Transcription factor EB‑mediated autophagy promotes dermal fibroblast differentiation and collagen production by regulating endoplasmic reticulum stress and autophagy‑dependent secretion

Zhou,Ling; Liu,Zeming; Chen,Sichao; Qiu,Jing; Li,Qianqian; Wang,Shipei; Zhou,Wei; Chen,Danyang; Yang,Guang; Guo,Liang

doi:10.3892/ijmm.2020.4814

Transcription factor EB‑mediated autophagy promotes dermal fibroblast differentiation and collagen production by regulating endoplasmic reticulum stress and autophagy‑dependent secretion

Authors:
- Ling Zhou
- Zeming Liu
- Sichao Chen
- Jing Qiu
- Qianqian Li
- Shipei Wang
- Wei Zhou
- Danyang Chen
- Guang Yang
- Liang Guo
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Affiliations: Department of Plastic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Geriatrics, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, Hubei 430070, P.R. China
Published online on: December 9, 2020 https://doi.org/10.3892/ijmm.2020.4814
Pages: 547-560
Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Autophagy is reported to be involved in the formation of skin hypertrophic scar (HTS). However, the role of autophagy in the process of fibrosis remains unclear, therefore an improved understanding of the molecular mechanisms associated with autophagy may accelerate the development of effective therapeutic strategies against HTS. The present study evaluated the roles of autophagy mediated by transcription factor EB (TFEB), a pivotal regulator of lysosome biogenesis and autophagy, in transforming growth factor‑β1 (TGF‑β1)‑induced fibroblast differentiation and collagen production. Fibroblasts were treated with TGF‑β1, TGF‑β1 + tauroursodeoxycholic acid (TUDCA) or TGF‑β1 + TFEB‑small interfering RNA (siRNA). TGF‑β1 induced phenotypic transformation of fibroblasts, as well as collagen synthesis and secretion in fibroblasts in a dose‑dependent manner. Western blotting and immunofluorescence analyses demonstrated that TGF‑β1 upregulated the expression of autophagy‑related proteins through the endoplasmic reticulum (ER) stress pathway, whereas TUDCA reversed TGF‑β1‑induced changes. Reverse transcription‑quantitative PCR (RT‑qPCR), western blotting and RFP‑GFP‑LC3 double fluorescence analyses demonstrated that knockdown of TFEB by TFEB‑siRNA decreased autophagic flux, upregulated the expression of proteins involved in the apoptotic pathway, such as phosphorylated‑α subunit of eukaryotic initiation factor 2, C/EBP homologous protein and cysteinyl aspartate specific proteinase 3, and also downregulated the expression of α‑smooth muscle actin and collagen I (COL I) in fibroblasts. Immunofluorescence confocal analyses and enzyme‑linked immunosorbent assay indicated that TGF‑β1 increased the colocalization of COL I with lysosomal‑associated membrane protein 1 and Ras‑related protein Rab‑8A, a marker of secretory vesicles, in fibroblasts, as well as the secretion of pro‑COL Iα1 in culture supernatants. Meanwhile, these effects were abolished by TFEB knockdown. The present results suggested that autophagy reduced ER stress, decreased cell apoptosis and maintained fibroblast activation not only through degradation of misfolded or unfolded proteins, but also through promotion of COL I release from the autolysosome to the extracellular environment.

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1	Benzaquen M, Collet-Villette AM and Delaporte E: Combined treatment of hypertrophic and keloid scars with intralesional injection of corticosteroids and laser-assisted corticosteroid delivery. Dermatol Ther. 32:e131262019. View Article : Google Scholar : PubMed/NCBI
2	Del Toro D, Dedhia R and Tollefson TT: Advances in scar management: Prevention and management of hypertrophic scars and keloids. Curr Opin Otolaryngol Head Neck Surg. 24:322–329. 2016. View Article : Google Scholar : PubMed/NCBI
