[논문]Insect peptide CopA3 promotes proliferation and PAX7 and MYOD expression in porcine muscle satellite cells

Jeongeun, Lee; Jinryoung, Park; Hosung, Choe; Kwanseob, Shim

doi:10.5187/jast.2022.e81

Insect peptide CopA3 promotes proliferation and PAX7 and MYOD expression in porcine muscle satellite cells 원문보기

Journal of animal science and technology : JAST, v.64 no.6, 2022년, pp.1132 - 1143

Jeongeun, Lee (Department of Agricultural Convergence Technology, Jeonbuk National University) , Jinryoung, Park (Department of Stem Cell and Regenerative Biotechnology, Konkuk University) , Hosung, Choe (Department of Animal Biotechnology, Jeonbuk National University) , Kwanseob, Shim (Department of Agricultural Convergence Technology, Jeonbuk National University)

Abstract ▼ AI-Helper

Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 ㎍/mL of CopA3). At a CopA3 concentration of 5 ㎍/mL and 10 ㎍/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 ㎍/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 ㎍/mL and 10 ㎍/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.

주제어

표/그림 (5)

표 Table 1. Primer sequences for qRT-PCR
그림 Fig. 1. Effects of CopA3 on MSCs proliferation. (A) Cell viability of muscle satellite cells (MSCs) after CopA3 treatment. (B) Comparison of MSCs morphology among different concentrations of CopA3 at 24, 48 and 72 h. Scale bar: 100 μm. (C) Proliferation of MSCs in the presence and absence of various concentrations of CopA3 on days 0, 1, 2, and 3. n = 5, Values are presented as mean ± SE. ^a-dDifferent superscripts represent statistically significant differences (p < 0.01).
그림 Fig. 2. Effects of CopA3 on cell cycle distribution in MSCs. (A) Schematic diagram of results of cell cycle analyses based on PI staining and FACS. Percentage of MSCs in (B) G0/G1, (C) S, and (D) G2/M phases, under different concentrations of CopA3. Cell cycle distribution was analyzed using PI staining and FACS. n = 3, Values are presented as mean ± SE. ^a-cDifferent superscripts represent statistically significant differences (p < 0.01). MSCs, muscle satellite cells; PI, propidium iodide; FACS, fluorescence activated cell sorting.
그림 Fig. 3. Effect of CopA3 on Apoptosis was analyzed via flow cytometry. (A) MSCs were stained using PI and Annexin V. Percentage of (B) live, (C) early apoptotic, and (D) late apoptotic cells in MSCs after FACS analyses. n = 3, Values are presented as mean ± SE. ^a-cDifferent superscripts represent statistically significant differences (p < 0.01). MSCs, muscle satellite cells; PI, propidium iodide; FACS, fluorescence activated cell sorting.
그림 Fig. 4. Comparative analyses of myogenesis transcription factors expressed under different CopA3 treatments. (A) Immunocytochemistry of cells stained with DAPI (blue), PAX7 (green) and MYOD (red). Scale bar: 50 μm. (B) Protein levels of PAX7, MYOD, MYOG and GAPDH. (C) PAX7 protein expression levels normalized by GAPDH. (D) MYOD protein expression levels normalized by GAPDH. (E) Gene expression levels of PAX7, MYOD1, and MYOG. n = 3, Values are presented as mean ± SE. ^a-cDifferent superscripts represent statistically significant differences (p < 0.01). ^A,BDifferent superscripts represent statistically significant differences (p < 0.05).

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데이터처리

One-way analysis of variance (ANOVA) was followed by Duncan’s Multiple Range Test to compare statistically significant differences in each group

