Exosomal miR-17-5p derived from epithelial cells is involved in aberrant epithelium-fibroblast crosstalk and induces the development of oral submucosal fibrosis

Abstract

Oral submucous fibrosis (OSF) is a chronic and inflammatory mucosal disease caused by betel quid chewing, which belongs to oral potentially malignant disorders. Abnormal fibroblast differentiation leading to disordered collagen metabolism is the core process underlying OSF development. The epithelium, which is the first line of defense against the external environment, can convert external signals into pathological signals and participate in the remodeling of the fibrotic microenvironment. However, the specific mechanisms by which the epithelium drives fibroblast differentiation remain unclear. In this study, we found that Arecoline-exposed epithelium communicated with the fibrotic microenvironment by secreting exosomes. MiR-17-5p was encapsulated in epithelial cell-derived exosomes and absorbed by fibroblasts, where it promoted cell secretion, contraction, migration and fibrogenic marker (α-SMA and collagen type I) expression. The underlying molecular mechanism involved miR-17-5p targeting Smad7 and suppressing the degradation of TGF-β receptor 1 (TGFBR1) through the E3 ubiquitination ligase WWP1, thus facilitating downstream TGF-β pathway signaling. Treatment of fibroblasts with an inhibitor of miR-17-5p reversed the contraction and migration phenotypes induced by epithelial-derived exosomes. Exosomal miR-17-5p was confirmed to function as a key regulator of the phenotypic transformation of fibroblasts. In conclusion, we demonstrated that Arecoline triggers aberrant epithelium-fibroblast crosstalk and identified that epithelial cell-derived miR-17-5p mediates fibroblast differentiation through the classical TGF-β fibrotic pathway, which provided a new perspective and strategy for the diagnosis and treatment of OSF.

