Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

doi:10.1016/S2095-3119(13)60574-2

Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (11): 2479-2487.DOI: 10.1016/S2095-3119(13)60574-2

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

JU Xiang-hong, XU Han-jin, YONG Yan-hong, AN Li-long, XU Ying-mei, JIAO Pei-rong , LIAO Ming

1、Department of Veterinary Medicine, Guangdong Ocean University, Zhanjiang 524088, P.R.China
2、Key Laboratory of Animal Disease Control and Prevention, Ministry of Agriculture/College of Veterinary Medicine, South China Agricultural
University, Guangzhou 510642, P.R.China
3、Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, P.R.China

收稿日期:2013-05-14 出版日期:2014-11-06 发布日期:2014-11-14
通讯作者: LIAO Ming, Tel/Fax: +86-20-85280240, E-mail: mliaoscau@gmail.com
作者简介:JU Xiang-hong, Mobile: 13553523590, E-mail: juxh77@163.com
基金资助:
This research was supported by grants from the National Natural Science Foundation of China (31101862), the China Postdoctor Science Foundation and Guangdong Ocean University Doctor Seed Grant, China (0712107).

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

JU Xiang-hong, XU Han-jin, YONG Yan-hong, AN Li-long, XU Ying-mei, JIAO Pei-rong , LIAO Ming

1、Department of Veterinary Medicine, Guangdong Ocean University, Zhanjiang 524088, P.R.China
2、Key Laboratory of Animal Disease Control and Prevention, Ministry of Agriculture/College of Veterinary Medicine, South China Agricultural
University, Guangzhou 510642, P.R.China
3、Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, P.R.China

Received:2013-05-14 Online:2014-11-06 Published:2014-11-14
Contact: LIAO Ming, Tel/Fax: +86-20-85280240, E-mail: mliaoscau@gmail.com
About author:JU Xiang-hong, Mobile: 13553523590, E-mail: juxh77@163.com
Supported by:
This research was supported by grants from the National Natural Science Foundation of China (31101862), the China Postdoctor Science Foundation and Guangdong Ocean University Doctor Seed Grant, China (0712107).

摘要/Abstract

摘要： Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity of gene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 (TLR4) in Bama miniature pigs (Sus scrofa domestica) following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a nonclassical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 (RPL4) and TATA box-binding protein (TBP) were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear cells (PBMCs) during heat shock. The mRNA level of the TLR4 gene (both classical and spliced) in stressed pigs increased significantly (P<0.05). Further, the expression levels of the alternative spliced variant of TLR4 (TLR4-ASV) showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

关键词: alternative splicing variant , Bama miniature pig , toll-like receptor 4 , heat stress

Abstract: Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity of gene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 (TLR4) in Bama miniature pigs (Sus scrofa domestica) following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a nonclassical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 (RPL4) and TATA box-binding protein (TBP) were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear cells (PBMCs) during heat shock. The mRNA level of the TLR4 gene (both classical and spliced) in stressed pigs increased significantly (P<0.05). Further, the expression levels of the alternative spliced variant of TLR4 (TLR4-ASV) showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

Key words: alternative splicing variant , Bama miniature pig , toll-like receptor 4 , heat stress

JU Xiang-hong, XU Han-jin, YONG Yan-hong, AN Li-long, XU Ying-mei, JIAO Pei-rong , LIAO Ming. Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs[J]. Journal of Integrative Agriculture, 2014, 13(11): 2479-2487.