성능/효과

All samples were analyzed via fluorescence activated cell sorting (FACS) calibur flow cytometry (Becton, Dickinson Company, San Diego, CA, USA) and BD Cell Quest Pro software. A total of 10,000 events were collected per sample to manually determine the percentage of G1, S and G2/M phases.
After washing with TBST, immunoblots were visualized using an enhanced chemiluminescene kit (Thermo Fisher), and the images were acquired with iBright CL 100 Imaging system (Thermo Fisher). All proteins were normalized with GAPDH.
Among all the groups, the proportion of the S phase was significantly higher (p < 0.01) in the 5 and 10 μg/mL groups and the lowest in the control (Fig
Cell cycle was analyzed via flow cytometry to determine whether the treatment of CopA3 affected the cell cycle of MSCs (Fig. 2A).
CopA3 significantly decreased (p < 0.01) the percentage of the G0/G1 phase (Fig
Furthermore, compared to the control group, the CopA3 treated groups showed significantly increased (p < 0.01) the S and G2/M phases (Figs
However, cell viability of the 50 μg/mL group reduced significantly (p < 0.01) compared to that of the control group.
1B). However, differences in cell proliferation rates among the groups were observed starting 24 h after CopA3 treatment (48 h). On the third day of culture, we observed a higher number of cells in the 5 and 10 μg/mL groups than in 25 μg/mL and control groups (Fig.
4E). However, no significant differences in PAX7 expression were observed among all groups (Fig. 4E).
However, the percentage of late apoptotic cells in 10 μg/mL groups were slightly increased (p < 0.01) than control group (Fig
In addition, cell viabilities of the 10 and 25 μg/mL groups were not significantly different from that of the control group
4A). In addition, results of western blot analyses were consistent with those of immunocytochemistry assays (Fig. 4B).
In conclusion, our results suggest that CopA3 treatment at concentrations of 5 and 10 μg/mL increases MSCs proliferation by promoting the transition from G1 to S phase, as well as upregulating PAX7 and MYOD expression
Moreover, the G2/M phase ratio was found to be the highest (p < 0.01) in the 25 μg/mL group (Fig
Results of mRNA expression analyses found that the expression level of MYOD1 was significantly higher (p < 0.01) in 5 and 10 μg/mL groups and that the highest (p < 0.05) expression of MYOG was observed in the 25 μg/mL (Fig
Results of the immunocytochemistry assays of PAX7 and MYOD showed that the expression of PAX7 was upregulated in the 5 and 10 μg/mL groups (Fig
The expressions of PAX7 and MYOD proteins were significantly upregulated (p < 0.01) in both 5 and 10 μg/mL groups (Figs
The number of early apoptotic cells in the 10 μg/mL groups was lower than that in the 25 μg/mL and control (Fig
The proliferation rate of MSCs significantly increased (p < 0.01) in the 5 and 10 μg/mL groups compared to that in other groups, and the MSC proliferation rate of the 25 μg/mL group was not significantly different from that of the control group (Fig
The results showed that treatment with 5 μg/mL CopA3 increased (p < 0.01) the number of live cells (Fig
We treated CopA3 (5, 10, 25, and 50 μg/mL) in MSCs to measure the viability and determined the optimal concentration (Fig

후속연구

In conclusion, our results suggest that CopA3 treatment at concentrations of 5 and 10 μg/mL increases MSCs proliferation by promoting the transition from G1 to S phase, as well as upregulating PAX7 and MYOD expression. Further studies are needed to identify the precise molecular mechanisms underlying these phenomena. This study may have implications for the application of insect-derived peptides in increasing the productivity of the livestock industry as well as the field of cultured meat.
Further studies are needed to identify the precise molecular mechanisms underlying these phenomena. This study may have implications for the application of insect-derived peptides in increasing the productivity of the livestock industry as well as the field of cultured meat.

참고문헌 (47)

1. Casciaro B Cappiello F Loffredo MR Mangoni ML Methods for the in vitro examination of the antibacterial and cytotoxic activities of antimicrobial peptides Sandrelli F Tettamanti G Immunity in insects. New York, NY Humana 2020 p 147 62 10.1007/978-1-0716-0259-1_9
2. Stork NE How many species of insects and other terrestrial arthropods are there on Earth Annu Rev Entomol. 2018 63 31 45 10.1146/annurev-ento-020117-043348 28938083

상세보기
3. Li Y Xiang Q Zhang Q Huang Y Su Z Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application Peptides. 2012 37 207 15 10.1016/j.peptides.2012.07.001 22800692

상세보기
4. Wu Q Patočka J Kuča K Insect antimicrobial peptides, a mini review Toxins. 2018 10 461 10.3390/toxins10110461 30413046

상세보기
5. Xiao H Shao F Wu M Ren W Xiong X Tan B The application of antimicrobial peptides as growth and health promoters for swine J Anim Sci Biotechnol. 2015 6 19 10.1186/s40104-015-0018-z 26019864

상세보기
6. Kim HJ Kim DH Lee JY Hwang JS Lee JH Lee SG Study of anti-inflammatory effect of CopA3 peptide derived from Copris tripartitus J Life Sci. 2013 23 38 43 10.5352/JLS.2013.23.1.38

원문보기 상세보기
7. Lee J Kim I Shin Y Park H Lee Y Lee I Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells Tumor Biol. 2016 37 3237 45 10.1007/s13277-015-4162-z 26432335

상세보기
8. Kim IW Kim S Kwon YN Yun EY Ahn MY Kang DC Effects of the synthetic coprisin analog peptide, CopA3 in pathogenic microorganisms and mammalian cancer cells J Microbiol Biotechnol. 2012 22 156 8 10.4014/jmb.1109.09014 22297233

