimura et al 2006). A 21 kDA IGFBP increased during gradual seawater adaptation in rainbow trout; however, cortisol increased only slightly and transiently (Shepherd et al 2005).…”

“…However, until recently, definitive identification of these proteins has been lacking due to the absence of molecular sequence data. Recent studies have shown that liver IGFBP-1 gene transcription is induced by hypoxia in adult gobies (Gillichthyes mirabilis) and zebrafish (Danio reiro) (Gracey et al 2001, Maures & Duan 2002, that the induction of IGFBP-1 partially underlies hypoxiainduced growth retardation in zebrafish embryos (Kajimura et al 2005) and that the zebrafish IGFBP-1 promoter contains a functional hypoxia-response element (Kajimura et al 2006). These findings suggest that both the growth-inhibitory role and the regulation of IGFBP-1 may be highly conserved among vertebrates.…”

HRE Finder: A Tool for Quarrying Hypoxia-Response Element in Genomic Sequences

“…The frequent occurrence of functional HREs involving in transcriptional response was observed in the 1kb upstream stream from the transcription start site while more than 70% hypoxia responsive genes were found without undetectable HIF-binding site in proximal promoters of a gene (Benita, 2006). Further, the frequently distributed HREs (5'-RCGTG-3') are functionally activated when its downstream contains an adjacent hypoxia ancillary sequence (HAS) in the space of 7-15 nucleotide (Kajimura et al, 2006;Liu et al, 1995). HRE and HAS are five nucleotide motifs and their consensus patterns and {5'-CA(G|C)(A|G)(T|G|C)-3'}] were reported in several hypoxia-inducible genes in mammals and IGFBP1 gene in fish (Kimura et al, 2001;Kajimura et al, 2006).…”