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Tissue distribution, developmental expression and up-regulation of p8 transcripts on stress in zebrafish
Fish & Shellfish Immunology 28 (2010) 549e554
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Tissue distribution, developmental expression and up-regulation of p8 transcripts on stress in zebrafish Yanling Sun, Zhenhui Liu*, Shicui Zhang Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2009 Received in revised form 10 December 2009 Accepted 12 December 2009 Available online 30 December 2009
The p8 is a transcription factor with a basic helix-loop-helix motif and a nuclear localization signal. A zebrafish p8 cDNA, which consists of 732 bp and encodes 75 amino acids, was identified in this paper. Sequence alignment showed that the bHLH region of p8 was well-conserved during the evolution. Phylogenetic analysis revealed that zebrafish p8 was close to its homologous protein in frog, together clustering to the clade of vertebrates. The zebrafish p8 mRNA expression levels varied much among the detected adult tissues, with the obvious higher expression in backbone and liver. During embryogenesis, the expression of zebrafish p8 mRNA was in higher levels in cleavage stage, decreased from blastula to segmentation stage, but sharply elevated at hatching stage. Quantitative real-time PCR assay suggested up-regulation expressions of zebrafish p8 on a wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value, implying an important role of p8 gene in response to stress. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Zebrafish p8 Starvation Temperature Osmotic pressure pH
1. Introduction P8 was first cloned from a rat acinar cDNA library in 1997 and found to encode a polypeptide of 80-amino acids in length [1]. The p8 expression was strongly activated in pancreatic acinar cells during the acute phase of pancreatitis in rat, as well as in human [2]. p8 encodes a transcription cofactor with a basic helix-loophelix (bHLH) domain and a canonical bipartite nuclear locating signal (NLS) motif [1e3], indicating its localization within nuclei. p8 shares the similar biochemical structure with the high mobility group (HMG) proteins [4], and is likely to be a DNA-binding protein [1,5,6], which interacts with DNA in a sequence-independent manner. It is also structurally similar to candidate of metastasis-1 (com1), a novel factor in human breast cancer [7]. It has been shown that the activation of p8 was not restricted to the pancreatic cells alone. p8 may run roles in regulation of multiple cellular functions, which are possiblely influenced by interaction with various partners. For example, p8 is both proapoptotic [8,9] and antiapoptotic [10,11]. Expression of p8 is up-regulated by stressors including lipopolysaccharide (LPS) and simply changing culture media [4,6,12]. It is also involved in the regulation of cellular * Corresponding author at: Department of Marine Biology, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China. Tel.: þ86 532 82032092. E-mail address: [email protected] (Z. Liu). 1050-4648/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.12.010
growth [1,2], cellular growth inhibition [8,13], tumor development and progression [7,14e16], animal embryo development [1,17] and cell defence [18]. P8 homologs have subsequently been cloned and/or characterized in vertebrates such as human [2], rat [1], mouse [5], frog [3], Atlantic salmon (GenBank accession number ACI66304), zebrafish (GenBank accession number BF717555), and invertebrates such as common urchin [19], shrimp [20] and fruit fly [21]. Recently, we have shown that up-regulation of amphioxus p8 transcripts on LPS and starvation challenge [12], suggesting a stress-related function. We are further interested that whether the p8 response to more wide range of cellular stressors such as temperature, osmotic pressure and pH value. Zebrafish is fast emerging as an excellent model organism widely used to study various biological processes, therefore zebrafish was selected to investigate the role of p8 response to stress in our study. The study of p8 gene in zebrafish is only restricted to the direct submission of the cDNA sequence to GenBank database, however the characterization and function of p8 in this animal remains untouched hitherto. The objective of our research is therefore to analyze its primary structure, tissue distribution, expression in embryonic developmental stages and response to stress including starvation, temperature, osmotic pressure and pH value challenge, which will provide an important foundation to further understand the biological function of p8 in zebrafish.
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2. Materials and methods 2.1. Animal Adult zebrafish (body weight, each 0.33 0.03 g) were obtained from local markets and maintained in containers under laboratory conditions with well-aerated tap water at 27 1 C. 2.2. Searching of p8 gene in zebrafish Using a p8 cDNA of Salmo salar (GenBank accession number ACI66304) as an information probe, homologous sequences of p8 were searched by tBlastx program in NCBI (http://www.ncbi.nlm. nih.gov). 2.3. Sequence comparison and phylogenetic analysis p8 protein sequences were aligned with the MegAlign program by the CLUSTAL method in DNASTAR [22]. The maximum parsimony and neighbor-joining methods in the Philip 3.5c software package [23] were used to construct phylogenetic trees based on p8 protein sequences. Statistical significance of groups within inferred trees was evaluated using the 1000 bootstrap replicates. 2.4. Sample collection 2.4.1. Zebrafish tissues Various adult zebrafish tissues including gill, brain, intestine, backbone, liver and muscle were dissected from fresh fish. For backbone, for example, we firstly dissect the fish from the anus to chin tip with sterile scissor, then lop the head, excise the closelybinding muscle quickly and separated the backbone lightly by using sterile forceps. They were grinded in RNAiso plus (TaKaRa, Japan), respectively, and stored at 80 C until RNA extraction. 2.4.2. Zebrafish embryos Zebrafish were cultured in 14 h light and 10 h darkness for one week, then the female and male zebrafish in 2:1 were choosed to produce embryos. Embryos were collected and cultured in temperature humidity incubator (CIMO, HPX-300BSH-III, Shanghai, China). 2.4.3. Zebrafish with starvation, temperature, osmotic pressure and pH value challenge Before beginning of the experiment, the fish (body weight, each 0.33 0.03 g) were allowed to acclimatize to container conditions for 7 days. During the period, the fish were fed twice daily at 8:00 am and 6:00 pm with a commercial diet (crude protein > 49.0%, crude fat > 5.0%, crude fibre < 3.0%, crude ash < 12.0%, moisture < 10.0%) until satiation. For starvation challenge, 24 zebrafish were divided into two groups. Fish of the experimental group were not fed (starvation) for 2 days, and fish of the control group were fed normally during the course of the experiment. For each group, fish backbone were immediately sampled in RNAiso plus from 4 fish at 0.5, 1 and 2 days, respectively, stored at 80 C until RNA extraction. For temperature challenge, 12 zebrafish were divided into three groups, and cultured under the temperature of 18, 27 (natural condition, as control) and 34 C for 12 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group. For osmotic pressure challenge, 12 zebrafish were divided into three groups. They were cultured in water which additional NaCl was added to each group at a concentration of 0 (as control), 0.25 and 0.5 g$per liter water for 12 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group.
