Browse > Article

Regulation of toll-like receptors expression in muscle cells by exercise-induced stress  

Park, Jeong-Woong (Department of Animal Science, College of Natural Resources and Life Sciences, Pusan National University)
Kim, Kyung-Hwan (Department of Animal Science, College of Natural Resources and Life Sciences, Pusan National University)
Choi, Joong-Kook (Division of Biochemistry, College of Medicine, Chungbuk National University)
Park, Tae Sub (Institute of Green-Bio Science and Technology, Seoul National University)
Song, Ki-Duk (The Animal Molecular Genetics and Breeding Center, Jeonbuk National University)
Cho, Byung-Wook (Department of Animal Science, College of Natural Resources and Life Sciences, Pusan National University)
Publication Information
Animal Bioscience / v.34, no.10, 2021 , pp. 1590-1599 More about this Journal
Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H2O2), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H2O2, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H2O2, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.
Exercise Stress; Horse Fetal Muscle Cells; Peripheral Blood Mononuclear Cell Migration; Toll-like Receptor;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Pourteymour S, Eckardt K, Holen T, et al. Global mRNA sequencing of human skeletal muscle: search for novel exercise-regulated myokines. Mol Metab 2017;6:352-65.   DOI
2 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8.   DOI
3 Park JW, Choi JY, Hong SA, et al. Exercise induced upregulation of glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modifier subunit gene expression in Thoroughbred horses. Asian-Australas J Anim Sci 2017;30:728-35.   DOI
4 Martin SJ. Cell death and inflammation: the case for IL-1 family cytokines as the canonical DAMPs of the immune system. FEBS J 2016;283:2599-615.   DOI
5 Chen BP, Liang G, Whelan J, Hai T. ATF3 and ATF3 delta Zip. Transcriptional repression versus activation by alternatively spliced isoforms. J Biol Chem 1994;269:15819-26.   DOI
6 Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101-9.   DOI
7 Robertson TA, Maley MAL, Grounds MD, Papadimitriou JM. The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res 1993;207:321-31.   DOI
8 Hai T, Hartman MG. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 2001;273:1-11.   DOI
9 Kim H, Lee T, Park W, et al. Peeling back the evolutionary layers of molecular mechanisms responsive to exercise-stress in the skeletal muscle of the racing horse. DNA Res 2013;20:287-98.   DOI
10 Imai Y, Kuba K, Neely GG, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 2008;133:235-49.   DOI
11 Catoire M, Mensink M, Kalkhoven E, Schrauwen P, Kersten S. Identification of human exercise-induced myokines using secretome analysis. Physiol Genomics 2014;46:256-67.   DOI
12 Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm 2010;2010:Article ID 672395.   DOI
13 Park KD, Park J, Ko J, et al. Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq. BMC Genomics 2012;13:473.   DOI
14 Eivers SS, McGivney BA, Fonseca RG, et al. Alterations in oxidative gene expression in equine skeletal muscle following exercise and training. Physiol Genomics 2010;40:83-93.   DOI
15 Park JW, Song KD, Kim NY, et al. Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene. Asian-Australas J Anim Sci 2017;30:1471-7.   DOI
16 Lee HG, Choi JY, Park JW, et al. Effects of exercise on myokine gene expression in horse skeletal muscles. Asian-Australas J Anim Sci 2019;32:350-6.   DOI
17 Lee HG, Khummuang S, Youn HH, et al. The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse. Asian-Australas J Anim Sci 2019;32:1095-103.   DOI
18 Schaefer L. Complexity of danger: the diverse nature of damage-associated molecular patterns. J Biol Chem 2014;289:35237-45.   DOI
