- Email: [email protected]
Characterization, expression, and response to stress of p8 gene in amphioxus
Fish & Shellfish Immunology 27 (2009) 407–413
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Characterization, expression, and response to stress of p8 gene in amphioxus Zhenhui Liu a, *, Yanling Sun a, Naiguo Liu a, b, Ningning Fan a, Shicui Zhang a a b
Key Laboratory of Marine Genetics and Gene Resource Exploitation of Ministry of Education (MaGGR), Ocean University of China, Qingdao 266003, China Department of Biology, Binzhou Medical College, Binzhou 256603, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2009 Received in revised form 2 June 2009 Accepted 4 June 2009 Available online 26 June 2009
The amphioxus Branchiostoma belcheri tsingtaunese homolog of p8, Bbp8, was identified from the gut cDNA library. The full-length Bbp8 cDNA consists of 1032 bp, which is clearly longer than those of p8 in human, mouse, rat, frog, zebrafish and fruit fly. The genomic DNA sequences of amphioxus p8 contain three exons and two introns, which is similar to the exon/intron organization of p8 homologues in vertebrates such as human, mouse and zebrafish, while it is sharply different to the organization of p8 gene in fruit fly, which has only one exon. Sequence alignment and phylogenetic analysis showed that the basic helix-loop-helix (bHLH) region of p8 is well conserved during the long process of evolution, and Bbp8 is more close to its homologous proteins in the invertebrates than to those in the vertebrates. RTPCR and In situ hybridization histochemistry demonstrated the expression of Bbp8 in all the tissues assayed, with relatively higher expression in the gut, gill and ovaries. Quantitative real-time PCR assay revealed quick up-regulation of Bbp8 transcripts on lipopolysaccharide (LPS) challenge and starvation, implying a stress-related function for Bbp8. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Amphioxus p8 Expression LPS Starvation
1. Introduction The p8, also known as NUPR1 (nuclear protein 1) and com1 (candidate of metastasis 1), encodes a transcription cofactor with a basic helix-loop-helix (bHLH) domain and a canonical bipartite nuclear locating signal (NLS) motif [1–3]. It was firstly cloned from a rat pancreatic cDNA library in 1997 [1], and found to encode a polypeptide 80-amino acids in length that is activated in pancreatic tissues during the acute phase of pancreatitis, pancreatic development, and regeneration [1]. p8 shares the similar biochemical structure with the HMG (high mobility group) proteins [4], and is likely to be a DNA-binding protein [1,5,6]. Recent research has shown that p8 activation is not restricted to pancreatic cells alone. p8 genes are also expressed in other cells in response to a wide range of cellular stressors, which may range from a simple change of culture media to LPS challenge [4,6], and in regulation of multiple cellular functions such as cellular growth [1,2], cellular growth inhibition [7,8], cell apoptosis [8,9], tumor development and progression [10–13], animal embryo development [1,14] and cell defence [15]. The p8 cDNAs have been cloned from both 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 [16], shrimp [17] and fruit fly [18]. Human, frog and mouse p8 homologues are 82, 82 and 80-amino acid long, respectively, while it is only 69-amino acid long in fruit fly [2,5]. Amphioxus, a cephalochordate, has recently been repositioned to the base of the chordate phylum [19], and therefore is fast emerging as a model organism for the understanding of chordate evolution. However, the study of p8 gene in this animal remains untouched hitherto. In this present study, we therefore report the identification and expression of the amphioxus p8 gene. Additionally, the response of amphioxus p8 to stress such as LPS challenge and starvation was also detected. 2. Materials and methods 2.1. Animal Adult amphioxus B. belcheri tsingtauense (about 0.125 g each) were collected from the sandy bottom of the sea near Shazikou, Qingdao, China, and maintained in containers with continuous aeration at room temperature. They were starved for two days in sterilized filtered seawater before the experiment to remove all the food in the gut before the start of experiment. 2.2. cDNA library construction
* Corresponding author. 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/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.06.011
Total gut cDNA library of adult amphioxus was constructed with SMART cDNA Library Construction Kit (CLONTECH, Palo Alto, CA,
408
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
USA) according to the method described by Liu et al. [20]. Both strands of selected clones were sequenced with Megabace 1000 DNA Sequencer (Amersham). 2.3. Sequence comparison and phylogenetic analysis Multiple protein sequences were aligned with the MegAlign program by the CLUSTAL method in DNASTAR [21], and phylogenetic trees were constructed by the maximum parsimony and neighbor-joining method within the Philip 3.5c software package [22] using 1000 bootstrap replicates. 2.4. RT-PCR Total RNAs were extracted with Trizol (Gibco) from various tissues of amphioxus including muscles, gill, foregut including hepatic caecum, hindgut, ovaries, testes and notochord. For RT-PCR, the access RT-PCR system (Promega) was used, with 1 mg of total RNA in 50 ml of AMV/Tfl 1 reaction buffer containing 0.1 u/ml of AMV reverse transcriptase, 0.2 mM of dNTP mix, 1 mM of both sense primer (50 -CGTATCGTTTTCGCCATTTT-30 ) and antisense primer (50 GTTCAATGTCCGCAATCCTT-30 ), 0.1 u/ml of Tfl DNA polymerase and 1 mM. MgSO4 Reaction conditions consisted of one cycle each at 48 C for 45 min, 94 C 2 min, followed by 35 cycles at 94 C for 30 s, 55 C for 1 min, and 68 C for 2 min. After PCR, 5 ml of PCR product was loaded on 1% agarose gel for running. Normalization was carried out by amplification of amphioxus b-actin mRNA using a sense primer (50 -CTCCGGTATGTGCAAGGC-30 ) and an antisense primer (50 -GCTGGGCTGTTGAAGGTC-30 ) under the same conditions as described above. 2.5. In situ hybridization histochemistry This was performed according to the method described by Fan et al. [23]. Briefly, sexually-mature amphioxus was fixed in freshly prepared 4% paraformaldehyde solution in 100 mM phosphate buffered saline (PBS, pH 7.4) at 4 C for 8 h. The samples were dehydrated, embedded in paraffin, and sectioned at 8 mm. After pre-hybridization in a hybridization buffer containing 50% deionized formamide (v/v), 100 mg/ml heparin, 5 SSC, 0.1% Tween-20, 5 mM EDTA, 1Denhardt’s solution and 100 mg/ml salmon sperm RNA at 50 C for 3 h, they were hybridized in the same hybridization buffer with 1 mg/ml DIG-labeled Bbp8 riboprobes at 50 C for 12–16 h in a moist chamber. Subsequently, the sections were preincubated in 1% blocking reagent (Roche) in 100 mM Tris–HCl (pH 7.4) with 150 mM NaCl for 2 h at room temperature, and incubated with anti-Dig alkaline phosphatase conjugated antibody (Roche) diluted 1:1000 in 1% blocking reagent for 2 h at room temperature. After washing and staining, the sections were photographed under a BX51 Olympus microscope.
