Expression Levels of ACE2 and TMPRSS2 in Different Cell Lines

Year 2023, Volume: 6 Issue: 2, 260 - 268, 30.06.2023

Merve Gülsen Bal Albayrak , Sevinc Yanar , Murat Kasap , Gürler Akpınar

https://doi.org/10.53446/actamednicomedia.1253701

Cited By: 1

Abstract

Objective: ACE2 and TMPRSS2 proteins have received increased attention gained emphasis together with the pandemic COVID-19. These proteins have roles in respiratory and hypertension disorders as well as cardiovascular and renal diseases. The objective of this work was to examine the mRNA and protein levels of ACE2 and TMPRSS2 in cell lines derived from various tissue origins.
Methods: After the growth of 14 different cell lines, protein and mRNA were isolated from the cell pellets. The amounts of mRNAs and proteins were then determined and quantified using RT-PCR and ELISA.
Results: Findings showed that VERO, HEK293T, and VERO E6 cell lines significantly differed from others in the mRNA levels of both the ACE2 and TMPRSS2 genes. In protein levels obtained using ELISA, PNT1A cell line had the highest level of ACE2 protein expression, while for TMPRSS2, A549 had the highest level of protein expression.
Conclusion: It was showed in this study how the expressions of ACE2 and TMPRSS2 depend on the cell type. This may be an explanation for why virulence entrance differs in different types of tissues. It is thought that HEK293T cells with high levels of both genes may be a suitable option for studies at the RNA level by using these two genes. MCF7 may be a good option for studies at the protein level. Given the high levels of mRNA expression of both genes, it may be inferred that cells derived from the kidney were among those that were most susceptible to virus entry.

Keywords

ACE2, TMPRSS2, COVID19

ACE2 ve TMPRSS2 Genlerinin Farklı Hücre Hatlarındaki İfade Düzeyleri

Abstract

Amaç: ACE2 ve TMPRSS2 proteinleri, COVID-19 pandemisi ile birlikte önem kazanmıştır. Bu proteinlerin solunum ve hipertansiyon bozukluklarının yanı sıra kardiyovasküler ve renal hastalıklarda rolleri vardır. Bu çalışmanın amacı, çeşitli dokulardan üretilen hücre hatlarında ACE2 ve TMPRSS2'nin mRNA ve protein seviyelerini belirlemektir.
Yöntem: 14 hücre hattı kültürde çoğaltıldıktan sonra, hücre pelletlerinden protein ve mRNA izole edildi. Ardından mRNA seviyeleri RT-PCR ve protein seviyeleri ise ELISA metotları kullanılarak ölçüldü.
Bulgular: Çalışmanın sonuçları, ACE2 ve TMPRSS2 genlerinin mRNA seviyelerinin VERO, HEK293T ve VERO E6 hücre hatlarında, diğer hücre hatlarına kıyasla, önemli ölçüde yüksek olduğunu göstermiştir. ELISA kullanılarak elde edilen protein seviyelerinde, PNT1A hücre hattı en yüksek ACE2 protein ekspresyonuna sahipken, TMPRSS2 için A549 en yüksek protein ekspresyonuna sahip olarak bulunmuştur.
Sonuç: Bu çalışmada ACE2 ve TMPRSS2 gen ifadelerinin hücre tipine göre farklılaşabileceği gösterilmiştir. Bu sonuç, farklı doku tiplerinde virüs girişinin neden farklı olduğunun bir açıklaması olabilir. Her iki genin de yüksek düzeyde bulunduğu HEK293T hücrelerinin, bu iki gen kullanılarak RNA düzeyinde yapılacak çalışmalar için uygun bir seçenek olabileceği düşünülmektedir. MCF7 hücre hattı ise protein seviyesindeki çalışmalar için iyi bir seçenek olabilir. Her iki genin yüksek mRNA ekspresyon seviyeleri göz önüne alındığında, böbrekten üretilen hücre hatlarının virüs girişine en duyarlı olanlar arasında olduğu sonucuna varılabilir.

