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Characterization of chicken visfatin gene: cDNA cloning, tissue distribution, and promoter analysis
Characterization of chicken visfatin gene: cDNA cloning, tissue distribution, and promoter analysis J. Li,*1 F. Meng,* C. Song,* Y. Wang,* and F. C. Leung†1 *Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610064, PR China; and †School of Biological Sciences, The University of Hong Kong, PR China ABSTRACT Here we report the cloning and characterization of chicken visfatin (also called pre-B cell enhancing factor; PBEF, or nicotinamide phosphoribosyltransferase; Nampt) gene. Sequence analyses revealed that the coding region of visfatin is 1,482 bp in length and encodes a protein of 493 amino acids, which shares high amino acid sequence identity not only to visfatin of human (94%), rat (94%), carp (89%), and zebrafish (89%), but also to Nampt of sponge (58%) and cyanobacterium (48%). The reverse transcription PCR assay and Northern-blot analysis demonstrated that visfatin was widely expressed in all chicken tissues examined.
Using a dual luciferase reporter system, we further demonstrated that the cloned 1,372-bp fragment upstream of the putative translation start site (ATG) displayed the maximal promoter activity in cultured CHO, DF-1, and HEK293 cells, whereas the removal of its 5′-region (1,075 bp) or 3′-region (297 bp) could only partially reduce its promoter activity, implying that visfatin gene transcription was likely controlled by multiple promoters near the translation start site. Taken together, results from present study will contribute to our better understanding of the expression and roles of visfatin gene in chickens.
Key words: chicken, visfatin, pre-B cell enhancing factor, Nampt, promoter analysis 2012 Poultry Science 91:2885–2894 http://dx.doi.org/10.3382/ps.2012-02315
INTRODUCTION It is generally believed that the adipose tissues mainly function as an energy reservoir. However, increasing evidences support that adipose tissue may also serve as an endocrine organ by secreting many adipokines involved in multiple physiological processes and pathophysiology of obesity-related metabolic disorders. Visfatin, a novel potential adipokine, has drawn attention since its discovery due to its insulin-mimetric action. Originally, visfatin was reported to be preferentially expressed in visceral fat and its concentrations in plasma were highly correlated with mouse visceral fat mass. Moreover, this protein has been shown to be capable of binding to insulin receptor and mimicking insulin-like activity in lowering of plasma glucose levels in mice (Garten et al., 2009; Imai, 2009). Since then, visfatin has been investigated extensively in various animal models (Klöting and Kloting, 2005; McGlothlin et al., 2005; Chen et al., 2007). However, many contradictory findings have been reported and thus questioned its identity as a novel adipokine. For instance, Berndt and his coworkers ex©2012 Poultry Science Association Inc. Received March 18, 2012. Accepted July 17, 2012. 1 Corresponding authors: [email protected] and fcleung@hkucc. hku.hk
amined 187 subjects with obesity and found that there was no differential expression of visfatin gene between visceral and subcutaneous adipose tissues. In addition, in a subgroup of 73 individuals, in which visceral fat mass was calculated from CT scans, no correlation was detected between visceral fat mass and plasma visfatin concentrations (Berndt et al., 2005). Visfatin is initially identified from activated lymophocytes and named as pre-B cell enhancing factor (PBEF) owing to its synergistic role with interleukin-7 (IL-7) and stem cell factor (SCF) in promoting preB cell colony formation in vitro (Samal et al., 1994). The expression of PBEF can be induced by lectin and cycloheximide, and PBEF protein is detected in the culture medium of activated lymphocytes, supporting that it may also function as a cytokine (Ognjanovic and Bryant-Greenwood, 2002; Ye et al., 2005). In addition to being viewed as an adipokine or cytokine, visfatin was also reported to function as a cytosolic enzyme. Mouse visfatin/PBEF was found to show remarkable degree of amino acid sequence identity with a bacterial enzyme encoded by a NadV gene. The enzyme catalyzes the conversion of nicotinamide (NAM) to nicotinamide mononucleotide (NMN), which represents the rate-limiting step in NAD biosynthesis (Martin et al., 2001). Further studies confirmed that it could function as a nicotinamide phosphoribosyltransferase
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(Nampt) and catalyze the condensation of NAM with 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield NMN, an intermediate substrate for NAD biosynthesis (Rongvaux et al., 2002). Thus, visfatin/PBEF is also named as Nampt. To date, the biological function of visfatin/PBEF/ Nampt remains controversial. Biochemical, structural and physiological studies strongly support that Nampt is the rating-limiting enzyme for NAD biosynthesis which plays critical roles in a wide variety of physiological processes including metabolism, stress response, aging, and cell differentiation in mammals (Revollo et al., 2004; Wang et al., 2006; Imai, 2009). For instance, Revollo et al. (2007) reported that Nampt/visfatin/ PBEF could regulate glucose-stimulated insulin secretion in pancreatic β-cells and this action is likely attributed to Nampt-mediated NAD biosynthesis because the administration of NMN, a product of the Nampt enzymatic reaction, could effectively rescue the defective glucose-stimulated insulin secretion observed in Nampt+/− mice (Revollo et al., 2007; Brown et al., 2010). Becuase NAD is involved in various physiological processes, Nampt being a potential key rate-limiting enzyme for NAD synthesis is therefore viewed as a crucial link between NAD metabolism and diseases (Garten et al., 2009; Gallí et al., 2010). Although the roles of visfatin/PBEF/Nampt have been extensively investigated in mammals, it remains largely unknown in nonmammalian vertebrates including birds. In chickens, there is evidence showing that visfatin is expressed in various tissues and it is likely involved in the regulation of muscle growth and metabolism (Krzysik-Walker et al., 2008), food intake, and testicular functions (Cline et al., 2008; Ocón-Grove et al., 2010). However, the mechanisms underlying its action and the transcriptional regulation on its expression remain unclear in chickens. In this study, using chicken as an experimental model, we cloned the full-length cDNA of visfatin gene and reported its tissue expression pattern (Li et al., 2005). Moreover, the 5′-flanking region of this gene was also cloned and characterized. Our present study would contribute to our better understanding of the expression and roles of visfatin gene in avian species.
MATERIALS AND METHODS Chemicals and Reagents All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and restriction enzymes were obtained from Amersham Biosciences (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) unless stated otherwise.
Total RNA Extraction Adult chickens were killed and different tissues including small intestine, heart, kidney, liver, lung, muscle, ovary, pituitary, spleen, pancreas, testis, and whole
brain were collected for total RNA extraction. Total RNA was extracted from chicken tissues with Trireagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions and dissolved in DEPC-treated H2O. The experiments were carried out according to the guidelines provided by the Animal Ethics Committee of Sichuan University and the University of Hong Kong.
Cloning of the Full-Length cDNA for Chicken Visfatin Gene To obtain the full-length cDNA of chicken visfatin, 2 gene-specific primers flanking the start and stop codons were designed (Table 1) based on the information from chicken genome database (http://www.emsembl.org/Gallus_gallus) and EST sequences deposited in GenBank (Accession No.: BU288000, BU280677, BU135629, and BU362525). The full-length cDNA containing an open reading frame of visfatin were amplified from adult chicken liver using high-fidelity Taq DNA polymerase (Roche diagnostics, Basel, Switzerland). The PCR products were cloned into pBluescript II SK (+/−; Stratagene, La Jolla, CA). The full-length cDNA were finally determined by sequencing (ABI PRISM 3100 Genetic Analyzer, Perkin Elmer, Norwalk, CT) at least 3 independent clones containing whole open reading frames.
Reverse Transcription and PCR Reverse transcription (RT) was performed at 42°C for 2 h in a total volume of 10 μL consisting of 2 μg total RNA from different tissues, 1× single-strand buffer, 0.5 mM each deoxynucleotide triphosphate, 0.5 μg oligo-deoxythymidine, and 100 U MMLV reverse transcriptase (Promega, Madison, WI). All negative controls were carried out under the same condition without reverse transcriptase added in the 10-μL reaction mix. One microliter of the first-strand cDNA was used as the template for each PCR reaction. According to our previously established methods (Wang et al., 2008), RT-PCR assays were employed to examine the expression of visfatin in chicken tissues. The PCR was performed under the following conditions: 2 min at 95°C for denaturation, followed by 23 cycles (for β-actin: 30 s at 95°C, 30 s at 58°C, and 60 s at 72°C) and 25 (or 33) cycles (for visfatin: 30 s at 95°C, 30 s at 58°C, 60 s at 72°C) of reaction, ending with a 5-min extension at 72°C. The primers used for visfatin gene and β-actin gene amplification were listed (Table 1). The PCR products were visualized on a UV-transilluminator (Bio-Rad Laboratories Inc., Hercules, CA) after electrophoresis on 2% agarose gel containing ethidium bromide. The identity of PCR product was further confirmed by sequencing.