For pH value challenge, 12 zebrafish were divided into three groups, and treated at pH value of 6.38, 8.27 (natural condition in cultured container, as control) and 9.97 for 6 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group. 2.5. RNA extraction Zebrafish total RNAs were extracted with RNAiso plus and digested with RQ1 RNase-free DNase (Promega) to eliminate the genomic contamination. 2.6. Quantitative real-time PCR cDNAs were synthesized using Random primer (Promega) with a reverse transcription system (25 mM Random primer 1 ml, 10 ng/ml total RNAs 6 ml, 10 mM dNTPs 0.5 ml, 5 RT Buffer 2 ml, 50 u/ml ribonuclease inhibitor 0.25 ml, EasyScript RT (Rnase H-M-MLV) 0.25 ml). Reaction conditions consisted of 30 C for 10 min, then 42 C for 50 min, and followed by 70 C for 10 min. The cDNAs were used as the template of quantitative real-time PCR. Two PCR primer sets specific for p8 and ubiquitin (GenBank accession number Ub52 NM-001037113) were used to amplify products of 183 bp and 131 bp, respectively [24]. p8 sense primer was: 50 -cgcaaaggga ggacgaaga-30 , anti-sense primer was: 50 -ccgtttgccgcagagacac-30 , ubiquitin sense primer was: 50 -gagccttctctccgtcagttag-30 , and antisense primer was: 50 -cgcaggttgttggtgtgtc-30 . Real-time PCR was performed on ABI 7500 real-time PCR system. SYBR_ Premix Ex TaqÔ (Takara) was used for real-time PCR, with a primer concentration of 200 nM. Reaction conditions consisted of 95 C for 10 s, followed by 40 cycles of 95 C for 5 s, 60 C for 20 s, and 72 C for 34 s. Reaction of each sample was performed in triplicate. Ubiquitin gene was used as control to normalize the starting quantity of RNAs. Dissociation analysis of amplified products was performed at the end of each PCR to confirm that only one PCR product was amplified. After the PCR program, data were analyzed with ABI 7500 SDS software (Applied Biosystems), and quantified using the comparative CT method (2DDCT method) based on CT values for both p8 and zebrafish ubiquitin in order to calculate the fold increase [25]. All data are given in terms of relative mRNA expressed as means SD. The data obtained from real-time PCR analysis were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's two-sided test to determine differences in the mean values among the treatments, and significance was concluded at P < 0.05 or P < 0.01. 3. Results 3.1. Identification of p8 gene in zebrafish Using a p8 cDNA of S. salar (GenBank accession number ACI66304) as an information probe, homologous sequences of p8 were searched by tBlastx program in NCBI (http://www.ncbi.nlm. nih.gov). The cDNA of zebrafish (GenBank accession number BC122137) was found to have similarity to S. salar p8 with E value of 9e10. The full-length zebrafish p8 cDNA consists of 732 bp and encodes 75 amino acids with a molecular weight of 8.961 kDa and isolectric point of 8.433. There was a start codon (ATG) at the 50 end of the cDNA, and a stop codon (TGA) at its 30 end. The clone also contained a 125 bp 50 untranslated region (UTR) and a long 30 UTR with a typical polyadenylation signal (AATAAA) upstream the polyadenine stretch. These suggest that the cDNA contained the full coding sequence. BLASTP searching in GenBank revealed that the protein contained the conserved domain of DNA-binding nuclear
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phosphoprotein p8, and it showed an affinity to the p8 homologs. Thus, the p8 cDNA was identified in zebrafish. Using the PSORT II program (http://psort.nibb.ac.jp), zebrafish p8 was localized in the nucleus with NLS. The subcellular localization of the protein had been confirmed in human using specific antibodies and transient transfection of expression plasmids [2].
3.2. Sequence comparision and phylogenetic analysis We performed multiple sequences alignment based on representative p8 protein sequences from different species, and discovered that the bHLH domain was well-conserved during evolution (Fig. 1a). It was noted that four amino acids such as aspartic acid (D), glutamic acid (E), arginine (R) and lysine (K) were present in all the detected species here (Fig. 1a). This is in line with the generally accepted notion that the D, E, R and K occur more frequently in DNA-binding proteins [26e30], which the negatively charged residues D and E have the most negative propensities, whilst the positively charged R residue has the highest positive propensity .
