HEK293 İNSAN EMBRİYONİK BÖBREK HÜCRELERİNDE KATLANMAMIŞ PROTEİN YANITI SİNYALİ BİLEŞENLERİNİN SİRKADİYEN SALINIM MODELİNİN BELİRLENMESİ

Yıl 2024, , 949 - 961, 10.09.2024

Yalçın Erzurumlu , Hatice Kübra Doğan , Deniz Çataklı

https://doi.org/10.33483/jfpau.1487169

Öz

Amaç: Sirkadiyen ritim, yaklaşık 24 saatlik salınımlara sahip temel düzenleyici sistemlerden biridir. Vücut ısısı ve hormon salgılanması da dahil olmak üzere insan vücudundaki fizyolojik koşulların düzenlenmesinde çok önemli bir role sahiptir. Kanser ve diyabet de dahil olmak üzere çok sayıda rahatsızlık hücresel sirkadiyen ritmin bozulmasıyla ilişkilendirilmiştir. Bu çalışmada memeli hücrelerinde önemli fizyolojik mekanizmalardan biri olan ve son zamanlarda kanserde ilaç direnci, invazyon ve metastaz ile ilişkilendirilen katlanmamış protein yanıtı (UPR) sinyali ile sirkadiyen ritim arasındaki ilişkiyi araştırmayı amaçladık.
Gereç ve Yöntem: İnsan embriyonik böbrek hücre hattı HEK293 Amerikan Tipi Kültür Koleksiyonundan sağlandı ve hücreler %10 FBS ve büyüme bileşenleri içeren DMEM besi ortamı içinde çoğaltıldı. İn vitro sirkadiyen senkronizasyon için hücreler %50 at serumuna maruz bırakıldı ve ardından UPR ile ilişkili hedef genlerin gen ifadesi ve protein düzeyindeki salınım modeli sırasıyla agaroz jel elektroforezi ve immünoblotlama ile analiz edildi. Salınım modeli eğri uydurma analizi yoluyla değerlendirildi.
Sonuç ve Tartışma: Bulgularımız, IRE1α, XBP-1s, eIF2α, fosfo(Ser51)-eIF2α, PERK, ATF4, GADD34 ve ATF6 dahil olmak üzere UPR bileşenlerinin, sirkadiyen ritmin çekirdek komponentlerinden PER1 geni gibi 48 saatlik bir zaman ölçeğinde sirkadiyen ritim altında sıkı bir şekilde salınım modelleri sergilediğini gösterdi. Ayrıca endoplazmik retikulum (ER) stres genleri, BiP/GRP78 ve CHOP da sirkadiyen ritim altında UPR bileşenlerine benzer şekilde davrandı. Ek olarak UPR sinyalinin aktivasyonunun sirkadiyen ritimle uyumlu bir şekilde modüle edildiğini bulduk. Mevcut veriler, UPR bileşenlerinin ekspresyon seviyesinin sirkadiyen ritim altında katı salınım sergilediğini gösterdi. Bulgularımız, çeşitli patolojileri sirkadiyen ritme göre tedavi etmek için geliştirilecek yeni nesil UPR hedefli ilaçların deneysel çalışmalarına yol gösterebilir.

Anahtar Kelimeler

ER stres, katlanmamış protein yanıtı, sirkadiyen saat

Etik Beyan

Bu çalışma herhangi bir etik izin gerektirmemektedir

Proje Numarası

TSG-2021-8302, TAB-2020-8253

DETERMINATION OF THE CIRCADIAN OSCILLATION PATTERN OF UNFOLDED PROTEIN RESPONSE SIGNALING COMPONENTS IN HUMAN EMBRYONIC KIDNEY HEK293 CELLS