参考文献

Akira S, Takeda K. 2004. Toll-like receptor signalling. NatureReviews Immunology, 4, 499-511

Alvarez B, Revilla C, Chamorro S, Lopez-Fraga M, AlonsoF, Dominguez J, Ezquerra A. 2006. Molecular cloning,characterization and tissue expression of porcine Toll-likereceptor 4. Developmental & Comparative Immunology,30, 345-355

Andersen C, Jensen J, Rntoft T. 2004. Normalization of realtimequantitative reverse transcription-PCR data: A modelbasedvariance estimation approach to identify genes suitedfor normalization, applied to bladder and colon cancer datasets. Cancer Research, 64, 5245-5250

Bas A, Forsberg G, Hammarstr M S, Hammarstr M M. 2004.Utility of the housekeeping genes 18S rRNA, β-actinand glyceraldehyde 3 phosphate dehydrogenase fornormalization in real time quantitative reverse transcriptasepolymerase chain reaction analysis of gene expressionin human T lymphocytes. Scandinavian Journal ofImmunology, 59, 566-573

Bhatia P, Taylor W, Greenberg A, Wright J. 1994. Comparisonof glyceraldehyde-3-phosphate dehydrogenase and28S-ribosomal RNA gene expression as RNA loadingcontrols for northern blot analysis of cell lines of varyingmalignant potential Analytical Biochemistry, 216, 223-226.

Black D L. 2003. Mechanisms of alternative pre-messengerRNA splicing. Annual Review of Biochemistry, 72, 291-336

de Boer M, de Boer T, Mari N J, Timmermans M, Nota B,van Straalen N, Ellers J, Roelofs D. 2009. Referencegenes for QRT-PCR tested under various stress conditionsin Folsomia candida and Orchesella cincta (Insecta,Collembola). BMC Molecular Biology, 10, 54.

Cappelli K, Felicetti M, Capomaccio S, Spinsanti G, SilvestrelliM, Supplizi A V. 2008. Exercise induced stress in horses:selection of the most stable reference genes for quantitativeRT-PCR normalization. BMC Molecular Biology, 9, 49.

Chen W, Wang J, An H, Zhou J, Zhang L, Cao X. 2005. Heatshock up-regulates TLR9 expression in human B cellsthrough activation of ERK and NF-κB signal pathways.Immunology Letters, 98, 153-159

Desautes C, Sarrieau A, Caritez J C, Mormede P 1999.Behavior and pituitary-adrenal function in large whiteand Meishan pigs. Domestic Animal Endocrinology, 16,193-205.

Gaido K, Maness S, Leonard L, Greenlee W. 1992.2,3,7,8-Tetrachlorodibenzo-p-dioxin-dependentregulation of transforming growth factors-alpha and beta2 expression in a human keratinocyte cell line involves bothtranscriptional and post-transcriptional control. Journal ofBiological Chemistry, 267, 24591.

Garcia-Blanco M A, Baraniak A P, Lasda E L. 2004.Alternative splicing in disease and therapy. NatureBiotechnology, 22, 535-546

Haehnel V, Schwarzfischer L, Fenton M J, Rehli M. 2002.Transcriptional regulation of the human toll-like receptor2 gene in monocytes and macrophages. The Journal ofImmunology, 168, 5629.

Heckmann L, Connon R, Hutchinson T, Maund S, Sibly R,Callaghan A. 2006. Expression of target and referencegenes in Daphnia magna exposed to ibuprofen. BMCgenomics, 7, 175.

Hermann G, Tovar C A, Beck F M, Allen C, Sheridan J F.1993. Restraint stress differentially affects the pathogenesisof an experimental influenza viral infection in three inbredstrains of mice. Journal of Neuroimmunology, 47, 83-94

Horiuchi T, Aigaki T 2006. Alternative trans-splicing: anovel mode of pre-RNA processing. Biology of the Cell,98, 135-140

Iwami K, Matsuguchi T, Masuda A, Kikuchi T, MusikacharoenT, Yoshikai Y. 2000. Cutting edge: Naturally occurringsoluble form of mouse toll-like receptor 4 inhibitslipopolysaccharide signaling. The Journal of Immunology,165, 6682-6686

Ju X H, Yong Y H, Xu H J, An L l, Xu Y M. 2011a. Impactsof heat stress on baseline immune measures and a subsetof T cells in Bama miniature pigs. Livestock Science, 135,289-292

Ju X H, Yong Y H, Xu H J, An L l, Xu Y M, Jiao P R, LiaoM. 2011b. Selection of reference genes for gene expressionstudies in PBMC from Bama miniature pig under heatstress. Veterinary Immunology and Immunopathology,144, 160-166

Liew F Y, Xu D, Brint E K, O’Neill L A J. 2005. Negativeregulation of toll-like receptor-mediated immuneresponses. Nature Reviews Immunology, 5, 446-458

Liu Y, Zeng B H, Shang H T, Cen Y Y, Wei H. 2008. Bamaminiature pigs (Sus scrofa domestica) as a model for drugevaluation for humans: comparison of in vitro metabolismand in vivo pharmacokinetics of lovastatin. ComparativeMedicine, 58, 580.