Northern-Blot Analysis Northern-blot analysis was performed to examine the tissue distribution of chicken visfatin gene. Twenty
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Table 1. Primers used in present study Primer name/construct Primers for full-length cDNA amplification PBU1 PBL1 Primers for the 5′-flanking region amplification pPBU1 pPBL1 Primers for reverse-transcription PCR and realtime PCR assay PBU2 PBL2 β-actin Primers for preparing the promoter constructs p(−1372/−1)Luc p(−1372/−297)Luc p(−1372/−197)Luc p(−720/−1)Luc p(−310/−1)Luc p(−179/−1)Luc
Sense/antisense
Primer sequence (5′–3′)
Sense Antisense
5′-CAGCGCAGCCCGGTCCTGT-3′ 5′-TTAGTGAGACGCCGTTTCAT-3′
Sense Antisense
5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′
Sense Antisense Sense Antisense
5′-ACCAGATTCTGGGAATCCCCTT-3′ 5′-TCTCCAGCAGGTGTCCTATGCA-3′ 5′-CAATGGCTCCGGTATGTGCA-3′ 5′-AGGCATACAGGGACAGCACA-3′
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sence Antisense
5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGTGACACCGCGAGTGC-3′ 5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTCTGCGCGCCGATTG-3′ 5′-GAGCTCGGATCCACGCACCT-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCGCGGTGTCACACCG-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCGGCGCGCAGAAGCCGA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′
micrograms total RNA was separated in a 1.0% agarose gel containing 2.2 M formaldehyde. Gels were then transferred to Hybond nylon membranes (Amersham Pharmacia Biotech, Baie d’Urfe, QC, Canada). After UV crosslinking (Amersham Pharmacia Biotech), membranes were prehybridized for 2 h at 68°C in solution I (40% formamide, 5xSSC, 50 mM HEPES, pH 6.8, 5 mM EDTA, 2× Denhardt’s, 0.1% SDS, and 100 μg/mL salmon sperm DNA) and then hybridized in the same solution overnight at 68°C with digoxigeninlabeled antisense RNA probe prepared from the cloned cDNA by in vitro transcription (Promega). Membranes were washed and detected with chemiluminescent detection kit according to the manufacturer’s instructions (Roche Diagnostics).
Quantitative Measurements of Visfatin mRNA Expression in Chicken Fat Tissues In the present study, the subcutaneous fat tissues from chicken embryo (d 15), subcutaneous and visceral fat tissues from 3-wk-old and adult chickens were collected to evaluate the mRNA levels of visfatin gene. Quantitative real-time PCR was performed in a 20-μL reaction mix consisting of 300 nM forward primer, 300 nM reverse primer, 5 μL cDNA sample, 0.25 μL of Taq DNA polymerase (Invitrogen), and 1 μL EvaGreen (Biotium Inc., Hayward, CA). The PCR conditions were 2 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 30 s at 95°C, 30 s at 60°C, and 60 s at 72°C. The PCR amplification was performed on an iCycler iQ real-time PCR machine (Bio-Rad Laboratories). To confirm the specificity of the PCR reaction, melting curve analyses were performed at the end of PCR and the identity of PCR product was further confirmed by
Size (bp) 1,538 1,372 384 401 1,372 1,075 1,175 720 310 180
sequencing. All real-time PCR amplifications were performed in triplicate. The standard curves were established using serially diluted plasmids with known copy number as templates to calculate the copy numbers of visfatin and actin in chicken tissues.
Isolation of 5′-Flanking Region of Chicken Visfatin Gene The 5′-flanking region of chicken visfatin gene has been cloned in an effort to reveal its transcriptional regulatory mechanism. Based on the chicken genome database, the full-length of chicken visfatin cDNA sequence was aligned with chicken genome sequence (www.ensembl.org/Gallus_gallus/). Sense and antisense primers (Table 1) were then designed to amplify the 5′-flanking region of chicken visfatin gene using chicken genomic DNA as template. The PCR reactions were performed as follows: 2 min at 95°C for denaturation, followed by 30 cycles of 20 s at 94°C, 10 s at 58°C, 1 min 30 s at 72°C, and ending with 7 min extension at 72°C. The amplified PCR products were then subcloned into pBluescript SK (+/−) and subjected for sequence analyses.
Promoter Analysis of Chicken Visfatin Gene in Cultured Cells To test whether the 5′-flanking region of visfatin gene upstream the translation start site could display promoter activity, a set of constructs containing the 5′-flanking region of different lengths were prepared using PCR method. The primers for each construct were listed in Table 1. The amplified PCR products were first cloned into pBluescript SK (+/−) for sequence
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Figure 1. A) Nucleotide and deduced amino acid sequences of chicken visfatin gene (AY946242). The 2 putative N-linked glycosylation sites are boxed. B) Gene structure of chicken visfatin gene. Eleven exons are labeled accordingly (number in box) and the number in italic indicates the size of each exon (bp). The putative coding region of chicken visfatin is shaded. Multiple promoters (arrow indicated) responsible for visfatin gene transcription may exist in the 5′-flanking region based on the promoter analyses performed in present study.
confirmation and then subcloned into pGL3-Basic vector (Promega). The cell lines used in this experiment include a Chinese hamster ovary (CHO) cell line, a chicken embryo fibroblast cell line (DF-1), and a human embryo kidney cell line (HEK 293), which were cultured under conditions as described in our previous studies (Wang et al., 2010, 2012). For transfection, 1 × 105 cells were plated on each 48-well plate. A mixture containing 100 ng of promoter-luciferase construct, 5 ng of pRL-TK (Promega), and 1.5 μL of Lipofectamine (Invitrogen) was prepared in 20 μL of PBS. Following the manufacture’s instruction, luciferase activity of each promoter construct in the cultured cells was normalized to Renilla luciferase activity derived from the pRL-TK vector and then expressed as relative fold increase as compared with the control group (pGL3-basic vector). The data were analyzed by one-way ANOVA followed by Dunnett’s test using GraphPad Prism 4 (GraphPad Software, San Diego, CA). To validate our results, all experiments were repeated at least 2 times.
RESULTS Cloning Full-Length cDNA of Chicken Visfatin Gene Based on the related genome and EST sequences from chicken genome database, gene-specific primers were designed to amplify the full-length cDNA of chicken visfatin gene from chicken liver. The cloned full-length visfatin cDNA is 1,538 bp in length, with an open reading frame of 1,482 bp (accession No. AY946242; Figure 1). In addition, a 5′-untranslated region (5′-UTR) region of 314 bp (JQ406639) and a long 3′-UTR of 2,766 bp (JQ354940) were also obtained by RT-PCR and confirmed by sequencing in present study (Supplemental Figures S1 and S2; available online at ps.fass.org). Comparison of the cloned cDNA to chicken genome database revealed that the chicken visfatin gene spans across ~29 kb on chromosome 1 and consists of 11 exons and 10 introns (Figure 1). The putative chicken visfatin is 493 amino acids in length with a predicted
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Figure 2. Amino acid sequence alignment of chicken visfatin and that of human (NP_005737), rat (NP_808789), mouse (NP_067499), carp (BAA96290), zebrafish (NM_212668), sponge (Suberites domuncula; CAB65409), and cyanobacterium (Synechocystis sp. PCC 6803; NP_442623). The conserved amino acids responsible for nicotinamide binding (arrow heads) and ribose binding (boxed residues) have been shown respectively. The triangles (▼) denote the conserved amino acids responsible for enzyme catalysis.
molecular weight of 55 kDa. Two putative N-linked glycosylation sites (N-X-S, where X represents any amino acids except proline) were found at its N- and Cterminal respectively. Sequence analyses revealed that the N-terminal of the predicted visfatin lacks the typical signal peptide sequence, which is hydrophobic and characteristic for most secreted peptides. As shown in Figure 2, the putative chicken visfatin protein not only shares high amino acid sequence identity with that of human (Homo sapiens; 94%; Accession No.: NP_005737), rat (Rattus Rattus; 94%; Accession No. NP_808789), mouse (94%; Accession No. NP_067499), common carp (Cyprinus carpio; 89%; Accession No. BAA96290), and zebrafish (Danio rerio; 89%; Accession No. NM_212668) but also shares considerable degree of sequence identity with the Nampt of sponge (Suberites domuncula; 58%; Accession No.: CAB65409) and cyanobacterium (Synechocystis sp. PCC 6803; 46%; Accession No. NP_442623). Consistent with its function as Nampt to catalyze the reaction between NAM and PRPP yielding in NMN
synthesis, the amino acid residues of Nampt known to be critical for nicotinamide ring binding (Asp17, Tyr19, Phe194, Asp220, and Arg312) and ribose binding (Asp314, Gly354, Asp355, and Gly385) are fully conserved among visfatin proteins from chicken and other species (Figure 2; Kim et al., 2006; Burgos et al., 2009). In addition, a conserved triad catalysis site (Ser281-His248-Asp314) was also identified in chicken visfatin (Figure 2; Wang et al., 2006).