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To evaluate the evolutionary relationship of zebrafish p8 with its homologs in other species, we constructed a phylogenetic tree with maximum parsimony method using the Philip 3.5c software (Fig. 1b). It was found that zebrafish p8 was close to its homologous protein in frog, together clustering to the clade of vertebrates. Another phylogenetic tree constructed by the neighbor-joining method also supported the data (not shown). These results revealed that the amino acid sequences of zebrafish p8 had a closer similarity to its vertebrate counterparts than invertebrate ones, which is in agreement with the concept of traditional taxonomy.
3.3. Tissue distribution of p8 mRNA The expressions of p8 mRNA at different zebrafish tissues including gill, brain, intestine, backbone, liver and muscle were detected by real-time PCR (Fig. 2). Analysis of the dissociation curve of amplified products in all cases showed only a single peak, indicating that the amplifications were specific (data not shown). The p8 mRNA had a wide distribution in the tested tissues, with the
Fig. 1. p8 sequence comparison and phylogenetic analysis. (a) Alignment of p8 sequences including zebrafish p8 using the MegAlign program (DNASTAR) by the CLUSTALW method. Shaded (with solid black) residues are the amino acids that match the consensus. Gaps introduced into sequences to optimize alignment are represented by (). The conserved bHLH and NLS regions were marked by grey bold line and black thin line, respectively. Four conserved amino acid residues D, E, R and K were marked with asterisk. (b) The phylogenetic tree of zebrafish p8 and other representative p8 proteins constructed by the maximum parsimony method within the package Philip 3.5c (programs SEQBOOT, PROTPARS and CONSENSE) using 1000 bootstrap replicates. The numbers near the branches are bootstrap permillages supporting the given branching pattern, and fly p8 was used as the outgroup. The accession numbers of the analyzed sequences from GenBank are: Fruit fly, Drosophila melanogaster, NP_609539; Common urchin, Echinometra mathaei, FJ423772; Amphioxus, Branchiostoma belcheri tsingtauense, AAQ18145; Atlantic salmon, S. salar, ACI66304; Atlantic hailbut, Hippoglossus hippoglossus, ACJ36448; Shrimp, Artemia franciscana, DQ361275; Sea urchin, Strongylocentrotus purpuratus, XP_001177744; Dog, Canis lupus familiaris XP_536921; African clawed frog, Xenopus laevis AB056582; House mouse, Mus musculus NM_019738; Human, Homo sapiens, O60356.
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Fig. 2. Expression profile of p8 at different tissues as measured by quantitative realtime PCR. The mRNA expressions of p8 and ubiquitin were measured in tissues including gill, brain, intestine, backbone, liver and muscle. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3).
strongest expression in backbone, intermediate in liver, low in brain, gill, intestine and muscle. 3.4. Developmental expression of p8 mRNA The expressions of zebrafish p8 mRNA at different developmental stages were analyzed by real-time PCR (Fig. 3). The expression of p8 mRNA varied much from cleavage stage to hatching stage. p8 mRNA was expressed at high levels in cleavage stage, reduced from blastula to segmentation stage up to the lowest point (1.5 fold relative to the cleavage stage), but elevated sharply at hatching stage, even at a higher levels than the cleavage stage. 3.5. Response of zebrafish p8 to stress challenge 3.5.1. Response of p8 to starvation challenge Real-time PCR showed that fasting for 2 days caused a significant up-regulation (about 3 fold) of p8 mRNA expression relative to fasting 12 h in zebrafish backbone (Fig. 4), while no statistically significant difference in p8 mRNA levels in the control group. 3.5.2. Response of p8 to temperature challenge After treated zebrafish for 12 h at the temperature of 18 C, 27 C and 34 C, respectively, the p8 mRNA levels were detected by
Fig. 3. Expression profile of p8 at different stages of embryonic development as measured by quantitative real-time PCR. The mRNA expressions of p8 and ubiquitin were detected at cleavage stage, blastula stage, gastrula stage, segmentation stage and hatching stage. Fold difference was calculated as 2_DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3).
Fig. 4. Starvation-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 0.5, 1 or 2 days of fasting treatment was performed. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.01) across control are indicated by asterisks (**).