References

1. Wynn T. A.& Ramalingam, T. R. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease.Nat. Med. 18, 1028-1040 (2012).
2. Zhao, M.et al.Targeting fibrosis, mechanisms and cilinical trials.Signal Transduct Target Ther. 7, 206(2022).
3. Henderson, N. C., Rieder, F.& Wynn, T. A. Fibrosis: From mechanisms to medicines.Nature 587, 555-566 (2020).
4. Yuwanati, M.et al.Prevalence of oral submucous fibrosis among areca nut chewers: A systematic review and meta-analysis.Oral Dis. 29, 1920-1926 (2023).
5. Qin, X.et al.Oral submucous fibrosis: Etiological mechanism, malignant transformation, therapeutic approaches and targets.Int. J. Mol. Sci. 24, 4992(2023).
6. Ray, J. G., Chatterjee, R.& Chaudhuri, K. Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities.Periodontol 2000 80, 200-212 (2019).
7. Sharma, M.et al.Loss of oral mucosal stem cell markers in oral submucous fibrosis and their reactivation in malignant transformation.Int. J. Oral Sci. 12, 23(2020).
8. Ko A. M.S., Tu, H. P. & Ko, Y. C. Systematic review of roles of arecoline and arecoline N-oxide in oral cancer and strategies to block carcinogenesis.Cells 12, 1208(2023).
9. Plikus, M. V.et al.Fibroblasts: Origins, definitions, and functions in health and disease.Cell 184, 3852-3872 (2021).
10. Lynch M. D.& Watt, F. M. Fibroblast heterogeneity: Implications for human disease.J. Clin. Invest. 128, 26-35 (2018).
11. Wei, K., Nguyen, H. N.& Brenner, M. B. Fibroblast pathology in inflammatory diseases.J. Clin. Invest. 131, e149538(2021).
12. Buechler, M. B., Fu, W.& Turley, S. J. Fibroblast-macrophage reciprocal interactions in health, fibrosis, and cancer.Immunity 54, 903-915 (2021).
13. Lendahl, U., Muhl, L.& Betsholtz, C. Identification, discrimination and heterogeneity of fibroblasts.Nat. Commun 13, 3409(2022).
14. Pakshir, P.et al.The myofibroblast at a glance.J. Cell Sci. 133, jcs227900 (2020).
15. Ogawa M.,LaRue, A. C. & Drake, C. J. Hematopoietic origin of fibroblasts/myofibroblasts: Its pathophysiologic implications.Blood 108, 2893-2896 (2006).
16. Schuster, R.et al.The role of myofibroblasts in physiological and pathological tissue repair.Cold. Spring Harb. Perspect. Biol. 15, a041231(2023).
17. Gibb, A. A., Lazaropoulos, M. P.& Elrod, J. W. Myofibroblasts and fibrosis: Mitochondrial and metabolic control of cellular differentiation.Circ. Res. 127, 427-447 (2020).
18. Hinz B.& Lagares, D. Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases.Nat. Rev. Rheumatol 16, 11-31 (2020).
19. Waasdorp, M.et al.The bigger picture: Why oral mucosa heals better than skin.Biomolecules 11, 1165(2021).
20. Farid, H.et al.Oral manifestations of Covid-19-A literature review.Rev. Med. Virol. 32, e2248(2022).
21. Nikoloudaki, G., Creber, K.& Hamilton, D. W. Wound healing and fibrosis: A contrasting role for periostin in skin and the oral mucosa.Am. J. Physiol. Cell Physiol. 318, C1065-C1077 (2020).
22. Katsuno Y.& Derynck, R. Epithelial plasticity, epithelial-mesenchymal transition, and the TGF-beta family.Dev Cell 56, 726-746 (2021).
23. Théry, C., Zitvogel, L.& Amigorena, S. Exosomes: Composition, biogenesis and function.Nat. Rev. Immunol 2, 569-579 (2002).
24. Isaac, R.et al.Exosomes as mediators of intercellular crosstalk in metabolism.Cell Metab. 33, 1744-1762 (2021).
25. Li, S. R.et al.Tissue-derived extracellular vesicles in cancers and non-cancer diseases: Present and future.J. Extracell Vesicles 10, e12175(2021).
26. Xie, C.et al.The role of extracellular vesicles from different origin in the microenvironment of head and neck cancers.Mol. Cancer 18, 83(2019).
27. Mathieu, M.et al.Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.Nat. Cell Biol. 21, 9-17 (2019).
28. Kalluri R.& LeBleu, V. S. The biology, function, and biomedical applications of exosomes.Science 367, eaau6977 (2020).
29. Gao, S.et al.PKM2 promotes pulmonary fibrosis by stabilizing TGF-beta1 receptor I and enhancing TGF-beta1 signaling.Sci. Adv. 8, eabo0987 (2022).
30. Xu, H. Q.et al.Fibrotic matrix induces mesenchymal transformation of epithelial cells in oral submucous fibrosis.Am. J. Pathol 193, 1208-1222 (2023).
31. Hu, X.et al.Overexpression of DEC1 in the epithelium of OSF promotes mesenchymal transition via activating FAK/Akt signal axis.J. Oral Pathol Med. 51, 780-790 (2022).
32. Li, M.et al. Fibroblast activating protein promotes the proliferation, migration,activation of fibroblasts in oral submucous fibrosis. Oral Dis. Epub ahead of print. (2023).
33. Kuang, H.et al. DNA methyltransferase 3A induces the occurrence of oral submucous fibrosis by promoting the methylation of the von Hippel-Lindau. Oral Dis. Epub ahead of print. (2023).
34. Peng, C. Y.et al.Positive feedback loop of SNAIL-IL-6 mediates myofibroblastic differentiation activity in precancerous oral submucous fibrosis.Cancers (Basel) 12, 1611(2020).
35. Fang, C. Y.et al.Slug mediates myofibroblastic differentiation to promote fibrogenesis in buccal mucosa.J. Cell Physiol. 234, 6721-6730 (2019).
36. Xie, C.et al.Identification of a BRAF/PA28gamma/MEK1 signaling axis and its role in epithelial-mesenchymal transition in oral submucous fibrosis.Cell Death Dis. 13, 701(2022).
37. Dinh P. U.C. et al. Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis.Nat. Commun 11, 1064(2020).
38. Xu, H.et al.Exosomal microRNA-21 derived from bronchial epithelial cells is involved in aberrant epithelium-fibroblast cross-talk in COPD induced by cigarette smoking.Theranostics 8, 5419-5433 (2018).
39. Zhou, X.et al.Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo.Int. J. Biol. Sci. 17, 4021-4033 (2021).
40. Garcia-Martin, R. et al. MicroRNA sequence codes for small extracellular vesicle release and cellular retention.Nature 601, 446-451 (2022).
41. Zhao, S.et al.Tumor-derived exosomal miR-934 induces macrophage M2 polarization to promote liver metastasis of colorectal cancer.J. Hematol. Oncol. 13, 156(2020).
42. Chou, M. Y.et al.MiR-424/TGIF2-mediated pro-fibrogenic responses in oral submucous fibrosis.Int. J. Mol. Sci. 24, 5811(2023).
43. Chou, M. Y.et al.Depletion of miR-155 hinders the myofibroblast activities and reactive oxygen species generation in oral submucous fibrosis.J. Formos Med. Assoc. 121, 467-472 (2022).
44. Liao, Y. W.et al.miR-200b ameliorates myofibroblast transdifferentiation in precancerous oral submucous fibrosis through targeting ZEB2.J. Cell Mol. Med. 22, 4130-4138 (2018).
45. Du, W. W.et al.miR-17 extends mouse lifespan by inhibiting senescence signaling mediated by MKP7.Cell Death Dis. 5, e1355(2014).
46. Du, W. W.et al.The microRNA miR-17-3p inhibits mouse cardiac fibroblast senescence by targeting Par4.J. Cell Sci. 142, e0306-e0306 (2015).
47. Peng, D.et al.Targeting TGF-beta signal transduction for fibrosis and cancer therapy.Mol. Cancer 21, 104(2022).
48. Meng X.,Nikolic-Paterson, D. J. & Lan, H. Y. TGF-beta: The master regulator of fibrosis.Nat. Rev. Nephrol 12, 325-338 (2016).
49. Chung A. C.K. et al. Disruption of the Smad7 gene promotes renal fibrosis and inflammation in unilateral ureteral obstruction (UUO) in mice.Nephrol Dial Transpl. 24, 1443-1454 (2009).
50. Aragón, E.et al.Structural basis for the versatile interactions of Smad7 with regulator WW domains in TGF-beta Pathways.Structure 20, 1726-1736 (2012).
51. Fukasawa, H.et al.Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice.Proc. Natl. Acad. Sci. USA 101, 8687-8692 (2004).
52. Kit Leng Lui, S. et al. USP26 regulates TGF-beta signaling by deubiquitinating and stabilizing SMAD7.EMBO Rep. 18, 797-808 (2017).
53. Jing, X.et al.Exosome-transmitted miR-769-5p confers cisplatin resistance and progression in gastric cancer by targeting CASP9 and promoting the ubiquitination degradation of p53.Clin. Transl. Med. 12, e780(2022).
54. Takahashi, A.et al.Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.Nat. Commun 8, 15287(2017).
55. Chen, G.et al.Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.Nature 560, 382-386 (2018).