from the samples, and digested with RQ1 RNase-free DNase (Promega) to eliminate the genomic contamination. cDNAs were synthesized with a reverse transcription system using oligo d(T) primer, and used as templates. Real-time PCR was performed on ABI 7500 real-time PCR system. SYBRÒ Premix Ex TaqÔ (Takara) was used for real-time PCR reaction, with a primer concentration of 200 nM. Reaction conditions consisted of 95 C for 10 s, followed by 40 cycles each of 95 C for 5 s, 60 C for 20 s, and 72 C for 34 s. All reactions were performed in triplicate. Amphioxus b-actin 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 reaction to confirm that only one PCR product was amplified. Gene expression 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 Bbp8 and b-actin in order to calculate the fold increase [24]. 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. 3. Results 3.1. Isolation and identification of the amphioxus p8 gene The nucleotide acid sequences of clone L217 obtained from the amphioxus gut cDNA library contained 1032 base pairs (bp), and consisted of an open reading frame (ORF) of 240 bp (GenBank Accession No. AY280668), which corresponded to a deduced protein of 79 amino acid residues with a predicted molecular weight of about 9.27 kDa and an isoelectric point of 8.14 (Fig. 1). There was a start codon (ATG) at the 5’end of the cDNA, and a stop codon (TGA) at its 3’end. The clone also contained a 120 bp
2.6. Quantitative real-time PCR Real-time PCR was performed to investigate the response of Bbp8 to stress. Two PCR primers sets specific for Bbp8 and amphioxus b-actin genes were used to amplify products of 143 bp and 97 bp, respectively. Bbp8 sense primer was 50 -TCACAAAGTT CCAAAACACAGAAAG-30 , Bbp8 antisense primer was 50 -ACTTTTCT CGCCTCTTCCCAAC-30 , b-actin sense primer was 50 -GAGACCTTCAA CAGCCCAGC-30 , b-actin antisense primer was 50 -CTCCAGAGTCCAG CACGATAC-30 . Adult B. belcheri were bath-challenged with 10 mg/ml of LPS (Sigma, St. Louis, MO), and no food was supplied during the course (starvation). Then they were sampled at 0, 2, 4, 8, 16, and 24 h after challenge, respectively. Samples (0 h) not challenged with LPS served as control. Total RNA was extracted with Trizol
Fig. 1. The nucleotide and deduced amino acid sequences of amphioxus p8 cDNA (Accession number in GenBank: AY280668). The asterisk represents the stop codon. The start codon and polyadenylation signal were underlined. The numbering of the nucleotide and amino acid sequences was shown to the right.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
5’untranslated region (UTR) with a typical oligopyrimidine motif, and a long 3’UTR with a typical polyadenylation signal (AATAAA) upstream of the polyadenine stretch. These suggest that the cDNA contained the full coding sequence of an amphioxus p8 gene. BLASTP searching in GenBank revealed that the protein contained the conserved domain of DNA-binding nuclear phosphoprotein p8, and it showed an affinity to the p8 homologues, with black-legged tick p8 showing the most homology (70% similarity, 59% identity). Thus we named it Bbp8. However, unlike other p8 cDNAs available from human, mouse, rat, frog, zebrafish and fruit fly in GenBank, which all have a nucleotide length of less than 760 bp, Bbp8 cDNA was found to be longer at 1032 bp. It is interesting to detect the
409
evolution of the p8 gene in different species to explain the difference. Using the PSORT II program (http://psort.nibb.ac.jp), Bbp8 was predicted to be 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 comparison and phylogenetic analysis The Bbp8 amino acid sequence was aligned with known p8 proteins (Fig. 2a). The well-conserved bHLH regions in p8 proteins were also found to be conserved in Bbp8. To understand the
Fig. 2. (a) Alignment of p8 proteins including amphioxus 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 region of NLS were marked by grey bold line and black line, respectively. (b) Volumes of sequence similarity and sequence divergence of the aligned p8 proteins in Fig. 2a. Numbers in the table are calculated with the method of CLUSTAL in the software package DNASTAR. The accession numbers of the analyzed sequences from GenBank are: Fruit fly, Drosophila melanogaster NP_609539; Blacklegged tick, Ixodes scapularis AAY66886; Shrimp, Artemia franciscana, DQ361275; Common urchin, Paracentrotus lividus, FJ423772; Sea urchin, Strongylocentrotus purpuratus, XP_001177744; Zebrafish, Danio rerio, BC122137; Atlantic salmon, Salmo salar, ACI66304; African clawed frog, Xenopus laevis AB056582; Dog, Canis lupus familiaris XP_536921; Norway rat, Rattus norvegicus, NM_053611; House mouse, Mus musculus NM_019738; Human, Homo sapiens, O60356.