Mansur N, Meyer-Siegler K, Wurzer J, Sirover M. 1993.Cell cycle regulation of the glyceraldehyde-3-phosphatedehydrogenaseluracil DNA glycosylase gene in normalhuman cells Nucleic Acids Research, 21, 993.

Markham N, Zuker M. 2005. DINAMelt web server for nucleicacid melting prediction. Nucleic Acids Research, 33, W577.Miggin S M, Oeill L A J. 2006. New insights into the regulationof TLR signaling. Journal of Leukocyte Biology, 80, 220-226

Pasare C, Medzhitov R. 2005. Control of B-cell responses byToll-like receptors. Nature, 438, 364-368

Radoni A, Thulke S, Bae H, Müller M, Siegert W, Nitsche A.2005. Reference gene selection for quantitative real-timePCR analysis in virus infected cells: SARS corona virus,Yellow fever virus, Human Herpesvirus-6, Camelpox virusand Cytomegalovirus infections Virology Journal, 2, 7.

Rosochacki S J, Piekarzewska A B, Poloszynowicz J, SakowskiT. 2000. The influence of restraint immobilization stress onthe concentration qf bioamines and cortisol in plasma ofPietrain and Duroc pigs. Journal of Veterinary Medicine(Series A: Physiology Pathology Clinical Medicine), 47,231-242.

Thomas A V, Broers A D, Vandegaart H F, Desmecht D J M.2006. Genomic structure, promoter analysis and expressionof the porcine (Sus scrofa) TLR4 gene. MolecularLmmunology, 43, 653-659

Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M,Sawada K, Sakiyama S. 1987. Enhanced expression of aglyceraldehyde-3-phosphate dehydrogenase gene in humanlung cancers Cancer Research, 47, 5616-5619

Vandesompele J, de Preter K, Pattyn F, Poppe B, Van Roy N,de Paepe A, Speleman F. 2002. Accurate normalizationof real-time quantitative RT-PCR data by geometricaveraging of multiple internal control genes. GenomeBiology, 3, 7.

Wang W, Shi C, Zhang J, Gu W, Li T, Gen M, Chu W, HuangR, Liu Y, Hou Y. 2009. Molecular cloning, distributionand ontogenetic expression of the oligopeptide transporter PepT1 mRNA in Tibetan suckling piglets. Amino Acids,37, 593-601

Wheeler D, Bhagwat M. 2007. BLAST QuickStart: exampledrivenweb-based BLAST tutorial. Methods in MolecularBiology (Clifton, NJ), 395, 149.

Ying X Q, Zhai C Y, Li H T, Liu D. 2011. Identif ication ofalternative splicing isoforms of pig TLR4 mRNA. ActaVeterinaria et Zootechnica Sinica, 42, 1051-1056

Zhang X, Ding L, Sandford A J. 2005. Selection of referencegenes for gene expression studies in human neutrophils byreal-time PCR. BMC Molecular Biology, 6, 4.

Zhong H, Simons J. 1999. Direct comparison of GAPDH,β-actin, cyclophilin, and 28S rRNA as internal standardsfor quantifying RNA levels under hypoxia. Biochemicaland Biophysical Research Communications, 259, 523-526

Zhou J, An H, Xu H, Liu S, Cao X. 2005. Heat shock upregulatesexpression of Toll-like receptor-2 and Toll-likereceptor-4 in human monocytes via p38 kinase signalpathway. Immunology, 114, 522-530.

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

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