Korean Journal of Poultry Science
Korean Society of Poultry Science
Article

Molecular Characterization and Expression Analysis of Nucleoporin 210 (Nup210) in Chicken

Marc Ndimukaga1https://orcid.org/0000-0001-8061-7753, Godfrey Bigirwa2https://orcid.org/0000-0002-5584-4273, Seokhyun Lee3https://orcid.org/0000-0001-9985-5380, Raham Lee3https://orcid.org/0000-0002-4928-9517, Jae-Don Oh3,https://orcid.org/0000-0001-7756-1330
1Graduate Student, Department of Animal Biotechnology, College of Agricultural and Life Sciences, Chonbuk National University, Jeonju 54896, Republic of Korea
2Graduate Student, Department of Agricultural Convergences, College of Agricultural and Life Sciences, Chonbuk National University, Jeonju 54896, Republic of Korea
3Research Professor, Department of Animal Biotechnology, College of Agricultural and Life Sciences, Chonbuk National University, Jeonju 54896, Republic of Korea
To whom correspondence should be addressed : oh5ow@naver.com

© Copyright 2019, Korean Society of Poultry Science. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Aug 27, 2019 ; Revised: Sep 27, 2019 ; Accepted: Sep 27, 2019

Published Online: Sep 30, 2019

ABSTRACT

Nucleoporin 210 (Nup210) is associated with several physiological processes including muscle and neural cell differentiation, autoimmune diseases, and peripheral T cell homeostasis. Chicken Nup210 (chNup210) gene was originally identified as one of the differentially expressed genes (DEGs) in the kidney tissues of chicken. To elucidate the role of Nup210 in metabolic disease of chicken, we studied the molecular characteristics of chNup210 and analyzed its gene expression under the stimulation of Toll-like receptor 3 (TLR3) ligands. The Nup210 genomic DNA and amino acid sequences of various species including fowls, fishes, and mammals were retrieved from the Ensemble database and subjected to bioinformatics analyses. The expression of Nup210 from several chicken tissues was probed through qRT-PCR, and chicken fibroblast DF-1 cell line was used to determine the change in expression of chNup210 after stimulation with TLR3 ligand, polyinosinicpolycytidylic acid (poly (I:C)). The chNup210 gene was highly expressed in chicken lung and spleen tissues. Although highly conserved among the species, chNup210 was evolutionary clustered in the same clade as that of duck compared to other mammals. Furthermore, this study revealed that chNup210 is expressed in TLR3 signaling pathway and provides fundamental information on Nup210 expression in chicken. Future studies that offer insight into the involvement of chNup210 in the chicken innate immune response against viral infection are recommended.

Keywords: chicken; nucleoporin 210; toll-like receptor 3; gene expression; innate immune receptor signaling

INTRODUCTION

Nuclear pore complexes (NPCs) are multi-protein channels embedded in the nuclear envelope which mediate molecular transports between the nucleus and cytoplasm. For this nucleocytoplasmic trafficking, NPCs are composed of about 30 different nucleoporins (Nups) regulating the NPC function and small receptors called importin and exportin which coordinate the transportation of molecules to NPC (Schwoebel and Moore, 2000; Weis, 2003). By constituting NPC assembly, nucleoporins have differing roles such as establishing nuclear membrane protein localization, facilitating nuclear import and export process and providing the interaction sites for the nucleoplasmic transport (Walde and Kehlenbach, 2010; Hoelz et al., 2011; Solmaz et al., 2011; Busayavalasa et al., 2012). Besides the trafficking function, NPCs are known to be involved in many cellular processes that include transcriptional regulation, chromatin organization and cell differentiation (Gomez-Cavazos and Hetzer, 2012). Several studies reported that nucleoporins are expressed cell-specifically and the composition of NPC may vary among the expressing cells.