Visfatin Gene Expression in Chicken Tissues In this study, RT-PCR and Northern blot analysis were employed to examine the visfatin mRNA expression in adult chicken tissues including brain, heart, intestine, kidney, liver, lung, muscle, ovary, pituitary, spleen, testes, and pancreas. As shown in Figure 3A, strong hybridization signals were detected in nearly all chicken tissues examined except pancreas by Northernblot analysis. Three mRNA species could be easily de-
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Figure 3. Expression of visfatin gene in 12 adult chicken tissues. A) Reverse-transcription PCR detection of visfatin mRNA expression in adult chicken tissues. Numbers in brackets indicate the PCR cycles used. B) Northern-blot analysis of visfatin mRNA expression in adult chicken tissues. Twenty micrograms of total RNA from each tissue was hybridized with Dig-labeled antisense probe. Arrows indicate the 3 major mRNA species identified in chicken tissues, and arrow heads indicate the location of 28s and 18s rRNA.
tected in chicken tissues. Among them, the 2 larger mRNA species of sizes close to that of 28S rRNA could be detected in most chicken tissues examined, while the smallest mRNA species with a size similar to that of 18S rRNA seemed to be abundantly expressed in several tissues including spleen, muscle, and small intestine. The generation of multiple visfatin mRNA species is likely due to usage of the alternative polyadenylation signal(s) located within the long 3′-UTR (2,766 bp; Accession No.: JQ354940) identified in this study (Supplemental Figure S2; available online at ps.fass.org). Using a more sensitive RT-PCR assay, visfatin expression was consistently detected in all chicken tissues examined including pancreas, further confirming the ubiquitous expression of visfatin gene in chicken tissues.
Sequence Analyses of the 5′-Flanking Region of Chicken Visfatin Gene According to the sequence information from chicken genome database, the 5′-flanking region of chicken visfatin gene was cloned (Figure 4). The amplified 5′-flanking region is 1,372 bp in length and GC-rich (60% GC content). Using online software [Genomatix MatInspector (http://www.genomatix.de), TFSEARCH (http://
www.cbrc.jp/research/db/TFSEARCH.html), and TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess)], the putative transcription factor binding sites were predicted. Within the cloned 5′-flanking region, 6 putative Sp1 binding sites (GC-boxes) were predicted to be located near the translation start codon (ATG). In addition, several putative binding sites for various transcription factors, such as AP-1, AP-2, AP-4, NF-kB, NF-IL6, CREB, and glucocorticoid receptor (GR) were also found (Figure 4).
Promoter Activity of Chicken Visfatin Gene To investigate whether the 5′-flanking region of chicken visfatin gene displays promoter activity, we further subcloned the 5′-flanking region (1,372 bp) into the promoterless pGL3-basic vector and examined its promoter activity in cultured HEK293, CHO, and DF-1 cells. As shown in Figure 5, the cloned 1,372-bp fragment consistently displayed strong promoter activities in all 3 cell lines, suggesting that it is involved in controlling visfatin gene transcription in different chicken tissues. The strongest promoter activity of this fragment was detected in cultured chicken DF-1 cells (~250-fold increase), an embryonic fibroblast cell line. In contrast, the relatively
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Figure 4. Nucleotide sequence of the 5′-flanking region of chicken visfatin gene (1,372 bp). The putative binding sites (shaded or underlined) for the transcriptional factors: Sp1, AP-1, AP-2, AP-4, GATA-1, MyoD, NF-kB, glucocorticoid receptor (GR), cAMP response element binding protein (CREB), and the nuclear factor NF-IL6 have been predicted. The first nucleotide ‘C’ upstream translation start codon is designated as ‘-1’.
lower promoter activities were detected in HEK293 cells (90-fold increase) and CHO cells (16-fold increase). Using a promoter deletion approach, we also noted that the removal of the 5′-portion (from −1,372 to −180) of this 1,372-bp 5′-UTR region could significantly reduce its promoter activity when tested in the 3 chicken cell lines. Because the construct (−179/-1 Luc) displayed the promoter activity in the 3 cell lines, it is likely that a promoter may be located near the putative translation start codon (ATG). Interestingly, the constructs (−1,372/-179 Luc and −1,372/-297 Luc) without the first 179-bp region from the translation start codon still showed strong promoter activities in the cell lines, particularly in DF-1 cells (90-fold and 120-fold increase, respectively, in comparison with pGL3-basic vector). It strongly suggests that alternative promoter(s) distal to the translational start site may be present within the region between −1,372 and −297.
Visfatin mRNA Expression in Chicken Adipose Tissues The quantity of total RNA extracted from chicken adipose tissue was not enough for Northern-blot analysis; therefore, quantitative real-time PCR assay was employed to examine the visfatin mRNA expression in adipose tissues. As shown in Figure 6, the visfatin mRNA levels showed no significant difference between chicken subcutaneous and visceral fat tissues at
the same developmental stage (3 wk and adult stage). Moreover, no significant difference in visfatin mRNA levels was noted among different stages of subcutaneous fat tissues or visceral fat tissues.
DISCUSSION In the present study, the full-length cDNA of visfatin gene has been cloned from chicken liver. The RT-PCR and Northern-blot analysis clearly demonstrated that it was widely expressed in all adult chicken tissues examined. Promoter analysis suggested that visfatin gene transcription was likely controlled by multiple promoters located upstream of the translation start codon. To our knowledge, this study represents the first to identify a functional promoter region(s) of visfatin gene in avian species. The cloned full-length cDNA of chicken visfatin gene encodes a protein of 493 amino acids. Sequence analyses revealed that the chicken visfatin protein not only shares high amino acid sequence identity (89–94%) to visfatin from human, rat, mouse, common carp, and zebrafish but also shares considerable degree of sequence identity with the Nampt of sponge and cyanobacterium. Although visfatin has been viewed as an adipokine or pre-B cell enhancing factor, the amino acid residues responsible for NAM and ribose binding are fully conserved between chicken and other species (Kim et al., 2006; Burgos et al., 2009). In addition, the catalytic
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Figure 5. Promoter activities of the 5′-flanking region of chicken visfatin gene in Chinese hamster ovary (CHO) (A), chicken embryo fibroblast (DF-1) (B), and human embryo kidney (HEK293) (C) cell lines. The constructs p(−1372/−1)-Luc, p(−1372/−297)-Luc, p(−1372/−179)-Luc, p(−720/−1)-Luc, p(−310/−1)-Luc, and p(−179/−1)-Luc were transfected to CHO, DF-1, and HEK293 cells together with pRL-TK vector. The later vector was used to normalize the transfection efficiency. A promoterless pGL3-Basic vector (pGL3) was included in each experiment. The relative promoter activity of each construct is expressed as the fold increase over the pGL3-Basic vector, whose activity is set at 1, after being normalized to pRL-TK activity. The ATG in (A), (B), and (C) indicates the translation start codon of the visfatin gene. Each data point represents the mean ± SEM of 3 replicas. **P < 0.001 versus respective pGL3-Basic vector.
triad site has been found to also be conserved in putative chicken visfatin protein (Wang et al., 2006). These conserved residues and motifs strongly support that chicken visfatin may function as a cytosolic enzyme (Nampt) to catalyze the condensation of NAM with PRPP to yield NMN.