3	Zhang J, Li Y, Bai X, Li Y, Shi J and Hu D: Recent advances in hypertrophic scar. Histol Histopathol. 33:27–39. 2018.
4	Gras C, Ratuszny D, Hadamitzky C, Zhang H, Blasczyk R and Figueiredo C: miR-145 contributes to hypertrophic scarring of the skin by inducing myofibroblast activity. Mol Med. 21:296–304. 2015. View Article : Google Scholar : PubMed/NCBI
5	Darby IA, Zakuan N, Billet F and Desmoulière A: The myofibro-blast, a key cell in normal and pathological tissue repair. Cell Mol Life Sci. 73:1145–1157. 2016. View Article : Google Scholar
6	Hinz B: The role of myofibroblasts in wound healing. Curr Res Transl Med. 64:171–177. 2016. View Article : Google Scholar : PubMed/NCBI
7	Pakyari M, Farrokhi A, Maharlooei MK and Ghahary A: Critical role of transforming growth factor beta in different phases of wound healing. Adv Wound Care (New Rochelle). 2:215–224. 2013. View Article : Google Scholar
8	Zhang Y, Cheng C, Wang S, Xu M, Zhang D and Zeng W: Knockdown of FOXM1 inhibits activation of keloid fibro-blasts and extracellular matrix production via inhibition of TGF-β1/Smad pathway. Life Sci. 232:1166372019. View Article : Google Scholar
9	Alizadeh J, Glogowska A, Thliveris J, Kalantari F, Shojaei S, Hombach-Klonisch S, Klonisch T and Ghavami S: Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells. Biochim Biophys Acta Mol Cell Res. 1865:749–768. 2018. View Article : Google Scholar : PubMed/NCBI
10	Liang C, Xu J, Meng Q, Zhang B, Liu J, Hua J, Zhang Y, Shi S and Yu X: TGFB1-induced autophagy affects the pattern of pancreatic cancer progression in distinct ways depending on SMAD4 status. Autophagy. 16:486–500. 2020. View Article : Google Scholar :
11	Cuomo F, Altucci L and Cobellis G: Autophagy function and dysfunction: Potential drugs as anti-cancer therapy. Cancers (Basel). 11:14652019. View Article : Google Scholar
12	Fitzwalter BE and Thorburn A: Recent insights into cell death and autophagy. FEBS J. 282:4279–4288. 2015. View Article : Google Scholar : PubMed/NCBI
13	Lu C, Yang Y, Zhu Y, Lv S and Zhang J: An intervention target for myocardial fibrosis: Autophagy. Biomed Res Int. 2018:62159162018. View Article : Google Scholar : PubMed/NCBI
14	Maiuri L and Kroemer G: Autophagy delays progression of the two most frequent human monogenetic lethal diseases: Cystic fibrosis and Wilson disease. Aging (Albany NY). 10:3657–3661. 2018. View Article : Google Scholar
15	Tseng YJ, Dong L, Liu YF, Xu N, Ma W, Weng SQ, Janssen HLA and Wu SD: Role of autophagy in chronic liver inflammation and fibrosis. Curr Protein Pept Sci. 20:817–822. 2019. View Article : Google Scholar : PubMed/NCBI
16	Zhao XC, Livingston MJ, Liang XL and Dong Z: Cell apoptosis and autophagy in renal fibrosis. Adv Exp Med Biol. 1165:557–584. 2019. View Article : Google Scholar : PubMed/NCBI
17	Lodder J, Denaës T, Chobert MN, Wan J, El-Benna J, Pawlotsky JM, Lotersztajn S and Teixeira-Clerc F: Macrophage autophagy protects against liver fibrosis in mice. Autophagy. 11:1280–1292. 2015. View Article : Google Scholar : PubMed/NCBI
18	Cabrera S, Maciel M, Herrera I, Nava T, Vergara F, Gaxiola M, López-Otín C, Selman M and Pardo A: Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis. Autophagy. 11:670–684. 2015. View Article : Google Scholar : PubMed/NCBI
19	Takagaki Y, Lee SM, Dongqing Z, Kitada M, Kanasaki K and Koya D: Endothelial autophagy deficiency induces IL6-dependent endothelial mesenchymal transition and organ fibrosis. Autophagy. 16:1905–1914. 2020. View Article : Google Scholar : PubMed/NCBI