410
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
Fig. 3. The unrooted phylogenetic tree of amphioxus p8 and other known 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. Sequences of p8 refer to Fig. 2 legends.
evolution of p8 genes, the volumes of sequence similarity and sequence divergence of the aligned p8 proteins in Fig. 2a were analyzed (Fig. 2b). At the amino acid level, Bbp8 was found to share 31.6–40.8% similarity to those in vertebrates including human, mouse, rat, dog, frog, Atlantic salmon and zebrafish, while 41.1–52.2% similarity was observed when compared to those in invertebrates including fruit fly, black-legged tick and shrimp. It is notable that the amino acid sequences of p8 genes in common urchin and sea urchin exhibit less similarity to those of p8 homologues in other species. A phylogenetic tree was then constructed
with the maximum parsimony method based on the amino acid sequences of p8 proteins in Fig. 2a. It was found that Bbp8 formed a cluster together with the p8 proteins in invertebrates including fruit fly, black-legged tick, shrimp, common urchin and sea urchin (Fig. 3). Another phylogenetic tree constructed by the neighborjoining method also supported the hypothesis data (not shown). These results suggest that the amino acid sequences of Bbp8 have a closer similarity to its invertebrate counterparts than to vertebrate ones. A search of the recently completed draft assembly and automated annotation of Branchiostoma floridae genome revealed the presence of a single copy of Florida amphioxus p8 gene (Bf_V2_48). Sequence comparison demonstrated that Bbp8 shared 96.2% identity with the deduced protein encoded by Florida amphioxus p8 gene at the amino acid level, implying that Bbp8 is highly conserved in intra-species (Fig. 2). It is also highly likely that Bbp8 is a single copy gene in Branchiostoma belcheri. Analysis of the genomic structure exhibited that Florida amphioxus p8 gene consisted of three exons and two introns. The three exons of 219, 184 and 443 bp, respectively, were interspaced by two introns of 1489 and 115 bp, which all began with GT and ended with an AG dinucleotide, sequences thought to be necessary for correct RNA splicing of various other eukaryotic genes [25]. In addition, the nucleotide acid sequences of the exon1, exon2 and exon3 between Bbp8 and Florida amphioxus p8 gene shared 78.3%, 90.2% and 66.7% identity, respectively, implying that the exon2 of p8 is more conserved. This is possibly due to the facts that both the bHLH domain and the NLS motif of p8 reside in the exon2. Similarly, the p8 genes in zebrafish, dog, mouse and human all had three exons and two introns. However, the p8 gene in fruit fly possesses only one exon and has no intron (Fig. 4). This implies that the split of one exon into three exons for p8 genes had occurred at least 500 million years ago, before the cephalochordate/vertebrate divergence, after which it has remained highly conserved. 3.3. Tissue distribution of Bbp8 mRNA The tissue distribution of Bbp8 mRNA was investigated by RTPCR analysis. It showed that Bbp8 mRNA was abundant in muscle, notochord, ovaries, gill, gut including foregut and hindgut, while it was present at lower level in testes (Fig. 5). Also, In situ hybridization histochemistry revealed that Bbp8 transcripts were most abundant in the hepatic caecum, gill, and ovaries, and at a lower level in the testes, muscle, notochord and neural tube (Fig. 6). These
Fig. 4. Diagram of the genomic structures of p8 genes in human, house mouse, zebrafish, amphioxus and fruit fly. Solid boxes represent the sequences of exons of the gene, and thin lines indicate the sequences of introns. The number of nucleotides above the solid boxes and thin lines represents the size of the exons and introns, respectively. The evolutionary trend of p8 is from one exon to three exons.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
411
Fig. 5. The tissue distribution of Bbp8 mRNA detected by RT-PCR. 1, gill; 2, hindgut; 3, front gut; 4, total gut; 5, ovaries; 6, testis; 7, notochord; 8, muscle. The products of Bbp8 and b-actin are 474 bp and 354 bp, respectively.
results suggested Bbp8 mRNA to be widely present in multiple tissues of amphioxus although the expression level varied between the different tissues.
indicating that the amplifications were specific (data not shown). Bbp8 expression was up-regulated (1.6 fold) very early, at 2 h after challenge. The expression reached a peak (2.5 fold relative to control) at 8 h after post-challenge.
3.4. Response of Bbp8 to LPS and starvation challenge 4. Discussions In order to detect the response of Bbp8 to stress, the adult amphioxus was exposed to LPS at 10 mg/ml, and no food was supplied (starvation) during the course. The expression of p8 mRNA was analyzed by real-time PCR (Fig. 7). Analysis of the dissociation curve of amplified products in all cases showed only a single peak,
A p8 cDNA, Bbp8, was identified from the amphioxus B. belcheri cDNA library in this study. Bbp8 exhibited a conserved structure when compared with its homologues in human, mouse, rat, frog, zebrafish and fruit fly, with a domain of bHLH and a motif of NLS
Fig. 6. Bbp8 expression in different tissues of adult amphioxus by in situ hybridization histochemistry. (A) A low magnification view of tissues of a male amphioxus. Bbp8 mRNA expressed strongly in hepatic caecum (hc) and front gut (fg), but weakly in testis, muscle, notochord and neural tube. Scale Bar ¼ 200 mm (B1) The enlargement of the box 1 in Fig. 6(A). Bbp8 mRNA signal was seen in neural tube and notochord. Scale Bar ¼ 50 mm (B2) The enlargement of the box 2 in Fig. 6(A). Bbp8 mRNA expressed strongly in hepatic caecum (hc) and front gut (fg), but faintly in testis. Bar ¼ 50 mm (C) Bbp8 mRNA was obviously found in gill of ampioxus. Bar ¼ 100 mm (D) A section showing the presence of Bbp8 mRNA in ovarian oocytes. Scale Bar ¼ 100 mm.