Like the visfatin from other species, chicken visfatin also lacks a typical N-terminal signal peptide sequence. Several studies demonstrated that visfatin protein could be detected in chicken serum as reported in mammals (Krzysik-Walker et al., 2008; Ocón-Grove et al., 2010), implying that a nonclassical pathway may be
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responsible for visfatin secretion (Tanaka et al., 2007; Krzysik-Walker et al., 2008; Ocón-Grove et al., 2010). In the present study, strong hybridization signals of visfatin mRNA were detected in nearly all chicken tissues except pancreas by Northern-blot analysis, indicating the wide expression of chicken visfatin gene. This finding is consistent with the report from Ons et al. (2010). However, in contrast to their finding that only a single mRNA species of ~2.4 kb was detected in all tissues examined by Northern-blot analysis, 3 mRNA species were detected by Northern-blot analysis in the present study, suggesting the existence of multiple chicken visfatin mRNA species. As shown in Figure 3B, the 2 strong hybridization signals detected in all tissues were of sizes close to that of 28S rRNA, suggesting that the sizes of the 2 mRNA species are much larger than 2.4 kb as previously reported (Ognjanovic et al., 2001). Our finding was also supported by the fact that chicken visfatin gene contains an open reading frame of 1,482 bp and a long 3′-UTR of 2,766 bp. Moreover, in silico assembly of chicken visfatin EST retrieved from EST database (http://www.chick.manchester.ac.uk/) can fully cover the open reading frame and the long 3′-UTR we identified, also implying the existence of transcript(s) much longer than 2.4 kb. In spite of the discrepancy in Northern-blot results between the 2 studies, RT-PCR results from present study and Ons’ study clearly indicated that visfatin mRNA is widely distributed in adult chicken tissues. It strongly suggests the involvement of visfatin in a variety of physiological processes in chicken (Garten et al., 2009; Gallí et al., 2010). In the present study, visfatin mRNA expression was detected in chicken visceral and subcutaneous fat tissues, however, no significant difference in visfatin mRNA levels was noted either between the 2 types of adipose tissues or between the 2 developmental stag-
Figure 6. Detection of visfatin mRNA expression in chicken adipose tissues by quantitative real-time PCR. The mRNA levels of visfatin were first normalized by β-actin and then expressed as percentage of subcutaneous fat tissue at embryonic d 15 (Em-SF). Em-SF: embryo; 3w-SF: 3-wk-old chicken subcutaneous fat; 3w-VF: 3-wk-old chicken visceral fat; Ad-SF: adult chicken subcutaneous fat; Ad-VF: adult chicken visceral fat.
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es of the same type of adipose tissues. Our finding is in line with the report from Krzysik-Walker et al., in which visfatin mRNA and protein quantities both show no relationship with subcutanteous fat tissue and visceral fat accumulation (Krzysik-Walker et al., 2008). Therefore, the question whether visfatin can function as an adipokine in chickens remains to be clarified. As an initial step to uncover the transcriptional control of visfatin gene expression in chickens, the 5′-flanking region (1,372 bp) upstream of the start codon (ATG) was cloned in present study. Promoter analysis demonstrated that it displayed strong promoter activity in all 3 cell lines tested, suggesting its importance in regulating visfatin gene transcription in chickens. Because the removal of either the 5′-portion (from −1,372 to −311) or 3′-portion (from −296 to −1) of this 1,372-bp 5′-UTR region could significantly, yet partially, reduce the promoter activities, it strongly suggests that multiple promoters both proximal and distal to the translational start codon (ATG) are likely present herein. In agreement with this postulation, the transcript(s) with a long 5′-UTR of 314 bp (accession No.: JQ406639) was detected in chicken liver using RTPCR in this study, further confirming the existence of the alternative promoter(s) distal to the start codon. Whether additional promoter(s) far from this 1,372-bp region of visfatin gene exist awaits further studies. As shown in Figure 4, the proximal promoter region (from −310 to −1) is GC-rich (60% GC content) and no typical TATA box was found. However, multiple Sp1 binding sites were predicted to be located within this region, suggesting that Sp1 may be responsible for basal expression of chicken visfatin gene, as reported in genes with a TATA-less promoter (Samson and Wong, 2002). In addition to Sp1-binding sites, putative binding sites for other transcription factors, such as AP-1, AP-2, AP-4, CREB, NF-kB, NF-IL6, GATA-1 MyoD, and GR were also found within the 5′-flanking region of 1,372-bp 5′-UTR region, whether they are functional cis-regulatory elements and can confer to the tissue-or cell-specific expression of visfatin gene remains to be clarified. In summary, chicken visfatin gene was cloned in present study. Sequence analyses revealed that chicken putative visfatin protein shares high amino acid sequence identity with their homologs in other species including distant species such as cyanobacterium. Northern blot and RT-PCR assays demonstrated that visfatin was widely expressed in chicken tissues. In addition, quantitative real-time PCR showed no significant difference in visfatin mRNA expression between the visceral and subcutaneous fat tissues of the same developmental stage. In combination with the result from promoter analysis that chicken visfatin gene transcription is likely controlled by multiple promoters upstream of the translation start codon, our findings would help to elucidate the expression and physiological roles of chicken visfatin gene.
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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China for Juan Li (30700452, 30971569) and the grant for undergraduate student training (J1103518).
REFERENCES Berndt, J., N. Kloting, S. Kralisch, P. Kovacs, M. Fasshauer, M. R. Schon, M. Stumvoll, and M. Bluher. 2005. Plasma visfatin concentrations and fat depot-specific mRNA expression in humans. Diabetes 54:2911–2916. Brown, J. E., D. J. Onyango, M. Ramanjaneya, A. C. Conner, S. T. Patel, S. J. Dunmore, and H. S. Randeva. 2010. Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic beta-cells. J. Mol. Endocrinol. 44:171–178. Burgos, E. S., M. C. Ho, S. C. Almo, and V. L. Schramm. 2009. A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT. Proc. Natl. Acad. Sci. USA 106:13748–13753. Chen, H., T. Xia, L. Zhou, X. Chen, L. Gan, W. Yao, Y. Peng, and Z. Yang. 2007. Gene organization, alternate splicing and expression pattern of porcine visfatin gene. Domest. Anim. Endocrinol. 32:235–245. Cline, M. A., W. Nandar, B. C. Prall, C. N. Bowden, and D. M. Denbow. 2008. Central visfatin causes orexigenic effects in chicks. Behav. Brain Res. 186:293–297. Gallí, M., F. Van Gool, A. Rongvaux, F. Andris, and O. Leo. 2010. The nicotinamide phosphoribosyltransferase: A molecular link between metabolism, inflammation, and cancer. Cancer Res. 70:8–11. Garten, A., S. Petzold, A. Korner, S. Imai, and W. Kiess. 2009. Nampt: Linking NAD biology, metabolism and cancer. Trends Endocrinol. Metab. 20:130–138. Imai, S. 2009. Nicotinamide phosphoribosyltransferase (Nampt): A link between NAD biology, metabolism, and diseases. Curr. Pharm. Des. 15:20–28. Kim, M. K., J. H. Lee, H. Kim, S. J. Park, S. H. Kim, G. B. Kang, Y. S. Lee, J. B. Kim, K. K. Kim, S. W. Suh, and S. H. Eom. 2006. Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866. J. Mol. Biol. 362:66–77. Klöting, N., and I. Kloting. 2005. Visfatin: Gene expression in isolated adipocytes and sequence analysis in obese WOKW rats compared with lean control rats. Biochem. Biophys. Res. Commun. 332:1070–1072. Krzysik-Walker, S. M., O. M. Ocon-Grove, S. R. Maddineni, G. L. Hendricks 3rd, and R. Ramachandran. 2008. Is visfatin an adipokine or myokine? Evidence for greater visfatin expression in skeletal muscle than visceral fat in chickens. Endocrinology 149:1543–1550. Li, J., Y. Wang, and F. C. Leung. 2005. Cloning of chicken pre-Bcell colony-enhancing factor (PBEF1) and characterization of its spatial expression. Poult. Sci. 84(Suppl. 1):74. Martin, P. R., R. J. Shea, and M. H. Mulks. 2001. Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence. J. Bacteriol. 183:1168–1174. McGlothlin, J. R., L. Gao, T. Lavoie, B. A. Simon, R. B. Easley, S. F. Ma, B. B. Rumala, J. G. Garcia, and S. Q. Ye. 2005. Molecular cloning and characterization of canine pre-B-cell colonyenhancing factor. Biochem. Genet. 43:127–141.