20	Frangou E, Chrysanthopoulou A, Mitsios A, Kambas K, Arelaki S, Angelidou I, Arampatzioglou A, Gakiopoulou H, Bertsias GK, Verginis P, et al: REDD1/autophagy pathway promotes thromboinflammation and fibrosis in human systemic lupus erythematosus (SLE) through NETs decorated with tissue factor (TF) and interleukin-17A (IL-17A). Ann Rheum Dis. 78:238–248. 2019. View Article : Google Scholar :
21	San-Miguel B, Crespo I, Sánchez DI, González-Fernández B, Ortiz de Urbina JJ, Tuñón MJ and González-Gallego J: Melatonin inhibits autophagy and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis. J Pineal Res. 59:151–162. 2015. View Article : Google Scholar : PubMed/NCBI
22	Livingston MJ, Ding HF, Huang S, Hill JA, Yin XM and Dong Z: Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction. Autophagy. 12:976–998. 2016. View Article : Google Scholar : PubMed/NCBI
23	Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, Erdin SU, Huynh T, Medina D, Colella P, et al: TFEB links autophagy to lysosomal biogenesis. Science. 332:1429–1433. 2011. View Article : Google Scholar : PubMed/NCBI
24	Du F, Zhu L, Qian ZM, Wu XM, Yung WH and Ke Y: Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by up-regulation of HIF-1 alpha expression and binding activity. Biochim Biophys Acta. 1802:1048–1053. 2010. View Article : Google Scholar : PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
26	Cybulsky AV: The intersecting roles of endoplasmic reticulum stress, ubiquitin- proteasome system, and autophagy in the patho-genesis of proteinuric kidney disease. Kidney Int. 84:25–33. 2013. View Article : Google Scholar
27	McCaughey J and Stephens DJ: ER-to-golgi transport: A sizeable problem. Trends Cell Biol. 29:940–953. 2019. View Article : Google Scholar : PubMed/NCBI
28	Schnieder J, Mamazhakypov A, Birnhuber A, Wilhelm J, Kwapiszewska G, Ruppert C, Markart P, Wujak L, Rubio K, Barreto G, et al: Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores. Matrix Biol. 88:69–88. 2020. View Article : Google Scholar
29	Dolivo DM, Larson SA and Dominko T: Fibroblast growth factor 2 as an antifibrotic: Antagonism of myofibroblast differentiation and suppression of pro-fibrotic gene expression. Cytokine Growth Factor Rev. 38:49–58. 2017. View Article : Google Scholar : PubMed/NCBI
30	Meng XM, Nikolic-Paterson DJ and Lan HY: TGF-β: The master regulator of fibrosis. Nat Rev Nephrol. 12:325–338. 2016. View Article : Google Scholar : PubMed/NCBI
31	Kirkness MW, Lehmann K and Forde NR: Mechanics and structural stability of the collagen triple helix. Curr Opin Chem Biol. 53:98–105. 2019. View Article : Google Scholar : PubMed/NCBI
32	Saito K, Maeda M and Katada T: Regulation of the Sar1 GTPase cycle is necessary for large cargo secretion from the endoplasmic reticulum. Front Cell Dev Biol. 5:752017. View Article : Google Scholar : PubMed/NCBI
33	Gjaltema RA and Bank RA: Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease. Crit Rev Biochem Mol Biol. 52:74–95. 2017. View Article : Google Scholar
34	Sorushanova A, Delgado LM, Wu Z, Shologu N, Kshirsagar A, Raghunath R, Mullen AM, Bayon Y, Pandit A, Raghunath M and Zeugolis DI: The collagen suprafamily: From biosynthesis to advanced biomaterial development. Adv Mater. 31:e18016512019. View Article : Google Scholar
35	Ishikawa Y and Bächinger HP: A molecular ensemble in the rER for procollagen maturation. Biochim Biophys Acta. 1833:2479–2491. 2013. View Article : Google Scholar : PubMed/NCBI
36	Hetz C, Zhang K and Kaufman RJ: Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol. 21:421–438. 2020. View Article : Google Scholar : PubMed/NCBI
37	Shu S, Zhu J, Liu Z, Tang C, Cai J and Dong Z: Endoplasmic reticulum stress is activated in post-ischemic kidneys to promote chronic kidney disease. EBioMedicine. 37:269–280. 2018. View Article : Google Scholar : PubMed/NCBI
