Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension.

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O(2) for 1 h. The greatest accumulation of HIF-1alpha protein occurred at 5% O(2) (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.

Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss).

Differential expression of two rainbow trout CYP1A genes was measured in vivo and in vitro in response to treatment with the model CYP1A inducers beta-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, only in vitro). Originally described by Berndtson and Chen (Arch. Biochem. Biophys. 310, 187-195, 1994) as CYP1A1 and CYP1A2, these genes were renamed CYP1A3 and CYP1A1, respectively, by the P450 nomenclature committee. A significant, differential, inducer-dependent induction of the two CYP1A mRNAs, as measured by RNase protection assay, was observed in vivo. CYP1A3 and CYP1A1 mRNA levels in liver were significantly induced 50- and 18-fold, respectively, following ip injection with BNF. Conversely, CYP1A3 and CYP1A1 mRNA levels were significantly induced 5- and 66-fold, respectively, following ip injection with 3-MC. Isosafrole had no significant effect on in vivo induction of CYP1A mRNA levels. In primary cultures of hepatocytes, BNF, 3-MC, ISF, as well as TCDD all significantly induced CYP1A3 and CYP1A1 mRNA levels compared to controls. The differential induction of the two CYP1A genes was not as evident in vitro as in vivo. In addition, reanalysis and sequence comparison of the these two trout CYP1A genes with the first trout CYP1A cDNA described by Heilmann et al. (DNA 7, 379-387, 1988) indicate that the Heilmann cDNA is a hybrid of the two trout genes. The 5' portion of the cDNA sequence (212 bp) was determined by sequencing of a genomic clone and is 100% identical to the trout CYP1A3 gene. The majority of the cDNA sequence (2377 bp), however, was sequenced from a partial cDNA clone and is 99.2% identical to trout CYP1A1. Although the nomenclature of these two trout CYP1A genes is undergoing revision, these results demonstrate a differential, inducer-dependent response to model mammalian CYP1A inducers.

Tissue-specific expression of zebrafish (Danio rerio) heat shock factor 1 mRNAs in response to heat stress.

All organisms respond to environmental, chemical and physiological stresses by enhanced synthesis of an evolutionarily conserved family of proteins known as heat shock proteins (HSPs) or stress proteins. Certain HSPs are also expressed constitutively during cell growth and development, and they function as molecular chaperones. The transcriptional regulation of hsp genes is mediated by the heat shock transcription factor (HSF). The stress response has been studied mostly in mammalian cell lines or organisms normally maintained under constant laboratory conditions. There is much less information on the regulation of the stress response of animals, such as fish, that have to tolerate large fluctuations in environmental and internal conditions. To characterize the regulation of the heat shock response in fish, we have cloned the first heat shock transcription factor from fish, zebrafish Danio rerio. Phylogenetic analysis confirms that the isolated zebrafish HSF belongs to the HSF1 family and is therefore designated zHSF1. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) shows the presence of two zHSF1 mRNA forms that are expressed in a tissue-specific fashion upon exposure to heat stress. Both forms are expressed in gonads under all conditions; in liver and to a lesser extent in the gills, the longer splice form of zHSF1 disappears upon heat shock. We present evidence for a unique tissue-specific regulation of HSF1 upon exposure to elevated temperature.

Effects of heat shock and hypoxia on protein synthesis in rainbow trout (Oncorhynchus mykiss) cells.

We examined the effects of heat stress (from 18 degreesC to 26 degreesC) and low oxygen tension (1% O2=1 kPa) on protein synthesis in primary cultures of hepatocytes, gill epithelial cells and fibroblast-like RTG-2 cells of rainbow trout Oncorhynchus mykiss. All these cell types displayed elevated levels of 67, 69 and 92 kDa proteins, whereas a 104 kDa protein was induced only in RTG-2 cells. Hypoxia induced a cell-type-specific response, increasing the synthesis of 36, 39 and 51 kDa proteins in the gill epithelial cells. The regulation of the heat-shock response in fish hepatocytes showed that an HSF1-like factor is involved in the transcriptional induction of the hsp70 gene. Consequently, there was a pronounced accumulation of hsp70 mRNA. Furthermore, the kinetics of activation of DNA binding and the increase in hsp70 gene expression showed a remarkable correlation, indicating that hsp70 expression is regulated at the transcriptional level in these trout cells.

Uptake of taurocholic acid and cholic acid in isolated hepatocytes from rainbow trout.

The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.