Ocón-Grove, O. M., S. M. Krzysik-Walker, S. R. Maddineni, G. L. Hendricks 3rd, and R. Ramachandran. 2010. NAMPT (visfatin) in the chicken testis: Influence of sexual maturation on cellular localization, plasma levels and gene and protein expression. Reproduction 139:217–226. Ocón-Grove, O. M., S. M. Krzysik-Walker, S. R. Maddineni, G. L. Hendricks 3rd, and R. Ramachandran. 2010. NAMPT (visfatin) in the chicken testis: Influence of sexual maturation on cellular localization, plasma levels and gene and protein expression. Reproduction 139:217–226. Ognjanovic, S., S. Bao, S. Y. Yamamoto, J. Garibay-Tupas, B. Samal, and G. D. Bryant-Greenwood. 2001. Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes. J. Mol. Endocrinol. 26:107–117. Ognjanovic, S., and G. D. Bryant-Greenwood. 2002. Pre-B-cell colony-enhancing factor, a novel cytokine of human fetal membranes. Am. J. Obstet. Gynecol. 187:1051–1058. Ons, E., A. Gertler, J. Buyse, E. Lebihan-Duval, A. Bordas, B. Goddeeris, and S. Dridi. 2010. Visfatin gene expression in chickens is sex and tissue dependent. Domest. Anim. Endocrinol. 38:63–74. Revollo, J. R., A. A. Grimm, and S. Imai. 2004. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol. Chem. 279:50754–50763. Revollo, J. R., A. Korner, K. F. Mills, A. Satoh, T. Wang, A. Garten, B. Dasgupta, Y. Sasaki, C. Wolberger, R. R. Townsend, J. Milbrandt, W. Kiess, and S. Imai. 2007. Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme. Cell Metab. 6:363–375. Rongvaux, A., R. J. Shea, M. H. Mulks, D. Gigot, J. Urbain, O. Leo, and F. Andris. 2002. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis. Eur. J. Immunol. 32:3225–3234. Samal, B., Y. Sun, G. Stearns, C. Xie, S. Suggs, and I. McNiece. 1994. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol. Cell. Biol. 14:1431–1437. Samson, S. L., and N. C. Wong. 2002. Role of Sp1 in insulin regulation of gene expression. J. Mol. Endocrinol. 29:265–279. Tanaka, M., M. Nozaki, A. Fukuhara, K. Segawa, N. Aoki, M. Matsuda, R. Komuro, and I. Shimomura. 2007. Visfatin is released from 3T3–L1 adipocytes via a non-classical pathway. Biochem. Biophys. Res. Commun. 359:194–201. Wang, J., Y. Wang, X. Li, J. Li, and F. C. Leung. 2008. Cloning, tissue distribution, and functional characterization of chicken glucagon receptor. Poult. Sci. 87:2678–2688. Wang, T., X. Zhang, P. Bheda, J. R. Revollo, S. Imai, and C. Wolberger. 2006. Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme. Nat. Struct. Mol. Biol. 13:661–662. Wang, Y., J. Li, A. H. Yan Kwok, W. Ge, and F. C. Leung. 2010. A novel prolactin-like protein (PRL-L) gene in chickens and zebrafish: Cloning and characterization of its tissue expression. Gen. Comp. Endocrinol. 166:200–210. Wang, Y., C. Y. Wang, Y. Wu, G. Huang, J. Li, and F. C. Leung. 2012. Identification of the receptors for prolactin-releasing peptide (PrRP) and Carassius RFamide peptide (C-RFa) in chickens. Endocrinology 153:1861–1874. Ye, S. Q., L. Q. Zhang, D. Adyshev, P. V. Usatyuk, A. N. Garcia, T. L. Lavoie, A. D. Verin, V. Natarajan, and J. G. Garcia. 2005. Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation. Microvasc. Res. 70:142–151.
Using a dual luciferase reporter system, we further demonstrated that the cloned 1,372-bp fragment upstream of the putative translation start site (ATG) displayed the maximal promoter activity in cultured CHO, DF-1, and HEK293 cells, whereas the removal of its 5′-region (1,075 bp) or 3′-region (297 bp) could only partially reduce its promoter activity, implying that visfatin gene transcription was likely controlled by multiple promoters near the translation start site. Taken together, results from present study will contribute to our better understanding of the expression and roles of visfatin gene in chickens.
Key words: chicken, visfatin, pre-B cell enhancing factor, Nampt, promoter analysis 2012 Poultry Science 91:2885–2894 http://dx.doi.org/10.3382/ps.2012-02315
INTRODUCTION It is generally believed that the adipose tissues mainly function as an energy reservoir. However, increasing evidences support that adipose tissue may also serve as an endocrine organ by secreting many adipokines involved in multiple physiological processes and pathophysiology of obesity-related metabolic disorders. Visfatin, a novel potential adipokine, has drawn attention since its discovery due to its insulin-mimetric action. Originally, visfatin was reported to be preferentially expressed in visceral fat and its concentrations in plasma were highly correlated with mouse visceral fat mass. Moreover, this protein has been shown to be capable of binding to insulin receptor and mimicking insulin-like activity in lowering of plasma glucose levels in mice (Garten et al., 2009; Imai, 2009). Since then, visfatin has been investigated extensively in various animal models (Klöting and Kloting, 2005; McGlothlin et al., 2005; Chen et al., 2007). However, many contradictory findings have been reported and thus questioned its identity as a novel adipokine. For instance, Berndt and his coworkers ex©2012 Poultry Science Association Inc. Received March 18, 2012. Accepted July 17, 2012. 1 Corresponding authors: [email protected] and fcleung@hkucc. hku.hk
amined 187 subjects with obesity and found that there was no differential expression of visfatin gene between visceral and subcutaneous adipose tissues. In addition, in a subgroup of 73 individuals, in which visceral fat mass was calculated from CT scans, no correlation was detected between visceral fat mass and plasma visfatin concentrations (Berndt et al., 2005). Visfatin is initially identified from activated lymophocytes and named as pre-B cell enhancing factor (PBEF) owing to its synergistic role with interleukin-7 (IL-7) and stem cell factor (SCF) in promoting preB cell colony formation in vitro (Samal et al., 1994). The expression of PBEF can be induced by lectin and cycloheximide, and PBEF protein is detected in the culture medium of activated lymphocytes, supporting that it may also function as a cytokine (Ognjanovic and Bryant-Greenwood, 2002; Ye et al., 2005). In addition to being viewed as an adipokine or cytokine, visfatin was also reported to function as a cytosolic enzyme. Mouse visfatin/PBEF was found to show remarkable degree of amino acid sequence identity with a bacterial enzyme encoded by a NadV gene. The enzyme catalyzes the conversion of nicotinamide (NAM) to nicotinamide mononucleotide (NMN), which represents the rate-limiting step in NAD biosynthesis (Martin et al., 2001). Further studies confirmed that it could function as a nicotinamide phosphoribosyltransferase
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(Nampt) and catalyze the condensation of NAM with 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield NMN, an intermediate substrate for NAD biosynthesis (Rongvaux et al., 2002). Thus, visfatin/PBEF is also named as Nampt. To date, the biological function of visfatin/PBEF/ Nampt remains controversial. Biochemical, structural and physiological studies strongly support that Nampt is the rating-limiting enzyme for NAD biosynthesis which plays critical roles in a wide variety of physiological processes including metabolism, stress response, aging, and cell differentiation in mammals (Revollo et al., 2004; Wang et al., 2006; Imai, 2009). For instance, Revollo et al. (2007) reported that Nampt/visfatin/ PBEF could regulate glucose-stimulated insulin secretion in pancreatic β-cells and this action is likely attributed to Nampt-mediated NAD biosynthesis because the administration of NMN, a product of the Nampt enzymatic reaction, could effectively rescue the defective glucose-stimulated insulin secretion observed in Nampt+/− mice (Revollo et al., 2007; Brown et al., 2010). Becuase NAD is involved in various physiological processes, Nampt being a potential key rate-limiting enzyme for NAD synthesis is therefore viewed as a crucial link between NAD metabolism and diseases (Garten et al., 2009; Gallí et al., 2010). Although the roles of visfatin/PBEF/Nampt have been extensively investigated in mammals, it remains largely unknown in nonmammalian vertebrates including birds. In chickens, there is evidence showing that visfatin is expressed in various tissues and it is likely involved in the regulation of muscle growth and metabolism (Krzysik-Walker et al., 2008), food intake, and testicular functions (Cline et al., 2008; Ocón-Grove et al., 2010). However, the mechanisms underlying its action and the transcriptional regulation on its expression remain unclear in chickens. In this study, using chicken as an experimental model, we cloned the full-length cDNA of visfatin gene and reported its tissue expression pattern (Li et al., 2005). Moreover, the 5′-flanking region of this gene was also cloned and characterized. Our present study would contribute to our better understanding of the expression and roles of visfatin gene in avian species.