38	Kropski JA and Blackwell TS: Endoplasmic reticulum stress in the pathogenesis of fibrotic disease. J Clin Invest. 128:64–73. 2018. View Article : Google Scholar : PubMed/NCBI
39	Ishida Y and Nagata K: Autophagy eliminates a specific species of misfolded procollagen and plays a protective role in cell survival against ER stress. Autophagy. 5:1217–1219. 2009. View Article : Google Scholar : PubMed/NCBI
40	Nabar NR, Shi CS and Kehrl JH: Signaling by the toll-like receptors induces autophagy through modification of beclin 1 Molecular Mechanism. Immunology. Hayat MA: Academic Press; London: pp. 75–84. 2018, View Article : Google Scholar
41	Swart C, Du Toit A and Loos B: Autophagy and the invisible line between life and death. Eur J Cell Biol. 95:598–610. 2016. View Article : Google Scholar
42	Füllgrabe J, Klionsky DJ and Joseph B: The return of the nucleus: Transcriptional and epigenetic control of autophagy. Nat Rev Mol Cell Biol. 15:65–74. 2014. View Article : Google Scholar
43	Napolitano G and Ballabio A: TFEB at a glance. J Cell Sci. 129:2475–2481. 2016. View Article : Google Scholar : PubMed/NCBI
44	Kandel-Kfir M, Almog T, Shaish A, Shlomai G, Anafi L, Avivi C, Barshack I, Grosskopf I, Harats D and Kamari Y: Interleukin-1α deficiency attenuates endoplasmic reticulum stress-induced liver damage and CHOP expression in mice. J Hepatol. 63:926–933. 2015. View Article : Google Scholar : PubMed/NCBI
45	Oyadomari S and Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11:381–389. 2004. View Article : Google Scholar
46	Sanchez-Wandelmer J, Ktistakis NT and Reggiori F: ERES: Sites for autophagosome biogenesis and maturation? J Cell Sci. 128:185–192. 2015. View Article : Google Scholar : PubMed/NCBI
47	Ponpuak M, Mandell MA, Kimura T, Chauhan S, Cleyrat C and Deretic V: Secretory autophagy. Curr Opin Cell Biol. 35:106–116. 2015. View Article : Google Scholar : PubMed/NCBI
48	Kimura T, Jia J, Claude-Taupin A, Kumar S, Choi SW, Gu Y, Mudd M, Dupont N, Jiang S, Peters R, et al: Cellular and molecular mechanism for secretory autophagy. Autophagy. 13:1084–1085. 2017. View Article : Google Scholar : PubMed/NCBI
49	Bel S and Hooper LV: Secretory autophagy of lysozyme in Paneth cells. Autophagy. 14:719–721. 2018. View Article : Google Scholar : PubMed/NCBI
50	New J, Arnold L, Ananth M, Alvi S, Thornton M, Werner L, Tawfik O, Dai H, Shnayder Y, Kakarala K, et al: Secretory autophagy in cancer-associated fibroblasts promotes head and neck cancer progression and offers a novel therapeutic target. Cancer Res. 77:6679–6691. 2017. View Article : Google Scholar : PubMed/NCBI
51	Rabouille C: Pathways of unconventional protein secretion. Trends Cell Biol. 27:230–240. 2017. View Article : Google Scholar
52	Gonzalez CD, Resnik R and Vaccaro MI: Secretory autophagy and its relevance in metabolic and degenerative disease. Front Endocrinol (Lausanne). 11:2662020. View Article : Google Scholar
53	Cavalli G and Cenci S: Autophagy and protein secretion. J Mol Biol. 432:2525–2545. 2020. View Article : Google Scholar : PubMed/NCBI
54	Ejlerskov P, Rasmussen I, Nielsen TT, Bergström AL, Tohyama Y, Jensen PH and Vilhardt F: Tubulin polymerization-promoting protein (TPPP/p25α) promotes unconventional secretion of α-synuclein through exophagy by impairing autopha-gosome-lysosome fusion. J Biol Chem. 288:17313–17335. 2013. View Article : Google Scholar : PubMed/NCBI
55	Khoshnoodi J, Cartailler JP, Alvares K, Veis A and Hudson BG: Molecular recognition in the assembly of collagens: Terminal noncollagenous domains are key recognition modules in the formation of triple helical protomers. J Biol Chem. 281:38117–38121. 2006. View Article : Google Scholar : PubMed/NCBI