real-time PCR (Fig. 5). Both lower and higher temperature could induce a strong enhancement of p8 mRNA expression (about 7 fold relative to control). 3.5.3. Response of p8 to osmotic pressure challenge The adult zebrafish were cultured in water with different osmotic pressure (adding 0 g, 0.25 g and 0.5 g of NaCl per liter water) for 12 h. The expressions of p8 mRNA were assayed by realtime PCR. With theosmotic pressureincreasing, the p8 expressions were up-regulated, reaching the peak (2.5 fold relative to control) in the treatment of adding 0.5 g/l of NaCl (Fig. 6). 3.5.4. Response of p8 to pH value challenge After zebrafish were cultured in water with different pH value (pH 6.38, pH 8.27 and pH 9.97) for 6 h, real-time PCR was performed to investigate the response of p8 to pH value challenge. Lower and higher pH value than the normal one could both induce the p8 expressions up-regulate significantly (Fig. 7). 4. Discussions We identified a zebrafish p8 homolog in this paper. The zebrafish p8 shares the similar structure domain to other p8 proteins with a bHLH motif and an NCL signal. Our previous study had shown that the genomic structure of zebrafish p8 was also similar to its vertebrate homologs with three exons and two introns (12). To further characterize the p8 gene in zebrafish, the tissue distribution and developmental expression of p8 in zebrafish were
Fig. 5. Temperature-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 12 h of 18, 27 (as control), or 34 C treatment was performed. Fold difference was calculated as 2_DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
Y. Sun et al. / Fish & Shellfish Immunology 28 (2010) 549e554
Fig. 6. Osmotic pressure-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 12 h of different osmotic pressure treatment was performed. Additional 0 g (as control), 0.25 g, or 0.5 g of NaCl was added to per liter water to generate different osmotic pressurein this study. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
detected by real-time PCR. We found that p8 mRNAs were expressed widely in all the detected tissues, supporting the idea that p8 mRNA may participate in diverse functions in different cells [1e3]. Noteworthily, p8 had the higher expression in backbone and liver in zebrafish. In mammalian such as rat and human, the high level of p8 in liver was also detected, while it was low or undetectable in backbone [1,2]. In addition, we detected the p8 mRNA expression levels in different development stages, which were also varied much. In cleavage stage, p8 was in higher levels of expression, but suddenly significantly fell in blastula, gastrula and segmentation stages (Fig. 3), implying the materal p8 transcripts presence in these stages. In contrast, p8 mRNA expressions came back to high levels at hatching stage, even higher than p8 levels in cleavage stage, suggesting the expression of zygote p8 mRNA had started before this stage. Collectively, we speculated that p8 may involve actively in the progress of embryogensis, as previous reported in common urchin and mice [1,17,19,31e33]. It had been shown that p8 genes were expressed in cells in response to cellular stressors, which may range from a simple change of culture media to LPS challenge [4,6]. In amphioxus, we had also shown the up-regulation of p8 transcripts on LPS and starvation challenge [12], implicating a stress-related function for p8. In this paper, we showed that p8 mRNA expression was significantly higher
Fig. 7. Expression profile of p8 in backbone after challenged with different pH value. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 6 h of different pH value treatment was performed. No significant differences in the expressions of p8 among the different pH value (6.38, 8.27 and 9.97) treatment. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
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in backbone after fasted 2 days, further suggesting a potent role for p8 in the response to starvation. Accordingly, we predicted that p8 mRNA expression would be reduced in some degree if refed [34]. Fasting causes an emergency to starvation, which orexigenic signals play an important role in mediating the drive to eat [35]. The fasting induced increase in p8 mRNA expression hints that a possible orexigenic role for p8 in zebrafish [4,36]. In our present study, we have also observed that p8 mRNA expressions were up-regulated in the challenged state by other stressors such as temperature, osmotic pressure and pH value. Here we provided evidences that p8 could response to more wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value. We could assume that p8 mRNA up-regulated expression may be part of ubiquitous defence program in a way that helps the tissue countract cellular stress or cellular injury, as described in rat [2]. However, it is a long way to clarify the mechanism of p8 gene expression response to stress. 5. Conclusions In summary, a zebrafish p8 cDNA with the conserved bHLH domain and NLS motif was identified in our paper. The zebrafish p8 mRNA expression levels varied much among the detected adult tissues, with the obvious higher expression in backbone and liver. During embryogenesis, the expression of zebrafish p8 mRNA was in higher levels in cleavage stage, decreased from blastula to segmentation stage, but sharply elevated at hatching stage. We provided evidences that p8 could response to more wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value, further implying an important role of p8 gene in regulation to stress. Acknowledgements This work was supported by Program for New Century Excellent Talents in University to Zhenhui Liu (No. NCET-08-0501), the Ministry of Science and Technology (MOST) of China (No. 2008AA092603 and 2008AA09Z409) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 2009-1001). References [1] Mallo GV, Fiedler F, Calvo EL, Ortiz EM, Vasseur S, Keim V, et al. Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth. J Biol Chem 1997;272:32360e9. [2] Vasseur S, Mallo GV, Fiedler F, Bödeker H, Canepa E, Moreno S, et al. Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur J Biochem 1999;259:670e5. [3] Igarashi T, Kuroda H, Takahashi S, Asashima M. Cloning and characterization of the Xenopus laevis p8 gene. Dev Growth Differ 2001;43:693e8. [4] Hoffmeister A, Ropolo A, Vasseur S, Mallo GV, Bodeker H, Ritz-Laser B, et al. The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter. J Biol Chem 2002;277:22314e9. [5] Vasseur S, Mallo GV, Garcia-Montero A, Ortiz EM, Fiedler F, Canepa E, et al. Structural and functional characterization of the mouse p8 gene: romotion of transcription by the CAAT-enhancer binding protein (C/EBP) and C/EBPb transacting factors involves a C/EBP cis-acting element and other regions of the promoter. Biochem J 1999;343:377e83. [6] Garcia-Montero A, Vasseur S, Mallo GV, Soubeyran P, Dagorn JC, Iovanna JL. Expression of the stress-induced p8 mRNA is transiently activated after culture medium change. Eur J Cell Biol 2001;80:720e5. [7] Ree AH, Tvermyr M, Engebraaten O, Rooman M, Røsok O, Hovig E, et al. Expression of a novel factor in human breast cancer cells with metastatic potential. Cancer Res 1999;59:4675e80. [8] Vasseur S, Hoffmeister A, Garcia-Montero A, Mallo GV, Feil R, Kühbandner S, et al. p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis. Oncogene 2002;21:1685e94.