Nup210 (or gp210), a component of NPC assembly, is a membrane-spanning glycoprotein with single transmembrane domain and anchors NPC to the nuclear membrane (Hoelz et al., 2011). Nup210 is also found to be highly conservative across eukaryotes including its yeast homologue Pom152 (Upla et al., 2017) suggesting the evolutionary importance of this glycoprotein. Additionally, it was reported that Nup210 along with other nucleoporins Nup133 and Nup98 were involved in transportation of transcription factors involved in neural lineage specific differentiation in embryonic stem cells (Lupu et al., 2008; D'Angelo et al., 2012; Liang et al., 2013). Nup210 expression was increased during neuron cell differentiation while the knock-down of Nup210 disrupted the neural differentiation and the expression of differentiation associated genes. Recently, it was also reported that Nup210 was involved in muscle differentiation and calcium homeostasis. This was based on the fact that Nup210-deficiency caused stress response in the endoplasmic reticulum during muscle differentiation leading to cell apoptosis (Gomez-Cavazos and Hetzer, 2015). Moreover, Nup210 promoted the assembly of Mef2C transcription complex that was essential for the expression of NPC muscle structural genes which regulated muscle growth and maintenance in zebrafish (Raices et al., 2017).

There are several reports that Nup210 may be involved in autoimmune disease in human including primary biliary cirrhosis (PBC), rheumatic disease and systemic lupus erythematosus (Worman and Courvalin, 2003; Enarson et al., 2004; Duarte-Rey et al., 2012). Primary biliary cirrhosis (PBC) is an autoimmune disease that destructs bile ducts and causes liver cirrhosis. Autoantibody of Nup210 were found in the severe PBC patients and the abnormal expression of Nup210 which is the autoimmune target of PBC might cause this disease (Nakamura et al., 2006). In this study, we analyzed the amino acid sequence and the gene expression profile of chicken Nup210 in various chicken tissues and chicken DF-1 cells.

MATERIALS AND METHODS

1. Cell Culture

The chicken cell lines DF-1 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). DF-1 cells were cultured in DMEM (Biowest, USA) medium with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/mL of penicillin and streptomycin (Thermo Scientific, Logan, UT, USA), respectively, at 37℃ with 5% CO2 in humidified atmosphere.

2. Treatment of Poly (I:C) and Transcription Factor Inhibitors

Poly (I:C) was purchased from Invivogen (San Diego, CA, USA) and treated in chicken DF-1 cells at the concentrations of 0.1, 1, 5, and 10 μg/mL for 24 h. The time-dependent effect of Poly (I:C) was analyzed by treatment for 1, 3, 6, 12, and 24 h at 10 μg/mL prior to gene expression analysis. Transcription factor inhibitors (BAY 11-7085 for NFκB and Tanshinone IIA for AP-1) were purchased from Sigma-Aldrich (Louis, MO, USA). The inhibitors were added on DF-1 cells with concentrations of 5 μM for BAY 11-7085 and 25 μM for Tanshinone IIA at 3 h prior to the treatment with 5 μg/mL poly (I:C).

3. Bioinformatic Analysis

The mRNA and amino acid sequences of various species (chicken, duck, human, chimpanzee, mouse, rat, cow, pig, horse, dog, and cat) for Nup210 were retrieved from the Ensemble database (http://www.ensembl.org/) (Table 1). The amino acid sequences were aligned by ClustalW method in BioEdit software. The protein domains were predicted by using the SMART domain search program (http://smart.emblheidelberg.de/). Phylogenetic analyses were performed with MEGA7 software (Kumar et al., 2016).

Table 1. Ensembl and amino acid sequence ID in the Nup210 gene of various species
Species Scientific name Ensembl ID NCBI Reference Sequence ID
Chicken Gallus gallus ENSGALG00000005078 XM_414320.6
Duck Anas platyrhynchos ENSAPLG00000012355 XM_013091930.2
Human Homo sapiens ENSG00000132182 NM_024923.3
Chimpanzee Pan troglodytes ENSPTRT00000027394 XM_003309631.4
Mouse Mus musculus ENSMUSG00000030091 NM_018815.2
Rat Rattus norvegicus ENSRNOG00000005390 NM_053322.2
Cow Bos taurus ENSBTAT00000043850 NM_001191461.1
Pig Sus scrofa ENSSSCT00000054838 XM_021069388.1
Horse Equus caballus ENSECAG00000009171 XM_014731350.1
Dog Canis ENSCAFT00000007053 XM_005632290.3
Cat Felis catus ENSFCAT00000007507 XM_019825131.1
Zebrafish Danio rerio ENSDARG00000063333 XM_002667560.6
Download Excel Table
4. RNA Extraction and cDNA Synthesis

Total RNA was isolated from chicken DF-1 cells according to Trizol reagent (Life Technologies, USA) manufacturer's instructions. Total RNA were quantified at 260 nm absorbance and RNA integrity was evaluated with 1.0% (w/v) agarose gel. cDNA was reverse-transcribed from 2 μg of total RNA (amounts) using QuantiTect Reverse Transcription Kit (Toyobo, JAPAN) according to the manufacturer's instructions.

5. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) Analysis

The expression level of chNup210 gene was confirmed in DF-1 cells by qPCR method (Oh et al., 2019). Briefly, SYBR green supermix and CFX96TM IVD Real-time PCR System (Bio-rad, USA) were used for qPCR, and relative quantification Ct (2—ΔΔCT) method was applied to compare the gene expression levels (Livak and Schmittgen, 2001). The PCR were performed as follows: an initial step at 94℃ for 3 min; 39 cycles at 94℃ for 10 s, 60℃ for 30 s, and 72℃ for 30 s; and a final step at 72℃ for 10 min. Dissociation was performed at 0.5℃ increments from 55℃ to 95℃ for over 25 min. For the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to detect mRNA expression levels. The GAPDH primer sequences are as follows: 5’- TGC TGC CCA GAA CAT CAT CC -3’ for forward primer and 5’-ACG GCA GGT CAG GTC AAC AA -3’ for reverse primer.

6. Statistical Analysis

Statistical significance was assessed using Student's t-test and P<0.05 was considered significant compared to vehicle control. The difference among each value was determined by Tukey’s multiple comparison test with alpha <0.05 were considered significant.

RESULTS AND DISCUSSION

The amino acid sequence of chNup210 in chicken was analyzed and compared with other species. The chNup210 gene sequence was identified as a DEG from chicken kidney RNA-seq study (Park et al., 2017). The resultant amino acid sequence of chNup210 gene and the sequences of Nup210 from other animals including duck, horse, dog, cat, mouse, rat, pig, cow, human and chimpanzee that were retrieved from Ensemble database are shown in Fig. 1A. From the alignment with GeneDoc 2.7 program, it was found out that Nup210 was highly conserved among the species. Based on the prediction with SMART domain search program, chNup210 was found to comprise of bacterial Ig-like domain 2 (Big-2) and transmembrane domain as like other species. The evolutionary relationships between chNup210 and other Nup210 orthologs are shown by phylogenetic tree (Fig. 1B). Based on the amino acid sequence alignment, it was revealed that chNup210 was evolutionary clustered in the same clade as that of the duck than for other mammals.

kjps-46-3-185-g1
Fig. 1. Analysis of Nup210 amino acid sequences. (A) Comparison of the chicken Nup210 amino acid sequence with those of various other species. ChNup210 was predicted to comprise bacterial Ig-like domain 2 (Big-2) (solid box), Transmembrane domain (dotted box). (B) Phylogenetic tree for Nup210 from various species. Phylogenetic analyses were performed with the amino acid sequence of each species, using MEGA7 software. ChNup210 clustered in the same clade with duck. The bar indicates 2% amino acid divergence.
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The gene expression pattern of chicken Nup210 was investigated in various chicken tissues. From this study, it was revealed that chNup210 gene was highly expressed in chicken lung and spleen tissues (Fig. 2A). This result contradicts other studies where Nup210 gene expression is known to be related to muscle and neural cell differentiation and autoimmune diseases of bile and liver (Enarson et al., 2004; Duarte-Rey et al., 2012). Therefore, the high expression chNup210 in chicken lung and spleen under normal condition is somewhat intriguing.