Keywords

ACE2, TMPRSS2, COVID19

References

• 1. Ritchie H, Mathieu E, Rodés-Guirao L, et al. Coronavirus Pandemic (COVID-19). Accessed April 17, 2023. https://ourworldindata.org/coronavirus
• 2. Imai Y, Kuba K, Ohto-Nakanishi T, Penninger JM. Angiotensin-Converting Enzyme 2 (ACE2) in Disease Pathogenesis. Circ J. 2010;74(3):405-410. doi:10.1253/circj.cj-10-0045
• 3. Imai Y, Kuba K, Rao S, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436(7047):112-116. doi:10.1038/nature03712
• 4. Gurwitz D. Angiotensin receptor blockers as tentative SARS‐CoV‐2 therapeutics. Drug Develop Res. 2020;81(5):537-540. doi:10.1002/ddr.21656
• 5. Lu R, Zhao X, Li J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet Lond Engl. 2020;395(10224):565-574. doi:10.1016/s0140-6736(20)30251-8
• 6. Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol. 2020;94(7):e00127-20. doi:10.1128/jvi.00127-20
• 7. Rezaei M, Ziai SA, Fakhri S, Pouriran R. ACE2: Its potential role and regulation in severe acute respiratory syndrome and COVID‐19. J Cell Physiol. 2021;236(4):2430-2442. doi:10.1002/jcp.30041
• 8. Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biology. 2022;23(1):3-20. doi:10.1038/s41580-021-00418-x
• 9. Reindl-Schwaighofer R, Hödlmoser S, Eskandary F, et al. ACE2 Elevation in Severe COVID-19. Am J Resp Crit Care. 2021;203(9):1191-1196. doi:10.1164/rccm.202101-0142le
• 10. Beyerstedt S, Casaro EB, Rangel ÉB. COVID-19: angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection. Eur J Clin Microbiol. 2021;40(5):905-919. doi:10.1007/s10096-020-04138-6
• 11. Crackower MA, Sarao R, Oudit GY, et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature. 2002;417(6891):822-828. doi:10.1038/nature00786
• 12. Zhong J, Yan Z, Liu D, et al. Association of angiotensin-converting enzyme 2 gene A/G polymorphism and elevated blood pressure in Chinese patients with metabolic syndrome. J Laboratory Clin Medicine. 2006;147(2):91-95. doi:10.1016/j.lab.2005.10.001
• 13. Burrell LM, Risvanis J, Kubota E, et al. Myocardial infarction increases ACE2 expression in rat and humans. Eur Heart J. 2005;26(4):369-375. doi:10.1093/eurheartj/ehi114
• 14. Goulter AB, Goddard MJ, Allen JC, Clark KL. ACE2 gene expression is up-regulated in the human failing heart. Bmc Med. 2004;2(1):19-19. doi:10.1186/1741-7015-2-19
• 15. Marshall RP, Webb S, Bellingan GJ, et al. Angiotensin Converting Enzyme Insertion/Deletion Polymorphism Is Associated with Susceptibility and Outcome in Acute Respiratory Distress Syndrome. Am J Resp Crit Care. 2002;166(5):646-650. doi:10.1164/rccm.2108086
• 16. Sato T, Ueha R, Goto T, Yamauchi A, Kondo K, Yamasoba T. Expression of ACE2 and TMPRSS2 proteins in the upper and lower aerodigestive tracts of rats. Biorxiv. Published online 2020:2020.05.14.097204. doi:10.1101/2020.05.14.097204
• 17. Song H, Seddighzadeh B, Cooperberg MR, Huang FW. Expression of ACE2, the SARS-CoV-2 receptor, and TMPRSS2 in prostate epithelial cells. Biorxiv. Published online 2020:2020.04.24.056259. doi:10.1101/2020.04.24.056259
• 18. Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARScoronavirus. Nature. 2003;426(6965):450-454. doi:10.1038/nature02145
• 19. Xu J, Chu M, Zhong F, et al. Digestive symptoms of COVID-19 and expression of ACE2 in digestive tract organs. Cell Death Discov. 2020;6(1):76. doi:10.1038/s41420-020-00307-w
• 20. Kuba K, Imai Y, Rao S, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus–induced lung injury. Nat Med. 2005;11(8):875-879. doi:10.1038/nm1267