원문보기 상세보기
9. Kim S Kim IW Kwon YN Yun EY Hwang JS Synthetic Coprisin analog peptide, D-CopA3 has antimicrobial activity and pro-apoptotic effects in human leukemia cells J Microbiol Biotechnol. 2012 22 264 9 10.4014/jmb.1110.10071 22370360

원문보기 상세보기
10. Lee JH Kim IW Kim SH Yun EY Nam SH Ahn MY Anticancer activity of CopA3 dimer peptide in human gastric cancer cells BMB Rep. 2015 48 324 9 10.5483/BMBRep.2015.48.6.073 25047444

원문보기 상세보기
11. Dayton WR White ME Cellular and molecular regulation of muscle growth and development in meat animals J Anim Sci. 2008 86 E217 25 10.2527/jas.2007-0456 17709769

상세보기
12. Zammit PS Beauchamp JR The skeletal muscle satellite cell: stem cell or son of stem cell? Differentiation. 2001 68 193 204 10.1046/j.1432-0436.2001.680407.x 11776472

상세보기
13. Dhawan J Rando TA Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment Trends Cell Biol. 2005 15 666 73 10.1016/j.tcb.2005.10.007 16243526

상세보기
14. Marroncelli N Bianchi M Bertin M Consalvi S Saccone V De Bardi M HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes Sci Rep. 2018 8 3448 10.1038/s41598-018-21835-7 29472596

상세보기
15. Pavlath GK Horsley V Cell fusion in skeletal muscle: central role of NFATC2 in regulating muscle cell size Cell Cycle. 2003 2 420 3 10.4161/cc.2.5.497 12963831

상세보기
16. Mesires NT Doumit ME Satellite cell proliferation and differentiation during postnatal growth of porcine skeletal muscle Am J Physiol Cell Physiol. 2002 282 C899 906 10.1152/ajpcell.00341.2001 11880278

상세보기
17. Pallafacchina G Blaauw B Schiaffino S Role of satellite cells in muscle growth and maintenance of muscle mass Nutr Metab Cardiovasc Dis. 2013 23 S12 8 10.1016/j.numecd.2012.02.002 22621743

상세보기
18. Bentzinger CF Wang YX Rudnicki MA Building muscle: molecular regulation of myogenesis Cold Spring Harb Perspect Biol. 2012 4 a008342 10.1101/cshperspect.a008342 22300977
19. Moresi V Marroncelli N Adamo S New insights into the epigenetic control of satellite cells World J Stem Cells. 2015 7 945 55 10.4252/wjsc.v7.i6.945 26240681
20. Yin H Price F Rudnicki MA Satellite cells and the muscle stem cell niche Physiol Rev. 2013 93 23 67 10.1152/physrev.00043.2011 23303905

상세보기
21. Le Grand F Rudnicki MA Skeletal muscle satellite cells and adult myogenesis Curr Opin Cell Biol. 2007 19 628 33 10.1016/j.ceb.2007.09.012 17996437

상세보기
22. Oishi Y Hayashida M Tsukiashi S Taniguchi K Kami K Roy RR Heat stress increases myonuclear number and fiber size via satellite cell activation in rat regenerating soleus fibers J Appl Physiol. 2009 107 1612 21 10.1152/japplphysiol.91651.2008 19556452

상세보기
23. Kamanga-Sollo E Pampusch MS White ME Hathaway MR Dayton WR Effects of heat stress on proliferation, protein turnover, and abundance of heat shock protein messenger ribonucleic acid in cultured porcine muscle satellite cells J Anim Sci. 2011 89 3473 80 10.2527/jas.2011-4123 21742942

상세보기
24. Xiong X Yang HS Li L Wang YF Huang RL Li FN Effects of antimicrobial peptides in nursery diets on growth performance of pigs reared on five different farms Livest Sci. 2014 167 206 10 10.1016/j.livsci.2014.04.024

상세보기
25. Park J Lee J Song KD Kim SJ Kim DC Lee SC Growth factors improve the proliferation of Jeju black pig muscle cells by regulating myogenic differentiation 1 and growth-related genes Anim Biosci. 2021 34 1392 402 10.5713/ab.20.0585 33561926

원문보기 상세보기
26. 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 10.1006/meth.2001.1262 11846609

상세보기
27. Kang BR Kim H Nam SH Yun EY Kim SR Ahn MY CopA3 peptide from Copris tripartitus induces apoptosis in human leukemia cells via a caspase-independent pathway BMB Rep. 2012 45 85 90 10.5483/BMBRep.2012.45.2.85 22360885