412
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
Bbp8 was expressed in all the assayed amphioxus tissues, with a higher expression in gut, gill and ovaries especially. A quick upregulation of Bbp8 expression in response to LPS challenge and starvation was detected, indicating the roles of Bbp8 in the response to stress. Acknowledgements We thank Dr. Ravindra Pawar for his critical reading of the manuscript. 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 National Natural Science Foundation of China (No. 30500256). References
Fig. 7. Expression profile of Bbp8 after challenged with LPS and starvation as measured by quantitative real-time PCR. The mRNA expression of Bbp8 and b-actin was measured at 0, 2, 4, 8, 16 and 24 h following challenge. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
[1–3], indicating a similar function of p8 during the long process of evolution [1,2,6]. Phylogenetically, amphioxus p8 is closer to its invertebrate homologues, whereas it seems to have more homology with its vertebrate counterparts at the exon–intron organizational level, in that they all possess three exons and two introns. This is consistent with the generally accepted notion that amphioxus occupies an intermediate position between the invertebrates and vertebrates in the evolutionary path. Thus, the amphioxus p8 gene may be an important reference point in understanding the evolution of p8 homologues. RT-PCR and in situ hybridization analyses in the present study showed Bbp8 expression in all the detected tissues, with a relatively higher expression in gut, gill and ovaries. In human, p8 is also widely expressed although the expression levels vary among different tissues, with particularly higher expression levels in liver and the pancreas [2]. In rat, p8 was detected mainly in liver, stomach and salivary gland [1]. Recently, it has been shown for p8 to have the strongest expression in intestine of common urchin [16]. Collectively, a relatively higher expression of the p8 mRNA seems to be associated with gut-related organs or tissues in these species. It would be interesting to explore the reasons behind the high expression levels of p8 mRNA in these tissues and the functions it serves. p8 is a DNA-binding protein and its expression is up-regulated by a complex process associated with cellular stress and apoptosis [1]. Expression of p8 could be induced in several tissues of rat such as pancreas, kidney and liver in response to LPS treatment [26]. In cell lines, p8 mRNA expression was also induced in response to stress [1,5]. Here, we demonstrated that the expression of Bbp8 was quickly up-regulated after challenged with LPS and starvation, supporting the statement that p8 is a ubiquitous protein induced by cellular stress [14]. Further study need to be performed to detect the response of Bbp8 to LPS and starvation independently. 5. Conclusions The amphioxus B. belcheri tsingtaunese homolog of p8, Bbp8, was identified in this study. Phylogenetic analysis showed that the bHLH region of p8 is well conserved during the long process of evolution, and that Bbp8 is close to its homologues in invertebrates.
[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;272:32360–9. [2] Vasseur S, Mallo GV, Fiedler F, Bo¨deker H, Canepa E, Moreno S, et al. Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur J Biochem 1999;259:670–5. [3] Igarashi T, Kuroda H, Takahashi S, Asashima M. Cloning and characterization of the Xenopus laevis p8 gene. Dev Growth Differ 2001;43:693–8. [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;277:22314–9. [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;343:377–83. [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;80:720–5. [7] Burland TG. DNASTAR’s Lasergene sequence analysis software. Methods Mol Biol 2000;132:71–91. [8] Vasseur S, Hoffmeister A, Garcia-Montero A, Mallo GV, Feil R, Ku¨hbandner S, et al. p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis. Oncogene 2002;21:1685–94. [9] Su SB, Motoo Y, Iovanna JL, Berthe´ze`ne P, Xie MJ, Mouri H, et al. Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer. Clin Cancer Res 2001;7:1320–4. [10] 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;59:4675–80. [11] Su SB, Motoo Y, Iovanna JL, Xie MJ, Mouri H, Ohtsubo K, et al. Expression of p8 in human pancreatic cancer. Clin Cancer Res 2001;7:309–13. [12] Ito Y, Yoshida H, Motoo Y, Miyoshi E, Iovanna JL, Tomoda C, et al. Expression and cellular localization of p8 protein in thyroid neoplasms. Cancer Lett 2003;201:237–44. [13] Mohammad HP, Seachrist DD, Quirk CC, Nilson JH. Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing hormone. Mol Endocrinol 2004;18:2583–93. [14] Quirk CC, Seachrist DD, Nilson JH. Embryonic expression of the luteinizing hormone beta gene appears to be coupled to the transient appearance of p8, a high mobility group-related transcription factor. J Biol Chem 2003;278: 1680–5. [15] Vasseur S, Hoffmeister A, Garcia-Montero A, Barthet M, Saint-Michel L, Berthe´ze`ne P, et al. Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response. BMC Gastroenterol 2003;3:25. [16] Wang JQ, Han JC, Li DZ, Li LC. In silico cloning and characterization of p8 homolog cDNA from common urchin (Paracentrotus lividus). Mol Biol Rep 2009;. doi:10.1007/s11033-009-9474-x. [17] Qiu Z, MacRae TH. Developmentally regulated synthesis of p8, a stress-associated transcription cofactor, in diapause-destined embryos of Artemia franciscana. Cell Stress Chaperones 2007;12:255–64. [18] Zinke I, Schu¨tz CS, Katzenberger JD, Bauer M, Pankratz MJ. Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugardependent response. EMBO J 2002;21:6162–73. [19] Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 2006;439: 965–8.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413 [20] Liu Z, Zhang S, Yuan J, Sawant MS, Wei J, Xu A. Molecular cloning and phylogenetic analysis of AmphiUbf80, a new member of ubiquitin family from the amphioxus Branchiostoma belcheri tsingtauense. Curr Sci 2002;83:50–3. [21] Bratland A, Risberg K, Maelandsmo GM, Gu¨tzkow KB, Olsen OE, Moghaddam A, et al. Expression of a novel factor, com1, is regulated by 1,25dihydroxyvitamin D3 in breast cancer cells. Cancer Res 2000;60:5578–83. [22] Felsenstein J. PHYLIP (Phylogeny interference package), version 3.5c. Seattle, Washington, USA: Department of Genetics, University of Washington; 1993. [23] Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G. Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma
413
belcheri with implications to the origin of vertebrate liver. Int J Biochem Cell Biol 2007;39:450–61. [24] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCT Method. Methods 2001;25:402–8. [25] Breathnach R, Benoist C, O’Hare K, Cannon F, Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon– intron boundaries. Proc Natl Acad Sci U S A 1978;75:4853–7. [26] Jiang YF, Vaccaro MI, Fiedler F, Calvo EL, Iovanna JL. Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro. Biochem Biophys Res Commun 1999;260:686–90.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Characterization, expression, and response to stress of p8 gene in amphioxus Zhenhui Liu a, *, Yanling Sun a, Naiguo Liu a, b, Ningning Fan a, Shicui Zhang a a b