MATERIALS AND METHODS Chemicals and Reagents All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and restriction enzymes were obtained from Amersham Biosciences (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) unless stated otherwise.
Total RNA Extraction Adult chickens were killed and different tissues including small intestine, heart, kidney, liver, lung, muscle, ovary, pituitary, spleen, pancreas, testis, and whole
brain were collected for total RNA extraction. Total RNA was extracted from chicken tissues with Trireagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions and dissolved in DEPC-treated H2O. The experiments were carried out according to the guidelines provided by the Animal Ethics Committee of Sichuan University and the University of Hong Kong.
Cloning of the Full-Length cDNA for Chicken Visfatin Gene To obtain the full-length cDNA of chicken visfatin, 2 gene-specific primers flanking the start and stop codons were designed (Table 1) based on the information from chicken genome database (http://www.emsembl.org/Gallus_gallus) and EST sequences deposited in GenBank (Accession No.: BU288000, BU280677, BU135629, and BU362525). The full-length cDNA containing an open reading frame of visfatin were amplified from adult chicken liver using high-fidelity Taq DNA polymerase (Roche diagnostics, Basel, Switzerland). The PCR products were cloned into pBluescript II SK (+/−; Stratagene, La Jolla, CA). The full-length cDNA were finally determined by sequencing (ABI PRISM 3100 Genetic Analyzer, Perkin Elmer, Norwalk, CT) at least 3 independent clones containing whole open reading frames.
Reverse Transcription and PCR Reverse transcription (RT) was performed at 42°C for 2 h in a total volume of 10 μL consisting of 2 μg total RNA from different tissues, 1× single-strand buffer, 0.5 mM each deoxynucleotide triphosphate, 0.5 μg oligo-deoxythymidine, and 100 U MMLV reverse transcriptase (Promega, Madison, WI). All negative controls were carried out under the same condition without reverse transcriptase added in the 10-μL reaction mix. One microliter of the first-strand cDNA was used as the template for each PCR reaction. According to our previously established methods (Wang et al., 2008), RT-PCR assays were employed to examine the expression of visfatin in chicken tissues. The PCR was performed under the following conditions: 2 min at 95°C for denaturation, followed by 23 cycles (for β-actin: 30 s at 95°C, 30 s at 58°C, and 60 s at 72°C) and 25 (or 33) cycles (for visfatin: 30 s at 95°C, 30 s at 58°C, 60 s at 72°C) of reaction, ending with a 5-min extension at 72°C. The primers used for visfatin gene and β-actin gene amplification were listed (Table 1). The PCR products were visualized on a UV-transilluminator (Bio-Rad Laboratories Inc., Hercules, CA) after electrophoresis on 2% agarose gel containing ethidium bromide. The identity of PCR product was further confirmed by sequencing.
Northern-Blot Analysis Northern-blot analysis was performed to examine the tissue distribution of chicken visfatin gene. Twenty
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Table 1. Primers used in present study Primer name/construct Primers for full-length cDNA amplification PBU1 PBL1 Primers for the 5′-flanking region amplification pPBU1 pPBL1 Primers for reverse-transcription PCR and realtime PCR assay PBU2 PBL2 β-actin Primers for preparing the promoter constructs p(−1372/−1)Luc p(−1372/−297)Luc p(−1372/−197)Luc p(−720/−1)Luc p(−310/−1)Luc p(−179/−1)Luc
Sense/antisense
Primer sequence (5′–3′)
Sense Antisense
5′-CAGCGCAGCCCGGTCCTGT-3′ 5′-TTAGTGAGACGCCGTTTCAT-3′
Sense Antisense
5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′
Sense Antisense Sense Antisense
5′-ACCAGATTCTGGGAATCCCCTT-3′ 5′-TCTCCAGCAGGTGTCCTATGCA-3′ 5′-CAATGGCTCCGGTATGTGCA-3′ 5′-AGGCATACAGGGACAGCACA-3′
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sence Antisense
5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTGTGACACCGCGAGTGC-3′ 5′-GAGCTCCCAGTCTGCCACGTAA-3′ 5′-AAGCTTCTGCGCGCCGATTG-3′ 5′-GAGCTCGGATCCACGCACCT-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCGCGGTGTCACACCG-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′ 5′-GAGCTCGGCGCGCAGAAGCCGA-3′ 5′-AAGCTTGCCGCCAAGCCCCGAGCA-3′
micrograms total RNA was separated in a 1.0% agarose gel containing 2.2 M formaldehyde. Gels were then transferred to Hybond nylon membranes (Amersham Pharmacia Biotech, Baie d’Urfe, QC, Canada). After UV crosslinking (Amersham Pharmacia Biotech), membranes were prehybridized for 2 h at 68°C in solution I (40% formamide, 5xSSC, 50 mM HEPES, pH 6.8, 5 mM EDTA, 2× Denhardt’s, 0.1% SDS, and 100 μg/mL salmon sperm DNA) and then hybridized in the same solution overnight at 68°C with digoxigeninlabeled antisense RNA probe prepared from the cloned cDNA by in vitro transcription (Promega). Membranes were washed and detected with chemiluminescent detection kit according to the manufacturer’s instructions (Roche Diagnostics).
Quantitative Measurements of Visfatin mRNA Expression in Chicken Fat Tissues In the present study, the subcutaneous fat tissues from chicken embryo (d 15), subcutaneous and visceral fat tissues from 3-wk-old and adult chickens were collected to evaluate the mRNA levels of visfatin gene. Quantitative real-time PCR was performed in a 20-μL reaction mix consisting of 300 nM forward primer, 300 nM reverse primer, 5 μL cDNA sample, 0.25 μL of Taq DNA polymerase (Invitrogen), and 1 μL EvaGreen (Biotium Inc., Hayward, CA). The PCR conditions were 2 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 30 s at 95°C, 30 s at 60°C, and 60 s at 72°C. The PCR amplification was performed on an iCycler iQ real-time PCR machine (Bio-Rad Laboratories). To confirm the specificity of the PCR reaction, melting curve analyses were performed at the end of PCR and the identity of PCR product was further confirmed by
Size (bp) 1,538 1,372 384 401 1,372 1,075 1,175 720 310 180
sequencing. All real-time PCR amplifications were performed in triplicate. The standard curves were established using serially diluted plasmids with known copy number as templates to calculate the copy numbers of visfatin and actin in chicken tissues.
Isolation of 5′-Flanking Region of Chicken Visfatin Gene The 5′-flanking region of chicken visfatin gene has been cloned in an effort to reveal its transcriptional regulatory mechanism. Based on the chicken genome database, the full-length of chicken visfatin cDNA sequence was aligned with chicken genome sequence (www.ensembl.org/Gallus_gallus/). Sense and antisense primers (Table 1) were then designed to amplify the 5′-flanking region of chicken visfatin gene using chicken genomic DNA as template. The PCR reactions were performed as follows: 2 min at 95°C for denaturation, followed by 30 cycles of 20 s at 94°C, 10 s at 58°C, 1 min 30 s at 72°C, and ending with 7 min extension at 72°C. The amplified PCR products were then subcloned into pBluescript SK (+/−) and subjected for sequence analyses.