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Tissue distribution, developmental expression and up-regulation of p8 transcripts on stress in zebrafish Yanling Sun, Zhenhui Liu*, Shicui Zhang Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2009 Received in revised form 10 December 2009 Accepted 12 December 2009 Available online 30 December 2009
The p8 is a transcription factor with a basic helix-loop-helix motif and a nuclear localization signal. A zebrafish p8 cDNA, which consists of 732 bp and encodes 75 amino acids, was identified in this paper. Sequence alignment showed that the bHLH region of p8 was well-conserved during the evolution. Phylogenetic analysis revealed that zebrafish p8 was close to its homologous protein in frog, together clustering to the clade of vertebrates. The zebrafish p8 mRNA expression levels varied much among the detected adult tissues, with the obvious higher expression in backbone and liver. During embryogenesis, the expression of zebrafish p8 mRNA was in higher levels in cleavage stage, decreased from blastula to segmentation stage, but sharply elevated at hatching stage. Quantitative real-time PCR assay suggested up-regulation expressions of zebrafish p8 on a wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value, implying an important role of p8 gene in response to stress. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Zebrafish p8 Starvation Temperature Osmotic pressure pH
1. Introduction P8 was first cloned from a rat acinar cDNA library in 1997 and found to encode a polypeptide of 80-amino acids in length [1]. The p8 expression was strongly activated in pancreatic acinar cells during the acute phase of pancreatitis in rat, as well as in human [2]. p8 encodes a transcription cofactor with a basic helix-loophelix (bHLH) domain and a canonical bipartite nuclear locating signal (NLS) motif [1e3], indicating its localization within nuclei. p8 shares the similar biochemical structure with the high mobility group (HMG) proteins [4], and is likely to be a DNA-binding protein [1,5,6], which interacts with DNA in a sequence-independent manner. It is also structurally similar to candidate of metastasis-1 (com1), a novel factor in human breast cancer [7]. It has been shown that the activation of p8 was not restricted to the pancreatic cells alone. p8 may run roles in regulation of multiple cellular functions, which are possiblely influenced by interaction with various partners. For example, p8 is both proapoptotic [8,9] and antiapoptotic [10,11]. Expression of p8 is up-regulated by stressors including lipopolysaccharide (LPS) and simply changing culture media [4,6,12]. It is also involved in the regulation of cellular * Corresponding author at: Department of Marine Biology, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China. Tel.: þ86 532 82032092. E-mail address: [email protected] (Z. Liu). 1050-4648/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.12.010
growth [1,2], cellular growth inhibition [8,13], tumor development and progression [7,14e16], animal embryo development [1,17] and cell defence [18]. P8 homologs have subsequently been cloned and/or characterized in vertebrates such as human [2], rat [1], mouse [5], frog [3], Atlantic salmon (GenBank accession number ACI66304), zebrafish (GenBank accession number BF717555), and invertebrates such as common urchin [19], shrimp [20] and fruit fly [21]. Recently, we have shown that up-regulation of amphioxus p8 transcripts on LPS and starvation challenge [12], suggesting a stress-related function. We are further interested that whether the p8 response to more wide range of cellular stressors such as temperature, osmotic pressure and pH value. Zebrafish is fast emerging as an excellent model organism widely used to study various biological processes, therefore zebrafish was selected to investigate the role of p8 response to stress in our study. The study of p8 gene in zebrafish is only restricted to the direct submission of the cDNA sequence to GenBank database, however the characterization and function of p8 in this animal remains untouched hitherto. The objective of our research is therefore to analyze its primary structure, tissue distribution, expression in embryonic developmental stages and response to stress including starvation, temperature, osmotic pressure and pH value challenge, which will provide an important foundation to further understand the biological function of p8 in zebrafish.
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2. Materials and methods 2.1. Animal Adult zebrafish (body weight, each 0.33 0.03 g) were obtained from local markets and maintained in containers under laboratory conditions with well-aerated tap water at 27 1 C. 2.2. Searching of p8 gene in zebrafish Using a p8 cDNA of Salmo salar (GenBank accession number ACI66304) as an information probe, homologous sequences of p8 were searched by tBlastx program in NCBI (http://www.ncbi.nlm. nih.gov). 2.3. Sequence comparison and phylogenetic analysis p8 protein sequences were aligned with the MegAlign program by the CLUSTAL method in DNASTAR [22]. The maximum parsimony and neighbor-joining methods in the Philip 3.5c software package [23] were used to construct phylogenetic trees based on p8 protein sequences. Statistical significance of groups within inferred trees was evaluated using the 1000 bootstrap replicates. 2.4. Sample collection 2.4.1. Zebrafish tissues Various adult zebrafish tissues including gill, brain, intestine, backbone, liver and muscle were dissected from fresh fish. For backbone, for example, we firstly dissect the fish from the anus to chin tip with sterile scissor, then lop the head, excise the closelybinding muscle quickly and separated the backbone lightly by using sterile forceps. They were grinded in RNAiso plus (TaKaRa, Japan), respectively, and stored at 80 C until RNA extraction. 2.4.2. Zebrafish embryos Zebrafish were cultured in 14 h light and 10 h darkness for one week, then the female and male zebrafish in 2:1 were choosed to produce embryos. Embryos were collected and cultured in temperature humidity incubator (CIMO, HPX-300BSH-III, Shanghai, China). 2.4.3. Zebrafish with starvation, temperature, osmotic pressure and pH value challenge Before beginning of the experiment, the fish (body weight, each 0.33 0.03 g) were allowed to acclimatize to container conditions for 7 days. During the period, the fish were fed twice daily at 8:00 am and 6:00 pm with a commercial diet (crude protein > 49.0%, crude fat > 5.0%, crude fibre < 3.0%, crude ash < 12.0%, moisture < 10.0%) until satiation. For starvation challenge, 24 zebrafish were divided into two groups. Fish of the experimental group were not fed (starvation) for 2 days, and fish of the control group were fed normally during the course of the experiment. For each group, fish backbone were immediately sampled in RNAiso plus from 4 fish at 0.5, 1 and 2 days, respectively, stored at 80 C until RNA extraction. For temperature challenge, 12 zebrafish were divided into three groups, and cultured under the temperature of 18, 27 (natural condition, as control) and 34 C for 12 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group. For osmotic pressure challenge, 12 zebrafish were divided into three groups. They were cultured in water which additional NaCl was added to each group at a concentration of 0 (as control), 0.25 and 0.5 g$per liter water for 12 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group.