• 21. (NCATS) B (MD): NC for ATS. TMPRSS2 enzymatic activity. SARS-CoV-2 Assays [Internet]. Published 2020. Accessed September 29, 2022. https://www.ncbi.nlm.nih.gov/books/NBK579897/
• 22. Mohamed MS, Moulin TC, Schiöth HB. Sex differences in COVID-19: the role of androgens in disease severity and progression. Endocrine. 2021;71(1):3-8. doi:10.1007/s12020-020-02536-6
• 23. Shen LW, Mao HJ, Wu YL, Tanaka Y, Zhang W. TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections. Biochimie. 2017;142:1-10. doi:10.1016/j.biochi.2017.07.016
• 24. Singh H, Choudhari R, Nema V, Khan AA. ACE2 and TMPRSS2 polymorphisms in various diseases with special reference to its impact on COVID-19 disease. Microb Pathogenesis. 2021;150:104621-104621. doi:10.1016/j.micpath.2020.104621
• 25. Lin B, Ferguson C, White JT, et al. Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2. Cancer Res. 1999;59(17):4180-4184.
• 26. Afar DE, Vivanco I, Hubert RS, et al. Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia. Cancer Res. 2001;61(4):1686-1692.
• 27. Jacquinet E, Rao NV, Rao GV, Hoidal JR. Cloning, genomic organization, chromosomal assignment and expression of a novel mosaic serine proteinase: epitheliasin. Febs Lett. 2000;468(1):93-100. doi:10.1016/s0014-5793(00)01196-0
• 28. Zhang H, Rostami MR, Leopold PL, et al. Expression of the SARS-CoV-2 ACE2 Receptor in the Human Airway Epithelium. Am J Resp Crit Care. 2020;202(2):219-229. doi:10.1164/rccm.202003-0541oc
• 29. Dong M, Zhang J, Ma X, et al. ACE2, TMPRSS2 distribution and extrapulmonary organ injury in patients with COVID-19. Biomed Pharmacother. 2020;131:110678. doi:10.1016/j.biopha.2020.110678
• 30. Ma D, Chen CB, Jhanji V, et al. Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea. Eye. 2020;34(7):1212-1219. doi:10.1038/s41433-020-0939-4
• 31. Kam YW, Okumura Y, Kido H, Ng LFP, Bruzzone R, Altmeyer R. Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro. Plos One. 2009;4(11):e7870. doi:10.1371/journal.pone.0007870
• 32. Hikmet F, Méar L, Edvinsson Å, Micke P, Uhlén M, Lindskog C. The protein expression profile of ACE2 in human tissues. Biorxiv. Published online 2020:2020.03.31.016048. doi:10.1101/2020.03.31.016048
• 33. Takayama K. In vitro and Animal Models for SARS-CoV-2 research. Trends Pharmacol Sci. 2020;41(8):513-517. doi:10.1016/j.tips.2020.05.005
• 34. Elfiky AA. Anti-HCV, nucleotide inhibitors, repurposing against COVID-19. Life Sci. 2020;248:117477. doi:10.1016/j.lfs.2020.117477
• 35. Ji Y, Ma Z, Peppelenbosch MP, Pan Q. Potential association between COVID-19 mortality and health-care resource availability. Lancet Global Heal. 2020;8(4):e480. doi:10.1016/s2214-109x(20)30068-1
• 36. Harcourt J, Tamin A, Lu X, et al. Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient. Biorxiv. Published online 2020:2020.03.02.972935. doi:10.1101/2020.03.02.972935
• 37. Yanar S, Kasap M, Kanli A, Akpinar G, Sarihan M. Proteomics analysis of meclofenamic acid‐treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival. J Biochem Mol Toxicol. Published online 2022:e23289. doi:10.1002/jbt.23289
• 38. Albayrak MGB, Simsek T, Kasap M, Akpinar G, Canturk NZ, Guler SA. Tissue proteome analysis revealed an association between cancer, immune system response, and the idiopathic granulomatous mastitis. Med Oncol. 2022;39(12):238. doi:10.1007/s12032-022-01845-2
• 39. Vaarala MH, Porvari K, Kyllönen A, Lukkarinen O, Vihko P. The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: Detection of mutated TMPRSS2 form in a case of aggressive disease. Int J Cancer. 2001;94(5):705-710. doi:10.1002/ijc.1526