19 Capomaccio S, Cappelli K, Barrey E, Felicetti M, Silvestrelli M, Verini-Supplizi A. Microarray analysis after strenuous exercise in peripheral blood mononuclear cells of endurance horses. Anim Genet 2010;41:166-75.   DOI
20 Hai TW, Liu F, Coukos WJ, Green MR. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes dev 1989;3:2083-90.   DOI
21 Cho HW, Shin S, Park JW, et al. Molecular characterization and expression analysis of the peroxisome proliferator activated receptor delta (PPARδ) gene before and after exercise in horse. Asian-Australas J Anim Sci 2015;28:697-702.   DOI
22 Hindi SM, Kumar A. Toll-like receptor signalling in regenerative myogenesis: friend and foe. J Pathol 2016;239:125-8.   DOI
23 Kim DH, Lee HG, Nipin Sp, et al. Validation of exercise-response genes in skeletal muscle cells of Thoroughbred racing horses. Anim Biosci 2021;34:134-142.   DOI
24 Cristi MC, Sanchez CP, Veneroso C, Cuevas MJ, Gonzalez-Gallego J. Effect of an acute exercise bout on toll-like receptor 4 and inflammatory mechanisms in rat heart. Rev Med Chile 2012;140:1282-8.   DOI
25 Zhang X, Mosser DM. Macrophage activation by endogenous danger signals. J Pathol 2008;214:161-78.   DOI
26 Kerst B, Mennerich D, Schuelke M, et al. Heterozygous myogenic factor 6 mutation associated with myopathy and severe course of Becker muscular dystrophy. Neuromuscul Disord 2000;10:572-7.   DOI
27 Janeway JCA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197-216.   DOI
28 Shtil AA, Mandlekar S, Yu R, et al. Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells. Oncogene 1999;18:377-84.   DOI
29 Kim SY, Choi YJ, Joung SM, Lee BH, Jung YS, Lee JY. Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor. Immunology 2010;129:516-24.   DOI
30 Miner JH, Wold B. Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc Natl Acad Sci USA 1990;87:1089-93.   DOI
31 Gilchrist M, Thorsson V, Li B, et al. Systems biology approaches identify ATF3 as a negative regulator of toll-like receptor 4. Nature 2006;441:173-8.   DOI
32 Zou J, An H, Xu H, Liu S, Cao X. Heat shock up-regulates expression of toll-like receptor-2 and toll-like receptor-4 in human monocytes via p38 kinase signal pathway. Immunology 2005;114:522-30.   DOI
33 Ju XH, Xu HJ, Yong YH, An LL, Jiao PR, Liao M. Heat stress upregulation of Toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood mononuclear cell (PBMC) of Bama miniature pigs: an in vivo and in vitro study. Animal 2014;8:1462-8.   DOI
34 Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335-76.   DOI
35 Fernandez-Verdejo R, Vanwynsberghe AM, Essaghir A, et al. Activating transcription factor 3 attenuates chemokine and cytokine expression in mouse skeletal muscle after exercise and facilitates molecular adaptation to endurance training. FASEB J 2017;31:840-51. 987R   DOI
36 Sp N, Kang DY, Kim DH, et al. Methylsulfonylmethane inhibits cortisol-induced stress through p53-mediated SDHA/HPRT1 expression in racehorse skeletal muscle cells: a primary step against exercise stress. Exp Ther Med 2020;19:214-22.   DOI
37 Hsu JC, Laz T, Mohn KL, Taub R. Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver. Proc Natl Acad Sci USA 1991;88:3511-5.   DOI
38 Hai T, Wolfgang CD, Marsee DK, et al. ATF3 and stress responses. Gene Expr 1999;7:321-35.
39 Zimmermann J, Erdmann D, Lalande I, Grossenbacher R, Noorani M, Furst P. Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1. Oncogene 2000;19:2913-20.   DOI
40 Iyer VR, Eisen MB, Ross DT, et al. The transcriptional program in the response of human fibroblasts to serum. Science 1999;283:83-7.   DOI
41 Loetscher M, Gerber B, Loetscher P, et al. Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. J Exp Med 1996;184:963-9.   DOI
42 Weber M, Uguccioni M, Ochensberger B, Baggiolini M, Clark-Lewis I, Dahinden CA. Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3. J Immunol 1995;154:4166-72.
43 Pimkhaokham A, Shimada Y, Fukuda Y, et al. Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res 2000;91:1126-3.   DOI