For pH value challenge, 12 zebrafish were divided into three groups, and treated at pH value of 6.38, 8.27 (natural condition in cultured container, as control) and 9.97 for 6 h, respectively. The zebrafish backbone samples were obtained from 4 fish in each group. 2.5. RNA extraction Zebrafish total RNAs were extracted with RNAiso plus and digested with RQ1 RNase-free DNase (Promega) to eliminate the genomic contamination. 2.6. Quantitative real-time PCR cDNAs were synthesized using Random primer (Promega) with a reverse transcription system (25 mM Random primer 1 ml, 10 ng/ml total RNAs 6 ml, 10 mM dNTPs 0.5 ml, 5 RT Buffer 2 ml, 50 u/ml ribonuclease inhibitor 0.25 ml, EasyScript RT (Rnase H-M-MLV) 0.25 ml). Reaction conditions consisted of 30 C for 10 min, then 42 C for 50 min, and followed by 70 C for 10 min. The cDNAs were used as the template of quantitative real-time PCR. Two PCR primer sets specific for p8 and ubiquitin (GenBank accession number Ub52 NM-001037113) were used to amplify products of 183 bp and 131 bp, respectively [24]. p8 sense primer was: 50 -cgcaaaggga ggacgaaga-30 , anti-sense primer was: 50 -ccgtttgccgcagagacac-30 , ubiquitin sense primer was: 50 -gagccttctctccgtcagttag-30 , and antisense primer was: 50 -cgcaggttgttggtgtgtc-30 . Real-time PCR was performed on ABI 7500 real-time PCR system. SYBR_ Premix Ex TaqÔ (Takara) was used for real-time PCR, with a primer concentration of 200 nM. Reaction conditions consisted of 95 C for 10 s, followed by 40 cycles of 95 C for 5 s, 60 C for 20 s, and 72 C for 34 s. Reaction of each sample was performed in triplicate. Ubiquitin gene was used as control to normalize the starting quantity of RNAs. Dissociation analysis of amplified products was performed at the end of each PCR to confirm that only one PCR product was amplified. After the PCR program, data were analyzed with ABI 7500 SDS software (Applied Biosystems), and quantified using the comparative CT method (2DDCT method) based on CT values for both p8 and zebrafish ubiquitin in order to calculate the fold increase [25]. All data are given in terms of relative mRNA expressed as means SD. The data obtained from real-time PCR analysis were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's two-sided test to determine differences in the mean values among the treatments, and significance was concluded at P < 0.05 or P < 0.01. 3. Results 3.1. Identification of p8 gene in zebrafish Using a p8 cDNA of S. salar (GenBank accession number ACI66304) as an information probe, homologous sequences of p8 were searched by tBlastx program in NCBI (http://www.ncbi.nlm. nih.gov). The cDNA of zebrafish (GenBank accession number BC122137) was found to have similarity to S. salar p8 with E value of 9e10. The full-length zebrafish p8 cDNA consists of 732 bp and encodes 75 amino acids with a molecular weight of 8.961 kDa and isolectric point of 8.433. There was a start codon (ATG) at the 50 end of the cDNA, and a stop codon (TGA) at its 30 end. The clone also contained a 125 bp 50 untranslated region (UTR) and a long 30 UTR with a typical polyadenylation signal (AATAAA) upstream the polyadenine stretch. These suggest that the cDNA contained the full coding sequence. BLASTP searching in GenBank revealed that the protein contained the conserved domain of DNA-binding nuclear
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phosphoprotein p8, and it showed an affinity to the p8 homologs. Thus, the p8 cDNA was identified in zebrafish. Using the PSORT II program (http://psort.nibb.ac.jp), zebrafish p8 was localized in the nucleus with NLS. The subcellular localization of the protein had been confirmed in human using specific antibodies and transient transfection of expression plasmids [2].
3.2. Sequence comparision and phylogenetic analysis We performed multiple sequences alignment based on representative p8 protein sequences from different species, and discovered that the bHLH domain was well-conserved during evolution (Fig. 1a). It was noted that four amino acids such as aspartic acid (D), glutamic acid (E), arginine (R) and lysine (K) were present in all the detected species here (Fig. 1a). This is in line with the generally accepted notion that the D, E, R and K occur more frequently in DNA-binding proteins [26e30], which the negatively charged residues D and E have the most negative propensities, whilst the positively charged R residue has the highest positive propensity .
