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First published online August 8, 2008
Journal of Experimental Biology 211, 2700-2706 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019141

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Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Dan Wang, Xue-Ping Zhong, Zhi-Xian Qiao and Jian-Fang Gui^*

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China

View larger version (38K): [in this window] [in a new window]   Fig. 1. Nucleotide sequence and deduced amino acid sequence of CaHO-1. The nucleotides (upper row) and deduced amino acids (lower row) are numbered on the right. A putative transmembrane segment sequence is shaded, and a poly(A) signal (in the 3' UTR) is underlined. The start codon (ATG) is boxed and the stop codon (TGA) is indicated by an asterisk. An unstable motif (ATTTA) is doubly underlined. Heme oxygenase signature is in bold and underlined.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Multiple alignment of CaHO-1 amino acid sequence with that of other HO-1 proteins from fish, human, mouse, rat and chick. Ca, Carassius auratus L., accession number FD483976; Dr, Danio rerio, BC061954; Tr, Takifugu rubripes, AF022814; Hs, Homo sapiens, AY460337; Mm, Mus musculus, BC010757; Rn, Rattus norvegicus, BC091164; Gg, Gallus gallus, NM_205344. Missing amino acids are denoted by dashes (–). Shading is used to highlight regions with different levels of sequence identity: identical amino acids in at least four sequences are in black, and similar amino acids in at least four sequences are in gray. The heme oxygenase domain is indicated by lines above the aligned sequences. The heme oxygenase signature (HO signature) is boxed.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Inductive transcription of CaHO-1 in vitro. Two groups of CAB cells were treated with 2.5% O2 (A) and 1% O2 (B) for 3, 6, 12, 24, 48 and 72h. Cells were harvested immediately and total RNA was isolated as outlined in Materials and methods. The levels of CaHO-1 mRNA were determined by real-time PCR. Error bars represent standard deviations obtained by measuring each sample three times from three independent experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Gene transcription of CaHO-1 in vivo. (A) Total RNA from different tissues (posterior kidney, head kidney, heart, intestine, spleen, gill, brain and liver) of goldfish under normoxia (20% O2) was analyzed by real-time PCR. Error bars represent standard deviations obtained by measuring each tissue from three fish. (B) Inductive transcription of CaHO-1 in posterior kidney and gill. Total RNA from posterior kidney and gill of goldfish subjected to normoxia (as control) and hypoxia for 24h was analyzed by real-time PCR. Error bars represent standard deviations obtained by measuring each tissue from three fish. The data shown have been normalized to β-actin gene transcription. (C) Inductive transcription of CaHO-1 in goldfish larvae. Total CaHO-1 transcripts of one normoxic (N) and one hypoxic (H) goldfish larvae of 1.5 cm length, from a total of three in each group, following exposure to hypoxia (1.5% O2) for 1.5h, were analyzed by RT-PCR. Lane M, DL2000 DNA marker lane, kb (Takara). The data shown have been normalized to β-actin gene transcription.

View larger version (9K): [in this window] [in a new window]   Fig. 5. Subcellular localization of CaHO-1 by confocal microscopy. (A,B) CAB cells transfected with HO-1-GFP showing cytoplasmic (A) and plasma membrane (B) localization. (C) CAB cells transfected with empty vector pEGFP-N3.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Suppression of hypoxia-induced cell death by over-expression of CaHO-1. (A) Confirmation of the stably transfected CAB cells by semi-quantitative RT-PCR using T7 primer and CaHO-1-specific primer HO1-R4. A band (0.67 kb) was only detected in CAB cells stably transfected with plasmid pcDNA3.1 carrying the full-length ORF of HO-1 gene; β-actin was employed as an internal control. Lane M, DL2000 DNA marker; lane 1, CAB/pcDNA3.1 cell; lane 2, CAB/pcDNA3.1-HO-1 cell. (B) Over-expression of HO-1 in stably transfected CAB cells under hypoxic treatment. Total RNA was isolated from CAB/pcDNA3.1 and CAB/pcDNA3.1-HO-1 cells after 4 days of hypoxia (1% O2). The transcription of CaHO-1 was then evaluated by semi-quantitative RT-PCR (lane 2). CAB/pcDNA3.1 cells were employed as a control (lane 1). (C) Morphological observation of hypoxia-induced cell death under phase contrast microscope. (D) Viability of CAB/pcDNA3.1 and CAB/pcDNA3.1-HO-1 cells under the hypoxia–reoxygenation treatment. Cell viability was examined by a modified MTT assay using a CCK-8 kit each day (see Materials and methods). The experiments were repeated at least three times and the symbols represent the mean values of triplicate wells, with standard deviations.

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