• 40. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271-280.e8. doi:10.1016/j.cell.2020.02.052
• 41. Bertram S, Heurich A, Lavender H, et al. Influenza and SARS-Coronavirus Activating Proteases TMPRSS2 and HAT Are Expressed at Multiple Sites in Human Respiratory and Gastrointestinal Tracts. Plos One. 2012;7(4):e35876. doi:10.1371/journal.pone.0035876
• 42. Hamming I, Timens W, Bulthuis M, Lely A, Navis G, Goor H van. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathology. 2004;203(2):631-637. doi:10.1002/path.1570
• 43. Ren X, Glende J, Al-Falah M, et al. Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus. J Gen Virol. 2006;87(6):1691-1695. doi:10.1099/vir.0.81749-0
• 44. Ortiz ME, Thurman A, Pezzulo AA, et al. Heterogeneous expression of the SARS-Coronavirus-2 receptor ACE2 in the human respiratory tract. Ebiomedicine. 2020;60:102976. doi:10.1016/j.ebiom.2020.102976
• 45. Ou T, Mou H, Zhang L, Ojha A, Choe H, Farzan M. Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2. Plos Pathog. 2021;17(1):e1009212. doi:10.1371/journal.ppat.1009212
• 46. Iwata-Yoshikawa N, Okamura T, Shimizu Y, Hasegawa H, Takeda M, Nagata N. TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection. J Virol. 2019;93(6):e01815-18. doi:10.1128/jvi.01815-18
• 47. Ammerman NC, Beier‐Sexton M, Azad AF. Growth and Maintenance of Vero Cell Lines. Curr Protoc Microbiol. 2008;11(1):A.4E.1-A.4E.7. doi:10.1002/9780471729259.mca04es11
• 48. Shen CF, Guilbault C, Li X, et al. Development of suspension adapted Vero cell culture process technology for production of viral vaccines. Vaccine. 2019;37(47):6996-7002. doi:10.1016/j.vaccine.2019.07.003
• 49. Gkogkou E, Barnasas G, Vougas K, Trougakos IP. Expression profiling meta-analysis of ACE2 and TMPRSS2, the putative anti-inflammatory receptor and priming protease of SARS-CoV-2 in human cells, and identification of putative modulators. Redox Biol. 2020;36:101615. doi:10.1016/j.redox.2020.101615
• 50. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent Fusion of TMPRSS2 and ETS Transcription Factor Genes in Prostate Cancer. J Urology. 2006;175(5):1707. doi:10.1016/s0022-5347(06)00096-6
• 51. Wang Z, Wang Y, Zhang J, et al. Significance of the TMPRSS2:ERG gene fusion in prostate cancer. Mol Med Rep. 2017;16(4):5450-5458.doi:10.3892/mmr.2017.7281
• 52. Deng Q, Rasool R ur, Russell RM, Natesan R, Asangani IA. Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19. Iscience. 2021;24(3):102254. doi:10.1016/j.isci.2021.102254
• 53. Siciliano T, Sommer U, Beier AMK, et al. The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity. Curr Issues Mol Biol. 2022;44(2):597-608. doi:10.3390/cimb44020041
• 54. Minamiguchi K, Kawada M, Someno T, Ishizuka M. Androgen-independent prostate cancer DU145 cells suppress androgen-dependent growth of prostate stromal cells through production of inhibitory factors for androgen responsiveness. Biochem Bioph Res Co. 2003;306(3):629-636. doi:10.1016/s0006-291x(03)01023-4
• 55. Baratchian M, McManus JM, Berk MP, et al. Androgen regulation of pulmonary AR, TMPRSS2 and ACE2 with implications for sex-discordant COVID-19 outcomes. Sci Rep-uk. 2021;11(1):11130. doi:10.1038/s41598-021-90491-1
• 56. Mjaess G, Karam A, Aoun F, Albisinni S, Roumeguère T. COVID-19 and the male susceptibility: the role of ACE2, TMPRSS2 and the androgen receptor. Prog Urol. 2020;30(10):484-487. doi:10.1016/j.purol.2020.05.007
• 57. Carpenter EP, Beis K, Cameron AD, Iwata S. Overcoming the challenges of membrane protein crystallography. Curr Opin Struc Biol. 2008;18(5):581-586. doi:10.1016/j.sbi.2008.07.001