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To evaluate the evolutionary relationship of zebrafish p8 with its homologs in other species, we constructed a phylogenetic tree with maximum parsimony method using the Philip 3.5c software (Fig. 1b). It was found that zebrafish p8 was close to its homologous protein in frog, together clustering to the clade of vertebrates. Another phylogenetic tree constructed by the neighbor-joining method also supported the data (not shown). These results revealed that the amino acid sequences of zebrafish p8 had a closer similarity to its vertebrate counterparts than invertebrate ones, which is in agreement with the concept of traditional taxonomy.
3.3. Tissue distribution of p8 mRNA The expressions of p8 mRNA at different zebrafish tissues including gill, brain, intestine, backbone, liver and muscle were detected by real-time PCR (Fig. 2). Analysis of the dissociation curve of amplified products in all cases showed only a single peak, indicating that the amplifications were specific (data not shown). The p8 mRNA had a wide distribution in the tested tissues, with the
Fig. 1. p8 sequence comparison and phylogenetic analysis. (a) Alignment of p8 sequences including zebrafish p8 using the MegAlign program (DNASTAR) by the CLUSTALW method. Shaded (with solid black) residues are the amino acids that match the consensus. Gaps introduced into sequences to optimize alignment are represented by (). The conserved bHLH and NLS regions were marked by grey bold line and black thin line, respectively. Four conserved amino acid residues D, E, R and K were marked with asterisk. (b) The phylogenetic tree of zebrafish p8 and other representative p8 proteins constructed by the maximum parsimony method within the package Philip 3.5c (programs SEQBOOT, PROTPARS and CONSENSE) using 1000 bootstrap replicates. The numbers near the branches are bootstrap permillages supporting the given branching pattern, and fly p8 was used as the outgroup. The accession numbers of the analyzed sequences from GenBank are: Fruit fly, Drosophila melanogaster, NP_609539; Common urchin, Echinometra mathaei, FJ423772; Amphioxus, Branchiostoma belcheri tsingtauense, AAQ18145; Atlantic salmon, S. salar, ACI66304; Atlantic hailbut, Hippoglossus hippoglossus, ACJ36448; Shrimp, Artemia franciscana, DQ361275; Sea urchin, Strongylocentrotus purpuratus, XP_001177744; Dog, Canis lupus familiaris XP_536921; African clawed frog, Xenopus laevis AB056582; House mouse, Mus musculus NM_019738; Human, Homo sapiens, O60356.
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Fig. 2. Expression profile of p8 at different tissues as measured by quantitative realtime PCR. The mRNA expressions of p8 and ubiquitin were measured in tissues including gill, brain, intestine, backbone, liver and muscle. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3).
strongest expression in backbone, intermediate in liver, low in brain, gill, intestine and muscle. 3.4. Developmental expression of p8 mRNA The expressions of zebrafish p8 mRNA at different developmental stages were analyzed by real-time PCR (Fig. 3). The expression of p8 mRNA varied much from cleavage stage to hatching stage. p8 mRNA was expressed at high levels in cleavage stage, reduced from blastula to segmentation stage up to the lowest point (1.5 fold relative to the cleavage stage), but elevated sharply at hatching stage, even at a higher levels than the cleavage stage. 3.5. Response of zebrafish p8 to stress challenge 3.5.1. Response of p8 to starvation challenge Real-time PCR showed that fasting for 2 days caused a significant up-regulation (about 3 fold) of p8 mRNA expression relative to fasting 12 h in zebrafish backbone (Fig. 4), while no statistically significant difference in p8 mRNA levels in the control group. 3.5.2. Response of p8 to temperature challenge After treated zebrafish for 12 h at the temperature of 18 C, 27 C and 34 C, respectively, the p8 mRNA levels were detected by
Fig. 3. Expression profile of p8 at different stages of embryonic development as measured by quantitative real-time PCR. The mRNA expressions of p8 and ubiquitin were detected at cleavage stage, blastula stage, gastrula stage, segmentation stage and hatching stage. Fold difference was calculated as 2_DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3).
Fig. 4. Starvation-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 0.5, 1 or 2 days of fasting treatment was performed. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.01) across control are indicated by asterisks (**).