Key Laboratory of Marine Genetics and Gene Resource Exploitation of Ministry of Education (MaGGR), Ocean University of China, Qingdao 266003, China Department of Biology, Binzhou Medical College, Binzhou 256603, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2009 Received in revised form 2 June 2009 Accepted 4 June 2009 Available online 26 June 2009
The amphioxus Branchiostoma belcheri tsingtaunese homolog of p8, Bbp8, was identified from the gut cDNA library. The full-length Bbp8 cDNA consists of 1032 bp, which is clearly longer than those of p8 in human, mouse, rat, frog, zebrafish and fruit fly. The genomic DNA sequences of amphioxus p8 contain three exons and two introns, which is similar to the exon/intron organization of p8 homologues in vertebrates such as human, mouse and zebrafish, while it is sharply different to the organization of p8 gene in fruit fly, which has only one exon. Sequence alignment and phylogenetic analysis showed that the basic helix-loop-helix (bHLH) region of p8 is well conserved during the long process of evolution, and Bbp8 is more close to its homologous proteins in the invertebrates than to those in the vertebrates. RTPCR and In situ hybridization histochemistry demonstrated the expression of Bbp8 in all the tissues assayed, with relatively higher expression in the gut, gill and ovaries. Quantitative real-time PCR assay revealed quick up-regulation of Bbp8 transcripts on lipopolysaccharide (LPS) challenge and starvation, implying a stress-related function for Bbp8. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Amphioxus p8 Expression LPS Starvation
1. Introduction The p8, also known as NUPR1 (nuclear protein 1) and com1 (candidate of metastasis 1), encodes a transcription cofactor with a basic helix-loop-helix (bHLH) domain and a canonical bipartite nuclear locating signal (NLS) motif [1–3]. It was firstly cloned from a rat pancreatic cDNA library in 1997 [1], and found to encode a polypeptide 80-amino acids in length that is activated in pancreatic tissues during the acute phase of pancreatitis, pancreatic development, and regeneration [1]. p8 shares the similar biochemical structure with the HMG (high mobility group) proteins [4], and is likely to be a DNA-binding protein [1,5,6]. Recent research has shown that p8 activation is not restricted to pancreatic cells alone. p8 genes are also expressed in other cells in response to a wide range of cellular stressors, which may range from a simple change of culture media to LPS challenge [4,6], and in regulation of multiple cellular functions such as cellular growth [1,2], cellular growth inhibition [7,8], cell apoptosis [8,9], tumor development and progression [10–13], animal embryo development [1,14] and cell defence [15]. The p8 cDNAs have been cloned from both 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 [16], shrimp [17] and fruit fly [18]. Human, frog and mouse p8 homologues are 82, 82 and 80-amino acid long, respectively, while it is only 69-amino acid long in fruit fly [2,5]. Amphioxus, a cephalochordate, has recently been repositioned to the base of the chordate phylum [19], and therefore is fast emerging as a model organism for the understanding of chordate evolution. However, the study of p8 gene in this animal remains untouched hitherto. In this present study, we therefore report the identification and expression of the amphioxus p8 gene. Additionally, the response of amphioxus p8 to stress such as LPS challenge and starvation was also detected. 2. Materials and methods 2.1. Animal Adult amphioxus B. belcheri tsingtauense (about 0.125 g each) were collected from the sandy bottom of the sea near Shazikou, Qingdao, China, and maintained in containers with continuous aeration at room temperature. They were starved for two days in sterilized filtered seawater before the experiment to remove all the food in the gut before the start of experiment. 2.2. cDNA library construction
* Corresponding author. 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/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.06.011
Total gut cDNA library of adult amphioxus was constructed with SMART cDNA Library Construction Kit (CLONTECH, Palo Alto, CA,
408
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
USA) according to the method described by Liu et al. [20]. Both strands of selected clones were sequenced with Megabace 1000 DNA Sequencer (Amersham). 2.3. Sequence comparison and phylogenetic analysis Multiple protein sequences were aligned with the MegAlign program by the CLUSTAL method in DNASTAR [21], and phylogenetic trees were constructed by the maximum parsimony and neighbor-joining method within the Philip 3.5c software package [22] using 1000 bootstrap replicates. 2.4. RT-PCR Total RNAs were extracted with Trizol (Gibco) from various tissues of amphioxus including muscles, gill, foregut including hepatic caecum, hindgut, ovaries, testes and notochord. For RT-PCR, the access RT-PCR system (Promega) was used, with 1 mg of total RNA in 50 ml of AMV/Tfl 1 reaction buffer containing 0.1 u/ml of AMV reverse transcriptase, 0.2 mM of dNTP mix, 1 mM of both sense primer (50 -CGTATCGTTTTCGCCATTTT-30 ) and antisense primer (50 GTTCAATGTCCGCAATCCTT-30 ), 0.1 u/ml of Tfl DNA polymerase and 1 mM. MgSO4 Reaction conditions consisted of one cycle each at 48 C for 45 min, 94 C 2 min, followed by 35 cycles at 94 C for 30 s, 55 C for 1 min, and 68 C for 2 min. After PCR, 5 ml of PCR product was loaded on 1% agarose gel for running. Normalization was carried out by amplification of amphioxus b-actin mRNA using a sense primer (50 -CTCCGGTATGTGCAAGGC-30 ) and an antisense primer (50 -GCTGGGCTGTTGAAGGTC-30 ) under the same conditions as described above. 2.5. In situ hybridization histochemistry This was performed according to the method described by Fan et al. [23]. Briefly, sexually-mature amphioxus was fixed in freshly prepared 4% paraformaldehyde solution in 100 mM phosphate buffered saline (PBS, pH 7.4) at 4 C for 8 h. The samples were dehydrated, embedded in paraffin, and sectioned at 8 mm. After pre-hybridization in a hybridization buffer containing 50% deionized formamide (v/v), 100 mg/ml heparin, 5 SSC, 0.1% Tween-20, 5 mM EDTA, 1Denhardt’s solution and 100 mg/ml salmon sperm RNA at 50 C for 3 h, they were hybridized in the same hybridization buffer with 1 mg/ml DIG-labeled Bbp8 riboprobes at 50 C for 12–16 h in a moist chamber. Subsequently, the sections were preincubated in 1% blocking reagent (Roche) in 100 mM Tris–HCl (pH 7.4) with 150 mM NaCl for 2 h at room temperature, and incubated with anti-Dig alkaline phosphatase conjugated antibody (Roche) diluted 1:1000 in 1% blocking reagent for 2 h at room temperature. After washing and staining, the sections were photographed under a BX51 Olympus microscope.