원문보기 상세보기
28. Nam ST Kim DH Lee MB Nam HJ Kang JK Park MJ Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis Biochem Biophys Res Commun. 2013 437 35 40 10.1016/j.bbrc.2013.06.031 23791873

상세보기
29. Kim DH Hwang JS Lee IH Nam ST Hong J Zhang P The insect peptide CopA3 increases colonic epithelial cell proliferation and mucosal barrier function to prevent inflammatory responses in the gut J Biol Chem. 2016 291 3209 23 10.1074/jbc.M115.682856 26655716

상세보기
30. Graña X Reddy EP Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs) Oncogene. 1995 11 211 9 7624138

상세보기
31. Nigg EA Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle BioEssays. 1995 17 471 80 10.1002/bies.950170603 7575488

상세보기
32. Starostina NG Kipreos ET Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors Trends Cell Biol. 2012 22 33 41 10.1016/j.tcb.2011.10.004 22154077

상세보기
33. Sato J Nair K Hiddinga J Eberhardt NL Fitzpatrick LA Katusic ZS eNOS gene transfer to vascular smooth muscle cells inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis Cardiovasc Res. 2000 47 697 706 10.1016/S0008-6363(00)00137-1 10974218

상세보기
34. Bond M Sala-Newby GB Wu YJ Newby AC Biphasic effect of p21Cip1 on smooth muscle cell proliferation: role of PI 3-kinase and Skp2-mediated degradation Cardiovasc Res. 2006 69 198 206 10.1016/j.cardiores.2005.08.020 16212951

상세보기
35. Quasnichka H Slater SC Beeching CA Boehm M Sala-Newby GB George SJ Regulation of smooth muscle cell proliferation by β-catenin/T-cell factor signaling involves modulation of cyclin D1 and p21 expression Circ Res. 2006 99 1329 37 10.1161/01.RES.0000253533.65446.33 17122440

상세보기
36. Qin LL Li XK Xu J Mo DL Tong X Pan ZC Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors Mol Cell Biochem. 2012 370 221 30 10.1007/s11010-012-1413-9 22875667

상세보기
37. Li J Han S Cousin W Conboy IM Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells Stem Cells. 2015 33 951 61 10.1002/stem.1908 25447026

상세보기
38. Golias CH Charalabopoulos A Charalabopoulos K Cell proliferation and cell cycle control: a mini review Int J Clin Pract. 2004 58 1134 41 10.1111/j.1742-1241.2004.00284.x 15646411

상세보기
39. Chakravarthy MV Abraha TW Schwartz RJ Fiorotto ML Booth FW Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3′-kinase/AKT signaling pathway J Biol Chem. 2000 275 35942 52 10.1074/jbc.M005832200 10962000

상세보기
40. Belal SA Sivakumar AS Kang DR Cho S Choe HS Shim KS Modulatory effect of linoleic and oleic acid on cell proliferation and lipid metabolism gene expressions in primary bovine satellite cells Anim Cells Syst. 2018 22 324 33 10.1080/19768354.2018.1517824 30460114

상세보기
41. Gabrielli B Brooks K Pavey S Defective cell cycle checkpoints as targets for anti-cancer therapies Front Pharmacol. 2012 3 9 10.3389/fphar.2012.00009 22347187

상세보기
42. Schmidt M Schüler SC Hüttner SS von Eyss B von Maltzahn J Adult stem cells at work: regenerating skeletal muscle Cell Mol Life Sci. 2019 76 2559 70 10.1007/s00018-019-03093-6 30976839

상세보기
43. Olguin HC Olwin BB Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal Dev Biol. 2004 275 375 88 10.1016/j.ydbio.2004.08.015 15501225

상세보기
44. Wen Y Bi P Liu W Asakura A Keller C Kuang S Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells Mol Cell Biol. 2012 32 2300 11 10.1128/MCB.06753-11 22493066

상세보기
45. Dumont NA Wang YX Rudnicki MA Intrinsic and extrinsic mechanisms regulating satellite cell function Development. 2015 142 1572 81 10.1242/dev.114223 25922523

상세보기
46. Zammit PS Relaix F Nagata Y Ruiz AP Collins CA Partridge TA Pax7 and myogenic progression in skeletal muscle satellite cells J Cell Sci. 2006 119 1824 32 10.1242/jcs.02908 16608873

상세보기
47. Day K Paterson B Yablonka‐Reuveni Z A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis Dev Dyn. 2009 238 1001 9 10.1002/dvdy.21903 19301399

상세보기

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