Öz

Objective: The circadian rhythm is one of the primary regulatory systems with near 24-hour oscillations. It has a crucial role in regulating physiological conditions in the human body, including body temperature and the secretion of hormones. Numerous disorders, such as cancer and diabetes, have been linked to disruptions of the cellular circadian rhythm. Herein, we aimed to investigate the relationship between the circadian rhythm and unfolded protein response (UPR) signaling, which is one of the important physiological mechanisms in mammalian cells and has recently been associated with drug resistance, invasion and metastasis in cancer.
Material and Method: Human embryonic kidney cell line HEK293 was provided from the American Type Culture Collection and propagated in DMEM containing 10% FBS and growth ingredients. For in vitro circadian synchronization, cells were exposed to 50% and then the oscillation pattern of gene and protein expression of UPR-related target genes was analyzed by agarose gel electrophoresis and immunoblotting, respectively. The oscillation pattern was commented on through curve-fitting analysis.
Result and Discussion: Our findings demonstrated that UPR components, including IRE1α, XBP-1s, eIF2α, phospho(Ser51)-eIF2α, PERK, ATF4, GADD34 and ATF6, tightly exhibit oscillation patterns under a circadian rhythm on a 48-hour time scale like the PER1 gene that is a core component of the circadian rhythm. Moreover, endoplasmic reticulum (ER) stress genes, BiP/GRP78 and CHOP, were similar to UPR components under the circadian rhythm. Additionally, we found the activation of UPR signaling harmoniously modulated with the circadian rhythm. Present data indicated that the expression level of UPR components exhibited strict oscillation under the circadian rhythm. Our findings may guide experimental studies of new-generation UPR-targeted drugs to be developed to treat various pathologies in accordance with the circadian rhythm.

Anahtar Kelimeler

Circadian clock, ER stress, unfolded protein response

Etik Beyan

This study does not require any ethical permission

Destekleyen Kurum

This study was partially supported by Suleyman Demirel University internal funds (TSG-2021-8302, TAB-2020-8253).

Proje Numarası

TSG-2021-8302, TAB-2020-8253

Teşekkür

We thank Dr. Signem Oney Birol (Department of Molecular Biology and Genetics, Faculty of Science, Mehmet Akif Ersoy University) for allowing us to access the use of the chemiluminescence monitoring system.