from the samples, and digested with RQ1 RNase-free DNase (Promega) to eliminate the genomic contamination. cDNAs were synthesized with a reverse transcription system using oligo d(T) primer, and used as templates. Real-time PCR was performed on ABI 7500 real-time PCR system. SYBRÒ Premix Ex TaqÔ (Takara) was used for real-time PCR reaction, with a primer concentration of 200 nM. Reaction conditions consisted of 95 C for 10 s, followed by 40 cycles each of 95 C for 5 s, 60 C for 20 s, and 72 C for 34 s. All reactions were performed in triplicate. Amphioxus b-actin 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 reaction to confirm that only one PCR product was amplified. Gene expression 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 Bbp8 and b-actin in order to calculate the fold increase [24]. 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. 3. Results 3.1. Isolation and identification of the amphioxus p8 gene The nucleotide acid sequences of clone L217 obtained from the amphioxus gut cDNA library contained 1032 base pairs (bp), and consisted of an open reading frame (ORF) of 240 bp (GenBank Accession No. AY280668), which corresponded to a deduced protein of 79 amino acid residues with a predicted molecular weight of about 9.27 kDa and an isoelectric point of 8.14 (Fig. 1). There was a start codon (ATG) at the 5’end of the cDNA, and a stop codon (TGA) at its 3’end. The clone also contained a 120 bp
2.6. Quantitative real-time PCR Real-time PCR was performed to investigate the response of Bbp8 to stress. Two PCR primers sets specific for Bbp8 and amphioxus b-actin genes were used to amplify products of 143 bp and 97 bp, respectively. Bbp8 sense primer was 50 -TCACAAAGTT CCAAAACACAGAAAG-30 , Bbp8 antisense primer was 50 -ACTTTTCT CGCCTCTTCCCAAC-30 , b-actin sense primer was 50 -GAGACCTTCAA CAGCCCAGC-30 , b-actin antisense primer was 50 -CTCCAGAGTCCAG CACGATAC-30 . Adult B. belcheri were bath-challenged with 10 mg/ml of LPS (Sigma, St. Louis, MO), and no food was supplied during the course (starvation). Then they were sampled at 0, 2, 4, 8, 16, and 24 h after challenge, respectively. Samples (0 h) not challenged with LPS served as control. Total RNA was extracted with Trizol
Fig. 1. The nucleotide and deduced amino acid sequences of amphioxus p8 cDNA (Accession number in GenBank: AY280668). The asterisk represents the stop codon. The start codon and polyadenylation signal were underlined. The numbering of the nucleotide and amino acid sequences was shown to the right.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
5’untranslated region (UTR) with a typical oligopyrimidine motif, and a long 3’UTR with a typical polyadenylation signal (AATAAA) upstream of the polyadenine stretch. These suggest that the cDNA contained the full coding sequence of an amphioxus p8 gene. BLASTP searching in GenBank revealed that the protein contained the conserved domain of DNA-binding nuclear phosphoprotein p8, and it showed an affinity to the p8 homologues, with black-legged tick p8 showing the most homology (70% similarity, 59% identity). Thus we named it Bbp8. However, unlike other p8 cDNAs available from human, mouse, rat, frog, zebrafish and fruit fly in GenBank, which all have a nucleotide length of less than 760 bp, Bbp8 cDNA was found to be longer at 1032 bp. It is interesting to detect the
409
evolution of the p8 gene in different species to explain the difference. Using the PSORT II program (http://psort.nibb.ac.jp), Bbp8 was predicted to be 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 comparison and phylogenetic analysis The Bbp8 amino acid sequence was aligned with known p8 proteins (Fig. 2a). The well-conserved bHLH regions in p8 proteins were also found to be conserved in Bbp8. To understand the
Fig. 2. (a) Alignment of p8 proteins including amphioxus 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 region of NLS were marked by grey bold line and black line, respectively. (b) Volumes of sequence similarity and sequence divergence of the aligned p8 proteins in Fig. 2a. Numbers in the table are calculated with the method of CLUSTAL in the software package DNASTAR. The accession numbers of the analyzed sequences from GenBank are: Fruit fly, Drosophila melanogaster NP_609539; Blacklegged tick, Ixodes scapularis AAY66886; Shrimp, Artemia franciscana, DQ361275; Common urchin, Paracentrotus lividus, FJ423772; Sea urchin, Strongylocentrotus purpuratus, XP_001177744; Zebrafish, Danio rerio, BC122137; Atlantic salmon, Salmo salar, ACI66304; African clawed frog, Xenopus laevis AB056582; Dog, Canis lupus familiaris XP_536921; Norway rat, Rattus norvegicus, NM_053611; House mouse, Mus musculus NM_019738; Human, Homo sapiens, O60356.