Promoter Analysis of Chicken Visfatin Gene in Cultured Cells To test whether the 5′-flanking region of visfatin gene upstream the translation start site could display promoter activity, a set of constructs containing the 5′-flanking region of different lengths were prepared using PCR method. The primers for each construct were listed in Table 1. The amplified PCR products were first cloned into pBluescript SK (+/−) for sequence
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Figure 1. A) Nucleotide and deduced amino acid sequences of chicken visfatin gene (AY946242). The 2 putative N-linked glycosylation sites are boxed. B) Gene structure of chicken visfatin gene. Eleven exons are labeled accordingly (number in box) and the number in italic indicates the size of each exon (bp). The putative coding region of chicken visfatin is shaded. Multiple promoters (arrow indicated) responsible for visfatin gene transcription may exist in the 5′-flanking region based on the promoter analyses performed in present study.
confirmation and then subcloned into pGL3-Basic vector (Promega). The cell lines used in this experiment include a Chinese hamster ovary (CHO) cell line, a chicken embryo fibroblast cell line (DF-1), and a human embryo kidney cell line (HEK 293), which were cultured under conditions as described in our previous studies (Wang et al., 2010, 2012). For transfection, 1 × 105 cells were plated on each 48-well plate. A mixture containing 100 ng of promoter-luciferase construct, 5 ng of pRL-TK (Promega), and 1.5 μL of Lipofectamine (Invitrogen) was prepared in 20 μL of PBS. Following the manufacture’s instruction, luciferase activity of each promoter construct in the cultured cells was normalized to Renilla luciferase activity derived from the pRL-TK vector and then expressed as relative fold increase as compared with the control group (pGL3-basic vector). The data were analyzed by one-way ANOVA followed by Dunnett’s test using GraphPad Prism 4 (GraphPad Software, San Diego, CA). To validate our results, all experiments were repeated at least 2 times.
RESULTS Cloning Full-Length cDNA of Chicken Visfatin Gene Based on the related genome and EST sequences from chicken genome database, gene-specific primers were designed to amplify the full-length cDNA of chicken visfatin gene from chicken liver. The cloned full-length visfatin cDNA is 1,538 bp in length, with an open reading frame of 1,482 bp (accession No. AY946242; Figure 1). In addition, a 5′-untranslated region (5′-UTR) region of 314 bp (JQ406639) and a long 3′-UTR of 2,766 bp (JQ354940) were also obtained by RT-PCR and confirmed by sequencing in present study (Supplemental Figures S1 and S2; available online at ps.fass.org). Comparison of the cloned cDNA to chicken genome database revealed that the chicken visfatin gene spans across ~29 kb on chromosome 1 and consists of 11 exons and 10 introns (Figure 1). The putative chicken visfatin is 493 amino acids in length with a predicted
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Figure 2. Amino acid sequence alignment of chicken visfatin and that of human (NP_005737), rat (NP_808789), mouse (NP_067499), carp (BAA96290), zebrafish (NM_212668), sponge (Suberites domuncula; CAB65409), and cyanobacterium (Synechocystis sp. PCC 6803; NP_442623). The conserved amino acids responsible for nicotinamide binding (arrow heads) and ribose binding (boxed residues) have been shown respectively. The triangles (▼) denote the conserved amino acids responsible for enzyme catalysis.
molecular weight of 55 kDa. Two putative N-linked glycosylation sites (N-X-S, where X represents any amino acids except proline) were found at its N- and Cterminal respectively. Sequence analyses revealed that the N-terminal of the predicted visfatin lacks the typical signal peptide sequence, which is hydrophobic and characteristic for most secreted peptides. As shown in Figure 2, the putative chicken visfatin protein not only shares high amino acid sequence identity with that of human (Homo sapiens; 94%; Accession No.: NP_005737), rat (Rattus Rattus; 94%; Accession No. NP_808789), mouse (94%; Accession No. NP_067499), common carp (Cyprinus carpio; 89%; Accession No. BAA96290), and zebrafish (Danio rerio; 89%; Accession No. NM_212668) but also shares considerable degree of sequence identity with the Nampt of sponge (Suberites domuncula; 58%; Accession No.: CAB65409) and cyanobacterium (Synechocystis sp. PCC 6803; 46%; Accession No. NP_442623). Consistent with its function as Nampt to catalyze the reaction between NAM and PRPP yielding in NMN
synthesis, the amino acid residues of Nampt known to be critical for nicotinamide ring binding (Asp17, Tyr19, Phe194, Asp220, and Arg312) and ribose binding (Asp314, Gly354, Asp355, and Gly385) are fully conserved among visfatin proteins from chicken and other species (Figure 2; Kim et al., 2006; Burgos et al., 2009). In addition, a conserved triad catalysis site (Ser281-His248-Asp314) was also identified in chicken visfatin (Figure 2; Wang et al., 2006).
Visfatin Gene Expression in Chicken Tissues In this study, RT-PCR and Northern blot analysis were employed to examine the visfatin mRNA expression in adult chicken tissues including brain, heart, intestine, kidney, liver, lung, muscle, ovary, pituitary, spleen, testes, and pancreas. As shown in Figure 3A, strong hybridization signals were detected in nearly all chicken tissues examined except pancreas by Northernblot analysis. Three mRNA species could be easily de-
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Figure 3. Expression of visfatin gene in 12 adult chicken tissues. A) Reverse-transcription PCR detection of visfatin mRNA expression in adult chicken tissues. Numbers in brackets indicate the PCR cycles used. B) Northern-blot analysis of visfatin mRNA expression in adult chicken tissues. Twenty micrograms of total RNA from each tissue was hybridized with Dig-labeled antisense probe. Arrows indicate the 3 major mRNA species identified in chicken tissues, and arrow heads indicate the location of 28s and 18s rRNA.
tected in chicken tissues. Among them, the 2 larger mRNA species of sizes close to that of 28S rRNA could be detected in most chicken tissues examined, while the smallest mRNA species with a size similar to that of 18S rRNA seemed to be abundantly expressed in several tissues including spleen, muscle, and small intestine. The generation of multiple visfatin mRNA species is likely due to usage of the alternative polyadenylation signal(s) located within the long 3′-UTR (2,766 bp; Accession No.: JQ354940) identified in this study (Supplemental Figure S2; available online at ps.fass.org). Using a more sensitive RT-PCR assay, visfatin expression was consistently detected in all chicken tissues examined including pancreas, further confirming the ubiquitous expression of visfatin gene in chicken tissues.
Sequence Analyses of the 5′-Flanking Region of Chicken Visfatin Gene According to the sequence information from chicken genome database, the 5′-flanking region of chicken visfatin gene was cloned (Figure 4). The amplified 5′-flanking region is 1,372 bp in length and GC-rich (60% GC content). Using online software [Genomatix MatInspector (http://www.genomatix.de), TFSEARCH (http://
www.cbrc.jp/research/db/TFSEARCH.html), and TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess)], the putative transcription factor binding sites were predicted. Within the cloned 5′-flanking region, 6 putative Sp1 binding sites (GC-boxes) were predicted to be located near the translation start codon (ATG). In addition, several putative binding sites for various transcription factors, such as AP-1, AP-2, AP-4, NF-kB, NF-IL6, CREB, and glucocorticoid receptor (GR) were also found (Figure 4).
Promoter Activity of Chicken Visfatin Gene To investigate whether the 5′-flanking region of chicken visfatin gene displays promoter activity, we further subcloned the 5′-flanking region (1,372 bp) into the promoterless pGL3-basic vector and examined its promoter activity in cultured HEK293, CHO, and DF-1 cells. As shown in Figure 5, the cloned 1,372-bp fragment consistently displayed strong promoter activities in all 3 cell lines, suggesting that it is involved in controlling visfatin gene transcription in different chicken tissues. The strongest promoter activity of this fragment was detected in cultured chicken DF-1 cells (~250-fold increase), an embryonic fibroblast cell line. In contrast, the relatively
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Figure 4. Nucleotide sequence of the 5′-flanking region of chicken visfatin gene (1,372 bp). The putative binding sites (shaded or underlined) for the transcriptional factors: Sp1, AP-1, AP-2, AP-4, GATA-1, MyoD, NF-kB, glucocorticoid receptor (GR), cAMP response element binding protein (CREB), and the nuclear factor NF-IL6 have been predicted. The first nucleotide ‘C’ upstream translation start codon is designated as ‘-1’.
lower promoter activities were detected in HEK293 cells (90-fold increase) and CHO cells (16-fold increase). Using a promoter deletion approach, we also noted that the removal of the 5′-portion (from −1,372 to −180) of this 1,372-bp 5′-UTR region could significantly reduce its promoter activity when tested in the 3 chicken cell lines. Because the construct (−179/-1 Luc) displayed the promoter activity in the 3 cell lines, it is likely that a promoter may be located near the putative translation start codon (ATG). Interestingly, the constructs (−1,372/-179 Luc and −1,372/-297 Luc) without the first 179-bp region from the translation start codon still showed strong promoter activities in the cell lines, particularly in DF-1 cells (90-fold and 120-fold increase, respectively, in comparison with pGL3-basic vector). It strongly suggests that alternative promoter(s) distal to the translational start site may be present within the region between −1,372 and −297.