kjps-46-3-185-g2
Fig. 2. (A) Analysis of expression levels of chNup210 in different chicken tissues (liver, lung, kidney, spleen, and heart). (B) chNup210 in chicken DF-1 cells after poly (I:C) treatment. DF-1 cells were treated with 0.1, 1, 5, 10 μg/mL of poly (I:C) for 24 h. (C) DF-1 cells were treated with 10 μg/mL poly (I:C) for 1, 3, 6, 12 and 24 h. The experiment was repeated for three times (n=3). Significant differences were determined by Tukey’s multiple comparison test, and * means P<0.05.
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The transcriptional profile of chicken Nup210 was also investigated in chicken DF-1 cells under the stimulation with TLR-3/Mda-5 agonist, poly (I:C). As shown in Fig. 2B, the expressional level of chNup210 after stimulation with 10 μg/mL of poly (I:C) was highly elevated. This result suggests that the expression of chNup210 could be induced by viral infection in chicken cell and thus chNup210 may be related to TLR3-mediated immune responses. To further study the hypothesis that the up-regulation of chNup210 was due to TLR signaling, transcriptional inhibitors of TLR3 signal pathway were added in chicken DF-1 cell. The inhibitors of transcription factors NF-κB and AP-1 used were BAY 11-7085 (Bay) and Tanshinone IIA (Tan-II), respectively. The expressional level of chIL1B evaluated after poly (I:C) treatment in the presence of inhibitors Bay or Tan-II is shown in Fig. 3. In this study, the transcriptional levels of chNup210, along with chIL1B, were all up-regulated after treatment of 5 μg/mL of poly (I:C) without the inhibitors. However, under the presence of transcriptional inhibitors, the up-regulations of chNup210 and chIL1B genes were significantly suppressed compared to poly (I:C)-only treatment. This suggests that the expressions of chNup210 and chIL1B genes were all induced by transcriptional factors NF-κB and AP-1 under the stimulation of poly (I:C) and chNup210 expression seemed to be under control of TLR3 signaling pathway in chicken.

kjps-46-3-185-g3
Fig. 3. Effects of NFκB inhibitor (BAY11-7085; Bay) and AP-1 inhibitor (Tanshinone IIA; Tan-II) on the gene expression of chNup210 and chIL1B after stimulation with poly (I:C) in the cultured DF-1 fibroblast cells. Cultured DF-1 cells were treated with or without poly (I:C) (5 μg/mL, 6 h) in the presence or absence of Bay (5 μM) and Tan-II (25 μM). Values are mean±SE (n=3). Significant differences were determined by Tukey’s multiple comparison test, and bars with the same letter (a~d) on top are the cases that significant differences were not found (P>0.05).
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Although several studied have shown that the functions of Nup210 are related to cell differentiations and autoimmune diseases, this study revealed the expression of chNup210 in TLR3 signaling pathway in the fowl. As a result, chNup210 may play a significant role during viral inflammation in chicken. This study therefore provides the fundamental information about the expression Nup210 in chicken and further studies that can generate more insight on the involvement of chNup210 in the chicken innate immune response against viral infection are recommended.

적 요

Nucleoporin 210(Nup210)는 근육 및 신경세포의 분화, 자기 면역 질환, 말초 T세포 항상성 등 여러 생리작용에 관여한다. 닭의 Nup210 유전자는 닭 신장조직에서 칼슘 의존성 차별 발현 유전자로 발굴되었으며, 닭의 대사 이상 질환과 Nup210의 관련 연구를 위해 Nup210 유전자의 분자유전학적 특성을 구명하고, 톨-유사수용체 3(Toll-like receptor 3 (TLR3)) 자극에 의한 전사 조절을 연구하였다. 닭의 여러 조직과 배아 섬유아세포주인 DF-1 세포에서 Nup210 유전자의 전사 수준을 조사한 결과, 폐와 비장 조직에서 가장 높게 발현되었으며, Nup210의 발현은 TLR3 신호자극에 의해 증가함을 확인하였다. 또한 닭 Nup210 유전자가 코딩하는 단백질의 구조는 조류, 어류, 포유류를 포함한 여러 종과 매우 보존적이나 진화적으로 다른 포유류보다는 오리와 가장 가깝다고 추정되었다. 본 연구의 결과를 통해 닭 Nup210이 TLR3 신호시스템에 관여함을 확인하였고, 추가연구를 통해 바이러스 침입에 대한 닭 면역 메커니즘을 구명할 필요가 있다고 사료된다.

(색인어: 닭, Nup210, Toll-like receptor 3, 유전자 발현, 선천적 면역수용체 신호전달)

ACKNOWLEDGMENTS

This work was supported by a grant from the Next Generation BioGreen 21 Program (No. PJ01324201, PJ01315 101), Rural Development Administration, Republic of Korea.

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