410
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
Fig. 3. The unrooted phylogenetic tree of amphioxus p8 and other known 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. Sequences of p8 refer to Fig. 2 legends.
evolution of p8 genes, the volumes of sequence similarity and sequence divergence of the aligned p8 proteins in Fig. 2a were analyzed (Fig. 2b). At the amino acid level, Bbp8 was found to share 31.6–40.8% similarity to those in vertebrates including human, mouse, rat, dog, frog, Atlantic salmon and zebrafish, while 41.1–52.2% similarity was observed when compared to those in invertebrates including fruit fly, black-legged tick and shrimp. It is notable that the amino acid sequences of p8 genes in common urchin and sea urchin exhibit less similarity to those of p8 homologues in other species. A phylogenetic tree was then constructed
with the maximum parsimony method based on the amino acid sequences of p8 proteins in Fig. 2a. It was found that Bbp8 formed a cluster together with the p8 proteins in invertebrates including fruit fly, black-legged tick, shrimp, common urchin and sea urchin (Fig. 3). Another phylogenetic tree constructed by the neighborjoining method also supported the hypothesis data (not shown). These results suggest that the amino acid sequences of Bbp8 have a closer similarity to its invertebrate counterparts than to vertebrate ones. A search of the recently completed draft assembly and automated annotation of Branchiostoma floridae genome revealed the presence of a single copy of Florida amphioxus p8 gene (Bf_V2_48). Sequence comparison demonstrated that Bbp8 shared 96.2% identity with the deduced protein encoded by Florida amphioxus p8 gene at the amino acid level, implying that Bbp8 is highly conserved in intra-species (Fig. 2). It is also highly likely that Bbp8 is a single copy gene in Branchiostoma belcheri. Analysis of the genomic structure exhibited that Florida amphioxus p8 gene consisted of three exons and two introns. The three exons of 219, 184 and 443 bp, respectively, were interspaced by two introns of 1489 and 115 bp, which all began with GT and ended with an AG dinucleotide, sequences thought to be necessary for correct RNA splicing of various other eukaryotic genes [25]. In addition, the nucleotide acid sequences of the exon1, exon2 and exon3 between Bbp8 and Florida amphioxus p8 gene shared 78.3%, 90.2% and 66.7% identity, respectively, implying that the exon2 of p8 is more conserved. This is possibly due to the facts that both the bHLH domain and the NLS motif of p8 reside in the exon2. Similarly, the p8 genes in zebrafish, dog, mouse and human all had three exons and two introns. However, the p8 gene in fruit fly possesses only one exon and has no intron (Fig. 4). This implies that the split of one exon into three exons for p8 genes had occurred at least 500 million years ago, before the cephalochordate/vertebrate divergence, after which it has remained highly conserved. 3.3. Tissue distribution of Bbp8 mRNA The tissue distribution of Bbp8 mRNA was investigated by RTPCR analysis. It showed that Bbp8 mRNA was abundant in muscle, notochord, ovaries, gill, gut including foregut and hindgut, while it was present at lower level in testes (Fig. 5). Also, In situ hybridization histochemistry revealed that Bbp8 transcripts were most abundant in the hepatic caecum, gill, and ovaries, and at a lower level in the testes, muscle, notochord and neural tube (Fig. 6). These
Fig. 4. Diagram of the genomic structures of p8 genes in human, house mouse, zebrafish, amphioxus and fruit fly. Solid boxes represent the sequences of exons of the gene, and thin lines indicate the sequences of introns. The number of nucleotides above the solid boxes and thin lines represents the size of the exons and introns, respectively. The evolutionary trend of p8 is from one exon to three exons.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
411
Fig. 5. The tissue distribution of Bbp8 mRNA detected by RT-PCR. 1, gill; 2, hindgut; 3, front gut; 4, total gut; 5, ovaries; 6, testis; 7, notochord; 8, muscle. The products of Bbp8 and b-actin are 474 bp and 354 bp, respectively.
results suggested Bbp8 mRNA to be widely present in multiple tissues of amphioxus although the expression level varied between the different tissues.
indicating that the amplifications were specific (data not shown). Bbp8 expression was up-regulated (1.6 fold) very early, at 2 h after challenge. The expression reached a peak (2.5 fold relative to control) at 8 h after post-challenge.
3.4. Response of Bbp8 to LPS and starvation challenge 4. Discussions In order to detect the response of Bbp8 to stress, the adult amphioxus was exposed to LPS at 10 mg/ml, and no food was supplied (starvation) during the course. The expression of p8 mRNA was analyzed by real-time PCR (Fig. 7). Analysis of the dissociation curve of amplified products in all cases showed only a single peak,
A p8 cDNA, Bbp8, was identified from the amphioxus B. belcheri cDNA library in this study. Bbp8 exhibited a conserved structure when compared with its homologues in human, mouse, rat, frog, zebrafish and fruit fly, with a domain of bHLH and a motif of NLS
Fig. 6. Bbp8 expression in different tissues of adult amphioxus by in situ hybridization histochemistry. (A) A low magnification view of tissues of a male amphioxus. Bbp8 mRNA expressed strongly in hepatic caecum (hc) and front gut (fg), but weakly in testis, muscle, notochord and neural tube. Scale Bar ¼ 200 mm (B1) The enlargement of the box 1 in Fig. 6(A). Bbp8 mRNA signal was seen in neural tube and notochord. Scale Bar ¼ 50 mm (B2) The enlargement of the box 2 in Fig. 6(A). Bbp8 mRNA expressed strongly in hepatic caecum (hc) and front gut (fg), but faintly in testis. Bar ¼ 50 mm (C) Bbp8 mRNA was obviously found in gill of ampioxus. Bar ¼ 100 mm (D) A section showing the presence of Bbp8 mRNA in ovarian oocytes. Scale Bar ¼ 100 mm.
412
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413
Bbp8 was expressed in all the assayed amphioxus tissues, with a higher expression in gut, gill and ovaries especially. A quick upregulation of Bbp8 expression in response to LPS challenge and starvation was detected, indicating the roles of Bbp8 in the response to stress. Acknowledgements We thank Dr. Ravindra Pawar for his critical reading of the manuscript. 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 National Natural Science Foundation of China (No. 30500256). References
Fig. 7. Expression profile of Bbp8 after challenged with LPS and starvation as measured by quantitative real-time PCR. The mRNA expression of Bbp8 and b-actin was measured at 0, 2, 4, 8, 16 and 24 h following challenge. Vertical bars represent the mean SD (n ¼ 3). Significant differences (P < 0.05) across control are indicated by an asterisk (*).