Visfatin mRNA Expression in Chicken Adipose Tissues The quantity of total RNA extracted from chicken adipose tissue was not enough for Northern-blot analysis; therefore, quantitative real-time PCR assay was employed to examine the visfatin mRNA expression in adipose tissues. As shown in Figure 6, the visfatin mRNA levels showed no significant difference between chicken subcutaneous and visceral fat tissues at
the same developmental stage (3 wk and adult stage). Moreover, no significant difference in visfatin mRNA levels was noted among different stages of subcutaneous fat tissues or visceral fat tissues.
DISCUSSION In the present study, the full-length cDNA of visfatin gene has been cloned from chicken liver. The RT-PCR and Northern-blot analysis clearly demonstrated that it was widely expressed in all adult chicken tissues examined. Promoter analysis suggested that visfatin gene transcription was likely controlled by multiple promoters located upstream of the translation start codon. To our knowledge, this study represents the first to identify a functional promoter region(s) of visfatin gene in avian species. The cloned full-length cDNA of chicken visfatin gene encodes a protein of 493 amino acids. Sequence analyses revealed that the chicken visfatin protein not only shares high amino acid sequence identity (89–94%) to visfatin from human, rat, mouse, common carp, and zebrafish but also shares considerable degree of sequence identity with the Nampt of sponge and cyanobacterium. Although visfatin has been viewed as an adipokine or pre-B cell enhancing factor, the amino acid residues responsible for NAM and ribose binding are fully conserved between chicken and other species (Kim et al., 2006; Burgos et al., 2009). In addition, the catalytic
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Figure 5. Promoter activities of the 5′-flanking region of chicken visfatin gene in Chinese hamster ovary (CHO) (A), chicken embryo fibroblast (DF-1) (B), and human embryo kidney (HEK293) (C) cell lines. The constructs p(−1372/−1)-Luc, p(−1372/−297)-Luc, p(−1372/−179)-Luc, p(−720/−1)-Luc, p(−310/−1)-Luc, and p(−179/−1)-Luc were transfected to CHO, DF-1, and HEK293 cells together with pRL-TK vector. The later vector was used to normalize the transfection efficiency. A promoterless pGL3-Basic vector (pGL3) was included in each experiment. The relative promoter activity of each construct is expressed as the fold increase over the pGL3-Basic vector, whose activity is set at 1, after being normalized to pRL-TK activity. The ATG in (A), (B), and (C) indicates the translation start codon of the visfatin gene. Each data point represents the mean ± SEM of 3 replicas. **P < 0.001 versus respective pGL3-Basic vector.
triad site has been found to also be conserved in putative chicken visfatin protein (Wang et al., 2006). These conserved residues and motifs strongly support that chicken visfatin may function as a cytosolic enzyme (Nampt) to catalyze the condensation of NAM with PRPP to yield NMN.
Like the visfatin from other species, chicken visfatin also lacks a typical N-terminal signal peptide sequence. Several studies demonstrated that visfatin protein could be detected in chicken serum as reported in mammals (Krzysik-Walker et al., 2008; Ocón-Grove et al., 2010), implying that a nonclassical pathway may be
CHARACTERIZATION OF CHICKEN VISFATIN GENE
responsible for visfatin secretion (Tanaka et al., 2007; Krzysik-Walker et al., 2008; Ocón-Grove et al., 2010). In the present study, strong hybridization signals of visfatin mRNA were detected in nearly all chicken tissues except pancreas by Northern-blot analysis, indicating the wide expression of chicken visfatin gene. This finding is consistent with the report from Ons et al. (2010). However, in contrast to their finding that only a single mRNA species of ~2.4 kb was detected in all tissues examined by Northern-blot analysis, 3 mRNA species were detected by Northern-blot analysis in the present study, suggesting the existence of multiple chicken visfatin mRNA species. As shown in Figure 3B, the 2 strong hybridization signals detected in all tissues were of sizes close to that of 28S rRNA, suggesting that the sizes of the 2 mRNA species are much larger than 2.4 kb as previously reported (Ognjanovic et al., 2001). Our finding was also supported by the fact that chicken visfatin gene contains an open reading frame of 1,482 bp and a long 3′-UTR of 2,766 bp. Moreover, in silico assembly of chicken visfatin EST retrieved from EST database (http://www.chick.manchester.ac.uk/) can fully cover the open reading frame and the long 3′-UTR we identified, also implying the existence of transcript(s) much longer than 2.4 kb. In spite of the discrepancy in Northern-blot results between the 2 studies, RT-PCR results from present study and Ons’ study clearly indicated that visfatin mRNA is widely distributed in adult chicken tissues. It strongly suggests the involvement of visfatin in a variety of physiological processes in chicken (Garten et al., 2009; Gallí et al., 2010). In the present study, visfatin mRNA expression was detected in chicken visceral and subcutaneous fat tissues, however, no significant difference in visfatin mRNA levels was noted either between the 2 types of adipose tissues or between the 2 developmental stag-
Figure 6. Detection of visfatin mRNA expression in chicken adipose tissues by quantitative real-time PCR. The mRNA levels of visfatin were first normalized by β-actin and then expressed as percentage of subcutaneous fat tissue at embryonic d 15 (Em-SF). Em-SF: embryo; 3w-SF: 3-wk-old chicken subcutaneous fat; 3w-VF: 3-wk-old chicken visceral fat; Ad-SF: adult chicken subcutaneous fat; Ad-VF: adult chicken visceral fat.
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es of the same type of adipose tissues. Our finding is in line with the report from Krzysik-Walker et al., in which visfatin mRNA and protein quantities both show no relationship with subcutanteous fat tissue and visceral fat accumulation (Krzysik-Walker et al., 2008). Therefore, the question whether visfatin can function as an adipokine in chickens remains to be clarified. As an initial step to uncover the transcriptional control of visfatin gene expression in chickens, the 5′-flanking region (1,372 bp) upstream of the start codon (ATG) was cloned in present study. Promoter analysis demonstrated that it displayed strong promoter activity in all 3 cell lines tested, suggesting its importance in regulating visfatin gene transcription in chickens. Because the removal of either the 5′-portion (from −1,372 to −311) or 3′-portion (from −296 to −1) of this 1,372-bp 5′-UTR region could significantly, yet partially, reduce the promoter activities, it strongly suggests that multiple promoters both proximal and distal to the translational start codon (ATG) are likely present herein. In agreement with this postulation, the transcript(s) with a long 5′-UTR of 314 bp (accession No.: JQ406639) was detected in chicken liver using RTPCR in this study, further confirming the existence of the alternative promoter(s) distal to the start codon. Whether additional promoter(s) far from this 1,372-bp region of visfatin gene exist awaits further studies. As shown in Figure 4, the proximal promoter region (from −310 to −1) is GC-rich (60% GC content) and no typical TATA box was found. However, multiple Sp1 binding sites were predicted to be located within this region, suggesting that Sp1 may be responsible for basal expression of chicken visfatin gene, as reported in genes with a TATA-less promoter (Samson and Wong, 2002). In addition to Sp1-binding sites, putative binding sites for other transcription factors, such as AP-1, AP-2, AP-4, CREB, NF-kB, NF-IL6, GATA-1 MyoD, and GR were also found within the 5′-flanking region of 1,372-bp 5′-UTR region, whether they are functional cis-regulatory elements and can confer to the tissue-or cell-specific expression of visfatin gene remains to be clarified. In summary, chicken visfatin gene was cloned in present study. Sequence analyses revealed that chicken putative visfatin protein shares high amino acid sequence identity with their homologs in other species including distant species such as cyanobacterium. Northern blot and RT-PCR assays demonstrated that visfatin was widely expressed in chicken tissues. In addition, quantitative real-time PCR showed no significant difference in visfatin mRNA expression between the visceral and subcutaneous fat tissues of the same developmental stage. In combination with the result from promoter analysis that chicken visfatin gene transcription is likely controlled by multiple promoters upstream of the translation start codon, our findings would help to elucidate the expression and physiological roles of chicken visfatin gene.
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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China for Juan Li (30700452, 30971569) and the grant for undergraduate student training (J1103518).
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