real-time PCR (Fig. 5). Both lower and higher temperature could induce a strong enhancement of p8 mRNA expression (about 7 fold relative to control). 3.5.3. Response of p8 to osmotic pressure challenge The adult zebrafish were cultured in water with different osmotic pressure (adding 0 g, 0.25 g and 0.5 g of NaCl per liter water) for 12 h. The expressions of p8 mRNA were assayed by realtime PCR. With theosmotic pressureincreasing, the p8 expressions were up-regulated, reaching the peak (2.5 fold relative to control) in the treatment of adding 0.5 g/l of NaCl (Fig. 6). 3.5.4. Response of p8 to pH value challenge After zebrafish were cultured in water with different pH value (pH 6.38, pH 8.27 and pH 9.97) for 6 h, real-time PCR was performed to investigate the response of p8 to pH value challenge. Lower and higher pH value than the normal one could both induce the p8 expressions up-regulate significantly (Fig. 7). 4. Discussions We identified a zebrafish p8 homolog in this paper. The zebrafish p8 shares the similar structure domain to other p8 proteins with a bHLH motif and an NCL signal. Our previous study had shown that the genomic structure of zebrafish p8 was also similar to its vertebrate homologs with three exons and two introns (12). To further characterize the p8 gene in zebrafish, the tissue distribution and developmental expression of p8 in zebrafish were
Fig. 5. Temperature-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 12 h of 18, 27 (as control), or 34 C treatment was performed. Fold difference was calculated as 2_DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
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Fig. 6. Osmotic pressure-induced p8 up-regulation in backbone. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 12 h of different osmotic pressure treatment was performed. Additional 0 g (as control), 0.25 g, or 0.5 g of NaCl was added to per liter water to generate different osmotic pressurein this study. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
detected by real-time PCR. We found that p8 mRNAs were expressed widely in all the detected tissues, supporting the idea that p8 mRNA may participate in diverse functions in different cells [1e3]. Noteworthily, p8 had the higher expression in backbone and liver in zebrafish. In mammalian such as rat and human, the high level of p8 in liver was also detected, while it was low or undetectable in backbone [1,2]. In addition, we detected the p8 mRNA expression levels in different development stages, which were also varied much. In cleavage stage, p8 was in higher levels of expression, but suddenly significantly fell in blastula, gastrula and segmentation stages (Fig. 3), implying the materal p8 transcripts presence in these stages. In contrast, p8 mRNA expressions came back to high levels at hatching stage, even higher than p8 levels in cleavage stage, suggesting the expression of zygote p8 mRNA had started before this stage. Collectively, we speculated that p8 may involve actively in the progress of embryogensis, as previous reported in common urchin and mice [1,17,19,31e33]. It had been shown that p8 genes were expressed in cells in response to cellular stressors, which may range from a simple change of culture media to LPS challenge [4,6]. In amphioxus, we had also shown the up-regulation of p8 transcripts on LPS and starvation challenge [12], implicating a stress-related function for p8. In this paper, we showed that p8 mRNA expression was significantly higher
Fig. 7. Expression profile of p8 in backbone after challenged with different pH value. Quantitative real-time PCR analysis of p8 and ubiquitin mRNA levels in backbone following 6 h of different pH value treatment was performed. No significant differences in the expressions of p8 among the different pH value (6.38, 8.27 and 9.97) treatment. Fold difference was calculated as 2DDCt with zebrafish ubiquitin as a reference gene. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
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in backbone after fasted 2 days, further suggesting a potent role for p8 in the response to starvation. Accordingly, we predicted that p8 mRNA expression would be reduced in some degree if refed [34]. Fasting causes an emergency to starvation, which orexigenic signals play an important role in mediating the drive to eat [35]. The fasting induced increase in p8 mRNA expression hints that a possible orexigenic role for p8 in zebrafish [4,36]. In our present study, we have also observed that p8 mRNA expressions were up-regulated in the challenged state by other stressors such as temperature, osmotic pressure and pH value. Here we provided evidences that p8 could response to more wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value. We could assume that p8 mRNA up-regulated expression may be part of ubiquitous defence program in a way that helps the tissue countract cellular stress or cellular injury, as described in rat [2]. However, it is a long way to clarify the mechanism of p8 gene expression response to stress. 5. Conclusions In summary, a zebrafish p8 cDNA with the conserved bHLH domain and NLS motif was identified in our paper. The zebrafish p8 mRNA expression levels varied much among the detected adult tissues, with the obvious higher expression in backbone and liver. During embryogenesis, the expression of zebrafish p8 mRNA was in higher levels in cleavage stage, decreased from blastula to segmentation stage, but sharply elevated at hatching stage. We provided evidences that p8 could response to more wide range of cellular stressors such as starvation, temperature, osmotic pressure and pH value, further implying an important role of p8 gene in regulation to stress. Acknowledgements This work was supported by Program for New Century Excellent Talents in University to Zhenhui Liu (No. NCET-08-0501), the Ministry of Science and Technology (MOST) of China (No. 2008AA092603 and 2008AA09Z409) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 2009-1001). References [1] Mallo GV, Fiedler F, Calvo EL, Ortiz EM, Vasseur S, Keim V, et al. Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth. J Biol Chem 1997;272:32360e9. [2] Vasseur S, Mallo GV, Fiedler F, Bödeker H, Canepa E, Moreno S, et al. Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur J Biochem 1999;259:670e5. [3] Igarashi T, Kuroda H, Takahashi S, Asashima M. Cloning and characterization of the Xenopus laevis p8 gene. Dev Growth Differ 2001;43:693e8. [4] Hoffmeister A, Ropolo A, Vasseur S, Mallo GV, Bodeker H, Ritz-Laser B, et al. The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter. J Biol Chem 2002;277:22314e9. [5] Vasseur S, Mallo GV, Garcia-Montero A, Ortiz EM, Fiedler F, Canepa E, et al. Structural and functional characterization of the mouse p8 gene: romotion of transcription by the CAAT-enhancer binding protein (C/EBP) and C/EBPb transacting factors involves a C/EBP cis-acting element and other regions of the promoter. Biochem J 1999;343:377e83. [6] Garcia-Montero A, Vasseur S, Mallo GV, Soubeyran P, Dagorn JC, Iovanna JL. Expression of the stress-induced p8 mRNA is transiently activated after culture medium change. Eur J Cell Biol 2001;80:720e5. [7] Ree AH, Tvermyr M, Engebraaten O, Rooman M, Røsok O, Hovig E, et al. Expression of a novel factor in human breast cancer cells with metastatic potential. Cancer Res 1999;59:4675e80. [8] Vasseur S, Hoffmeister A, Garcia-Montero A, Mallo GV, Feil R, Kühbandner S, et al. p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis. Oncogene 2002;21:1685e94.
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