Abstract
Objective: Mandibular growth that is induced by functional appliances is closely associated with skeletal and neuromuscular adaptation. Accumulating evidence has proved that apoptosis and autophagy have a vital role in adaptation process. However, little is known about the underlying mechanisms. This study sought to determine whether ATF-6 is involved in stretch-induced apoptosis and autophagy in myoblast. The study also sought to uncover the potential molecular mechanism. Materials and methods: Apoptosis was assessed by TUNEL and Annexin V and PI staining. Autophagy was detected by transmission electron microscopy (TEM) analysis and immunofluorescent staining for autophagy-related protein light chain 3 (LC3). Real time-PCR and western blot were performed to evaluate the expression level of mRNA and proteins that were associated with endoplasmic reticulum stress (ERS), autophagy and apoptosis. Results: Cyclic stretch significantly decreased the cell viability and induced apoptosis and autophagy of myoblasts time-dependently. Stretching stimuli activated ATF-6 pathway and induced ERS-mediated apoptosis. Moreover, using 4-PBA significantly inhibited ERS-related apoptosis, as well as partially decreasing autophagy. In addition, inhibition of autophagy by 3-MA enhanced apoptosis by affecting the expression of CHOP and Bcl-2. However, it had no obvious effects on ERS-related proteins of GRP78 and ATF-6. More importantly, knockdown ATF-6 effectively weakened apoptosis and autophagy. It did so by regulating the expression of Bcl-2, Beclin1 and CHOP, but not cleaved Caspase-12, LC3II and p62 in stretched myoblast. Conclusion: ATF-6 pathway was activated in myoblast by mechanical stretch. ATF-6 may regulate the process of stretch-induced myoblast apoptosis and autophagy via CHOP, Bcl-2 and Beclin1 signaling.
Similar content being viewed by others
Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Change history
13 July 2023
A Correction to this paper has been published: https://doi.org/10.1007/s10495-023-01864-y
Abbreviations
- ATF-6:
-
activating transcription factor 6.
- Bcl-2:
-
B-cell lymphoma-2.
- CHOP:
-
CCAAT enhance-binding protein homologous protein.
- DAPI:
-
4′,6-Diamidino-2-phenylindole.
- ERS:
-
endoplasmic reticulum stress.
- FITC:
-
Fluorescein isothiocyanate.
- GRP78:
-
Glucose-regulated protein 78KD.
- LC3:
-
autophagy-related protein light chain 3.
- MTT:
-
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide.
- TEM:
-
transmission electron microscopy.
- TUNEL:
-
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
- UPR:
-
unfolded protein response.
- 3-MA:
-
3-Methyladenine.
- 4-PBA:
-
4-phenylbutyrate.
References
Dimberg L, Arnrup K, Bondemark L (2015) The impact of malocclusion on the quality of life among children and adolescents: a systematic review of quantitative studies. Eur J Orthod 37:238–247. https://doi.org/10.1093/ejo/cju046
Bilgic F, Gelgor IE, Celebi AA (2015) Malocclusion prevalence and orthodontic treatment need in central anatolian adolescents compared to european and other nations’ adolescents. Dent Press J Orthod 20:75–81. https://doi.org/10.1590/2177-6709.20.6.075-081.oar
Proske U, Morgan DL (2001) Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J Physiol 537:333–345. https://doi.org/10.1111/j.1469-7793.2001.00333.x
Hiyama S, Ono PT, Ishiwata Y, et al (2000) Neuromuscular and skeletal adaptations following mandibular forward positioning induced by the Herbst appliance. Angle Orthod 70:442–453. https://doi.org/10.1043/0003-3219(2000)070<0442:NASAFM>2.0.CO;2
Rando TA (2001) The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve 24:1575–1594. https://doi.org/10.1002/mus.1192
Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257. https://doi.org/10.1038/bjc.1972.33
Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol 10:173–194. https://doi.org/10.1146/annurev-pathol-012513-104649
Hu H, Tian M, Ding C, Yu S (2018) The C/EBP homologous protein (CHOP) transcription factor functions in endoplasmic reticulum Stress-Induced apoptosis and microbial infection. Front Immunol 9:3083. https://doi.org/10.3389/fimmu.2018.03083
Matsumoto H, Miyazaki S, Matsuyama S et al (2013) Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biol Open 2:1084–1090. https://doi.org/10.1242/bio.20135033
Cebollero E, Reggiori F, Kraft C (2012) Reticulophagy and ribophagy: regulated degradation of protein production factories. Int J Cell Biol 2012:182834. https://doi.org/10.1155/2012/182834
Song J, Zhang Q, Wang S et al (2018) Cleavage of caspase-12 at Asp94, mediated by endoplasmic reticulum stress (ERS), contributes to stretch-induced apoptosis of myoblasts. J Cell Physiol 233:9473–9487. https://doi.org/10.1002/jcp.26840