[1–3], indicating a similar function of p8 during the long process of evolution [1,2,6]. Phylogenetically, amphioxus p8 is closer to its invertebrate homologues, whereas it seems to have more homology with its vertebrate counterparts at the exon–intron organizational level, in that they all possess three exons and two introns. This is consistent with the generally accepted notion that amphioxus occupies an intermediate position between the invertebrates and vertebrates in the evolutionary path. Thus, the amphioxus p8 gene may be an important reference point in understanding the evolution of p8 homologues. RT-PCR and in situ hybridization analyses in the present study showed Bbp8 expression in all the detected tissues, with a relatively higher expression in gut, gill and ovaries. In human, p8 is also widely expressed although the expression levels vary among different tissues, with particularly higher expression levels in liver and the pancreas [2]. In rat, p8 was detected mainly in liver, stomach and salivary gland [1]. Recently, it has been shown for p8 to have the strongest expression in intestine of common urchin [16]. Collectively, a relatively higher expression of the p8 mRNA seems to be associated with gut-related organs or tissues in these species. It would be interesting to explore the reasons behind the high expression levels of p8 mRNA in these tissues and the functions it serves. p8 is a DNA-binding protein and its expression is up-regulated by a complex process associated with cellular stress and apoptosis [1]. Expression of p8 could be induced in several tissues of rat such as pancreas, kidney and liver in response to LPS treatment [26]. In cell lines, p8 mRNA expression was also induced in response to stress [1,5]. Here, we demonstrated that the expression of Bbp8 was quickly up-regulated after challenged with LPS and starvation, supporting the statement that p8 is a ubiquitous protein induced by cellular stress [14]. Further study need to be performed to detect the response of Bbp8 to LPS and starvation independently. 5. Conclusions The amphioxus B. belcheri tsingtaunese homolog of p8, Bbp8, was identified in this study. Phylogenetic analysis showed that the bHLH region of p8 is well conserved during the long process of evolution, and that Bbp8 is close to its homologues in invertebrates.
[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;272:32360–9. [2] Vasseur S, Mallo GV, Fiedler F, Bo¨deker H, Canepa E, Moreno S, et al. Cloning and expression of the human p8, a nuclear protein with mitogenic activity. Eur J Biochem 1999;259:670–5. [3] Igarashi T, Kuroda H, Takahashi S, Asashima M. Cloning and characterization of the Xenopus laevis p8 gene. Dev Growth Differ 2001;43:693–8. [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;277:22314–9. [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;343:377–83. [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;80:720–5. [7] Burland TG. DNASTAR’s Lasergene sequence analysis software. Methods Mol Biol 2000;132:71–91. [8] Vasseur S, Hoffmeister A, Garcia-Montero A, Mallo GV, Feil R, Ku¨hbandner S, et al. p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis. Oncogene 2002;21:1685–94. [9] Su SB, Motoo Y, Iovanna JL, Berthe´ze`ne P, Xie MJ, Mouri H, et al. Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer. Clin Cancer Res 2001;7:1320–4. [10] 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;59:4675–80. [11] Su SB, Motoo Y, Iovanna JL, Xie MJ, Mouri H, Ohtsubo K, et al. Expression of p8 in human pancreatic cancer. Clin Cancer Res 2001;7:309–13. [12] Ito Y, Yoshida H, Motoo Y, Miyoshi E, Iovanna JL, Tomoda C, et al. Expression and cellular localization of p8 protein in thyroid neoplasms. Cancer Lett 2003;201:237–44. [13] Mohammad HP, Seachrist DD, Quirk CC, Nilson JH. Reexpression of p8 contributes to tumorigenic properties of pituitary cells and appears in a subset of prolactinomas in transgenic mice that hypersecrete luteinizing hormone. Mol Endocrinol 2004;18:2583–93. [14] Quirk CC, Seachrist DD, Nilson JH. Embryonic expression of the luteinizing hormone beta gene appears to be coupled to the transient appearance of p8, a high mobility group-related transcription factor. J Biol Chem 2003;278: 1680–5. [15] Vasseur S, Hoffmeister A, Garcia-Montero A, Barthet M, Saint-Michel L, Berthe´ze`ne P, et al. Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response. BMC Gastroenterol 2003;3:25. [16] Wang JQ, Han JC, Li DZ, Li LC. In silico cloning and characterization of p8 homolog cDNA from common urchin (Paracentrotus lividus). Mol Biol Rep 2009;. doi:10.1007/s11033-009-9474-x. [17] Qiu Z, MacRae TH. Developmentally regulated synthesis of p8, a stress-associated transcription cofactor, in diapause-destined embryos of Artemia franciscana. Cell Stress Chaperones 2007;12:255–64. [18] Zinke I, Schu¨tz CS, Katzenberger JD, Bauer M, Pankratz MJ. Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugardependent response. EMBO J 2002;21:6162–73. [19] Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 2006;439: 965–8.
Z. Liu et al. / Fish & Shellfish Immunology 27 (2009) 407–413 [20] Liu Z, Zhang S, Yuan J, Sawant MS, Wei J, Xu A. Molecular cloning and phylogenetic analysis of AmphiUbf80, a new member of ubiquitin family from the amphioxus Branchiostoma belcheri tsingtauense. Curr Sci 2002;83:50–3. [21] Bratland A, Risberg K, Maelandsmo GM, Gu¨tzkow KB, Olsen OE, Moghaddam A, et al. Expression of a novel factor, com1, is regulated by 1,25dihydroxyvitamin D3 in breast cancer cells. Cancer Res 2000;60:5578–83. [22] Felsenstein J. PHYLIP (Phylogeny interference package), version 3.5c. Seattle, Washington, USA: Department of Genetics, University of Washington; 1993. [23] Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G. Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma
413
belcheri with implications to the origin of vertebrate liver. Int J Biochem Cell Biol 2007;39:450–61. [24] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCT Method. Methods 2001;25:402–8. [25] Breathnach R, Benoist C, O’Hare K, Cannon F, Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon– intron boundaries. Proc Natl Acad Sci U S A 1978;75:4853–7. [26] Jiang YF, Vaccaro MI, Fiedler F, Calvo EL, Iovanna JL. Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro. Biochem Biophys Res Commun 1999;260:686–90.