Zhang Q, Liu J, Chen S et al (2016) Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis 21:432–442. https://doi.org/10.1007/s10495-016-1217-6
Du Y, Yang F, Lv D et al (2019) MiR-147 inhibits cyclic mechanical stretch-induced apoptosis in L6 myoblasts via ameliorating endoplasmic reticulum stress by targeting BRMS1. Cell Stress Chaperones 24:1151–1161. https://doi.org/10.1007/s12192-019-01037-4
Fernández A, Ordóñez R, Reiter RJ et al (2015) Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis. J Pineal Res 59:292–307. https://doi.org/10.1111/jpi.12264
Cheng Y-C, Chang J-M, Chen C-A, Chen H-C (2015) Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: a protective role. Exp Biol Med (Maywood) 240:467–476. https://doi.org/10.1177/1535370214553772
Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752. https://doi.org/10.1038/nrm2239
Mukhopadhyay S, Panda PK, Sinha N et al (2014) Autophagy and apoptosis: where do they meet? Apoptosis 19:555–566. https://doi.org/10.1007/s10495-014-0967-2
Masud Alam M, Kariya R, Kawaguchi A et al (2016) Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress. Apoptosis 21:1191–1201. https://doi.org/10.1007/s10495-016-1277-7
Li X, Su J, Xia M et al (2016) Caspase-mediated cleavage of Beclin1 inhibits autophagy and promotes apoptosis induced by S1 in human ovarian cancer SKOV3 cells. Apoptosis 21:225–238. https://doi.org/10.1007/s10495-015-1197-y
Ye J, Rawson RB, Komuro R et al (2000) ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6:1355–1364. https://doi.org/10.1016/s1097-2765(00)00133-7
Wu J, Rutkowski DT, Dubois M et al (2007) ATF6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress. Dev Cell 13:351–364. https://doi.org/10.1016/j.devcel.2007.07.005
Yamamoto K, Sato T, Matsui T et al (2007) Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev Cell 13:365–376. https://doi.org/10.1016/j.devcel.2007.07.018
Tao J, Chen H, Li X, Wang J (2021) The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis. J Orthop Surg Res 16:1. https://doi.org/10.1186/s13018-020-02056-z
Yue L-L, Du X (2022) Thrombospondin 1 promotes endoplasmic reticulum stress and apoptosis in HK-2 cells by upregulating ATF6-CHOP. Curr Med Sci 42:341–347. https://doi.org/10.1007/s11596-022-2513-8
Ohoka N, Yoshii S, Hattori T et al (2005) TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J 24:1243–1255. https://doi.org/10.1038/sj.emboj.7600596
Czabotar PE, Lessene G, Strasser A, Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15:49–63. https://doi.org/10.1038/nrm3722
Liu J, Liu W, Lu Y et al (2018) Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models. Autophagy 14:845–861. https://doi.org/10.1080/15548627.2017.1390636
Liang XH, Kleeman LK, Jiang HH et al (1998) Protection against fatal Sindbis virus encephalitis by beclin, a novel bcl-2-interacting protein. J Virol 72:8586–8596. https://doi.org/10.1128/JVI.72.11.8586-8596.1998
Ren D, Liu R, Yan X et al (2022) Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through ca(2+) overloading. Apoptosis 27:929–945. https://doi.org/10.1007/s10495-022-01759-4
Marchiori GE, Sodré LO, da Cunha TCR et al (2015) Pleasantness of facial profile and its correlation with soft tissue cephalometric parameters: perception of orthodontists and lay people. Eur J Dent 9:352–355. https://doi.org/10.4103/1305-7456.163323
Seehra J, Fleming PS, Newton T, DiBiase AT (2011) Bullying in orthodontic patients and its relationship to malocclusion,self-esteem and oral health-related quality of life. J Orthod 38:247–256 quiz 294. https://doi.org/10.1179/14653121141641
Aggarwal P, Kharbanda OP, Mathur R et al (1999) Muscle response to the twin-block appliance: an electromyographic study of the masseter and anterior temporal muscles. Am J Orthod Dentofac Orthop Off Publ Am Assoc Orthod its Const Soc Am Board Orthod 116:405–414. https://doi.org/10.1016/S0889-5406(99)70225-8
Ma Y, Fu S, Lu L, Wang X (2017) Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways. Mol Cell Endocrinol 450:83–93. https://doi.org/10.1016/j.mce.2017.04.021
Formigli L, Sassoli C, Squecco R et al (2009) Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation. J Cell Sci 122:1322–1333. https://doi.org/10.1242/jcs.035402
Ruoslahti E (1997) Stretching is good for a cell. Science 276:1345–1346. https://doi.org/10.1126/science.276.5317.1345
Song J, Hu B, Qu H et al (2012) Mechanical stretch modulates microRNA 21 expression, participating in proliferation and apoptosis in cultured human aortic smooth muscle cells. PLoS ONE 7:e47657. https://doi.org/10.1371/journal.pone.0047657
Chen J, Liu Z, Zhong G et al (2016) Cyclic stretch enhances apoptosis in human lumbar ligamentum flavum cells via the induction of reactive oxygen species generation. J Spinal Cord Med 39:450–454. https://doi.org/10.1080/10790268.2016.1141470
Feng Y, Tian X-Y, Sun P et al (2018) Simultaneous study of Mechanical Stretch-Induced Cell Proliferation and apoptosis on C2C12 myoblasts. Cells Tissues Organs 205:189–196. https://doi.org/10.1159/000490239
Mayr M, Hu Y, Hainaut H, Xu Q (2002) Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells. FASEB J Off Publ Fed Am Soc Exp Biol 16:1423–1425. https://doi.org/10.1096/fj.02-0042fje
Wang F, Wei Z-L, Sun X-R et al (2017) Apoptosis inducing factor is involved in Stretch-Induced apoptosis of myoblast via a Caspase-9 independent pathway. J Cell Biochem 118:829–838. https://doi.org/10.1002/jcb.25759
Liu J, Wang Y, Yuan X et al (2010) Cyclic-stretch induces the apoptosis of myoblast by activation of Caspase-3 protease in a magnitude-dependent manner. Int J Biochem Cell Biol 42:2004–2011. https://doi.org/10.1016/j.biocel.2010.08.013
Clarke PG (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 181:195–213. https://doi.org/10.1007/BF00174615
Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306:990–995. https://doi.org/10.1126/science.1099993
Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721. https://doi.org/10.1126/science.290.5497.1717
Wirawan E, Vande Walle L, Kersse K et al (2010) Caspase-mediated cleavage of Beclin-1 inactivates beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis 1:e18. https://doi.org/10.1038/cddis.2009.16
Ravegnini G, Sammarini G, Nannini M et al (2017) Gastrointestinal stromal tumors (GIST): facing cell death between autophagy and apoptosis. Autophagy 13:452–463. https://doi.org/10.1080/15548627.2016.1256522
Yang L, Guan G, Lei L et al (2018) Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy. Cell Stress Chaperones 23:1283–1294. https://doi.org/10.1007/s12192-018-0936-8
Hsu Y-H, Chuang H-C, Lee Y-H et al (2019) Traffic-related particulate matter exposure induces nephrotoxicity in vitro and in vivo. Free Radic Biol Med 135:235–244. https://doi.org/10.1016/j.freeradbiomed.2019.03.008
Ciechomska IA, Gabrusiewicz K, Szczepankiewicz AA, Kaminska B (2013) Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death. Oncogene 32:1518–1529. https://doi.org/10.1038/onc.2012.174
Peiqi L, Rong H, Hongming D et al (2018) GDC-0152-induced autophagy promotes apoptosis in HL-60 cells. Mol Cell Biochem 445:135–143. https://doi.org/10.1007/s11010-017-3259-7
Park A, Koh HC (2019) NF-κB/mTOR-mediated autophagy can regulate diquat-induced apoptosis. Arch Toxicol 93:1239–1253. https://doi.org/10.1007/s00204-019-02424-7
Nakanishi K, Sudo T, Morishima N (2005) Endoplasmic reticulum stress signaling transmitted by ATF6 mediates apoptosis during muscle development. J Cell Biol 169:555–560. https://doi.org/10.1083/jcb.200412024
Yang H, Niemeijer M, van de Water B, Beltman JB (2020) ATF6 is a critical determinant of CHOP Dynamics during the unfolded protein response. iScience 23:100860. https://doi.org/10.1016/j.isci.2020.100860
Wang J, Kang R, Huang H et al (2014) Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression. Autophagy 10:766–784. https://doi.org/10.4161/auto.27954
Zhou Y, Zhang S, Dai C et al (2016) Quinocetone triggered ER stress-induced autophagy via ATF6/DAPK1-modulated mAtg9a trafficking. Cell Biol Toxicol 32:141–152. https://doi.org/10.1007/s10565-016-9323-3
Dang J, Bian X, Ma X et al (2017) ORMDL3 facilitates the survival of Splenic B cells via an ATF6α-Endoplasmic reticulum Stress-Beclin1 Autophagy Regulatory Pathway. J Immunol 199:1647–1659. https://doi.org/10.4049/jimmunol.1602124
Pattingre S, Tassa A, Qu X et al (2005) Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell 122:927–939. https://doi.org/10.1016/j.cell.2005.07.002
Xu H-D, Wu D, Gu J-H, et al (2013) The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions. PLoS One 8:e63232. https://doi.org/10.1371/journal.pone.0063232
Acknowledgements
The authors thank the laboratory members of Central Laboratory of Affiliated Hospital of Qingdao University for their help and instructions.
Funding
This work was supported by the National Natural Science Foundation of China [grant number 32171303].
Author information
Authors and Affiliations
Contributions
Conceptualization: Yuan X,Yan X; Experiment performance: ZQ,LG and LR; Data collection and analysis: LJ; ZQ,ZX and RD wrote the main manuscript text; ZQ prepared Figs. 1, 2 and 3 and LG prepared Figs. 4, 5 and 6; All authors reviewed the manuscript.
Corresponding authors
Ethics declarations
Conflict of Interest
All authors declare that they have no conflict of interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhang, Q., Liu, G., Liu, R. et al. Dual role of endoplasmic reticulum stress-ATF-6 activation in autophagy and apoptosis induced by cyclic stretch in myoblast. Apoptosis 28, 796–809 (2023). https://doi.org/10.1007/s10495-023-01825-5
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10495-023-01825-5