Kaynakça

• 1. Reppert, S.M., Weaver, D.R. (2002). Coordination of circadian timing in mammals. Nature, 418(6901), 935-941. [CrossRef]
• 2. Rosenwasser, A.M., Turek, F.W. (2015). Neurobiology of circadian rhythm regulation. Sleep Medicine Clinics, 10(4), 403-412. [CrossRef]
• 3. Patke, A., Young, M.W., Axelrod, S. (2020). Molecular mechanisms and physiological importance of circadian rhythms. Nature Reviews Molecular Cell Biology, 21(2), 67-84. [CrossRef]
• 4. Hastings, M., O’Neill, J.S., Maywood, E.S. (2007). Circadian clocks: Regulators of endocrine and metabolic rhythms. Journal of Endocrinology, 195(2), 187-198. [CrossRef]
• 5. Trott, A.J., Menet, J.S. (2018). Regulation of circadian clock transcriptional output by CLOCK:BMAL1. PLoS Genetics, 14(1), e1007156. [CrossRef]
• 6. Lee, Y., Field, J.M., Sehgal, A. (2021). Circadian rhythms, disease and chronotheraphy. Journal of Biological Rhythms, 36(6), 503-531. [CrossRef]
• 7. Lee, Y. (2021). Roles of circadian clocks in cancer pathogenesis and treatment. Experimental & Molecular Medicine, 53(10), 1529-1538. [CrossRef]
• 8. Crnko, S., Du Pré, B.C., Sluijter, J.P.G., Van Laake, L.W. (2019). Circadian rhythms and the molecular clock in cardiovascular biology and disease. Nature Reviews Cardiology, 16(7), 437-47. [CrossRef]
• 9. Erzurumlu, Y., Catakli, D., Dogan, H.K. (2023). Circadian oscillation pattern of endoplasmic reticulum quality control (ERQC) components in human embryonic kidney HEK293 cells. Journal of Circadian Rhythms, 21, 1. [CrossRef]
• 10. Borgese, N., Francolini, M., Snapp, E. (2006). Endoplasmic reticulum architecture: Structures in flux. Current Opinion in Cell Biology, 18(4), 358-364. [CrossRef]
• 11. Braakman, I., Hebert, D.N. (2013). Protein folding in the endoplasmic reticulum. Cold Spring Harbor Perspectives in Biology, 5(5), a013201. [CrossRef]
• 12. Fagone, P., Jackowski, S. (2009). Membrane phospholipid synthesis and endoplasmic reticulum function. Journal of Lipid Research, 50, 311-316. [CrossRef]
• 13. Hebert, D.N., Garman, S.C., Molinari, M. (2005). The glycan code of the endoplasmic reticulum: Asparagine-linked carbohydrates as protein maturation and quality-control tags. Trends in Cell Biology, 15(7), 364-370. [CrossRef]
• 14. Adams, C.J., Kopp, M.C., Larburu, N., Nowak, P.R., Ali, M.M.U. (2019). Structure and molecular mechanism of er stress signaling by the unfolded protein response signal activator IRE1. Frontiers in Molecular Biosciences, 6, 11. [CrossRef]
• 15. Hetz, C., Zhang, K., Kaufman, R.J. (2020). Mechanisms, regulation and functions of the unfolded protein response. Nature Reviews Molecular Cell Biology, 21(8), 421-438. [CrossRef]
• 16. Madden, E., Logue, S.E., Healy, S.J., Manie, S., Samali, A. (2019). The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance. Biology of the Cell, 111(1), 1-17. [CrossRef]
• 17. Scheuner, D., Song, B., McEwen, E., Liu, C., Laybutt, R., Gillespie, P., Saunders, T., Bonner-Weir, S., Kaufman, R.J. (2001). Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Molecular Cell, 7(6), 1165-1176. [CrossRef]
• 18. Cheng, G., Feng, Z., He, B. (2005). Herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase PERK and mediates eIF-2alpha dephosphorylation by the gamma(1)34.5 protein. Journal of Virology, 79(3), 1379–1388. [CrossRef]
• 19. Fels, D.R., Koumenis, C. (2006). The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biology & Therapy, 5(7), 723-728. [CrossRef]
• 20. Kakiuchi, C., Iwamoto, K., Ishiwata, M., Bundo, M., Kasahara, T., Kusumi, I., Tsujita, T., Okazaki, Y., Nanko, S., Kunugi, H., Sasaki, T., Kato, T. (2003). Impaired feedback regulation of XBP1 as a genetic risk factor for bipolar disorder. Nature Genetics, 35(2), 171-175. [CrossRef]
• 21. Balsalobre, A., Damiola, F., Schibler, U. (1998). A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell, 93(6), 929-937. [CrossRef]
• 22. Lambert, C.M., Weaver, D.R. (2006). Peripheral gene expression rhythms in a diurnal rodent. Journal of Biological Rhythms, 21(1), 77-79. [CrossRef]
• 23. Segers, A., Depoortere, I. (2021). Circadian clocks in the digestive system. Nature Reviews Gastroenterology & Hepatology, 18(4), 239-251. [CrossRef]
• 24. Scheiermann, C., Gibbs, J., Ince, L., Loudon, A. (2018). Clocking in to immunity. Nature Reviews Immunology, 18(7), 423-437. [CrossRef]
• 25. Chellappa, S.L., Vujovic, N., Williams, J.S., Scheer, F.A.J.L. (2019). Impact of circadian disruption on cardiovascular function and disease. Trends In Endocrinology and Metabolism: TEM, 30(10), 767-779. [CrossRef]
• 26. Logan, R.W., McClung, C.A. (2019). Rhythms of life: Circadian disruption and brain disorders across the lifespan. Nature Reviews Neuroscience, 20(1), 49-65. [CrossRef]
• 27. Chakraborti, S. (2022). Handbook of Oxidative Stress in Cancer: Therapeutic Aspects. Springer Nature, p.4078.
• 28. Masri, S., Sassone-Corsi, P. (2018). The emerging link between cancer, metabolism, and circadian rhythms. Nature medicine, 24(12), 1795-1803. [CrossRef]
• 29. Reddy, S., Reddy, V. Sharma, S. (2023). Physiology, Circadian Rhythm. In StatPearls. Treasure Island (FL): StatPearls Publishing.
• 30. Schwarz, D.S., Blower, M.D. (2016). The endoplasmic reticulum: Structure, function and response to cellular signaling. Cellular And Molecular Life Sciences, 73(1), 79-94. [CrossRef]
• 31. Ron, D., Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nature reviews. Molecular Cell Biology, 8(7), 519-529. [CrossRef]
• 32. Benham A.M. (2019). Endoplasmic Reticulum redox pathways: in sickness and in health. The FEBS Journal, 286(2), 311-321. [CrossRef]
• 33. Novoa, I., Zeng, H., Harding, H.P., Ron, D. (2001). Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. The Journal of Cell Biology, 153(5), 1011-1022. [CrossRef]
• 34. Zhu, T., Jiang, X., Xin, H., Zheng, X., Xue, X., Chen, J.L., Qi, B. (2021). GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis. Veterinary Research, 52(1), 148. [CrossRef]
• 35. Clarke, R. (2019). The Unfolded Protein Response in Cancer. Springer. p.220. (Book).
• 36. Bashir, S., Banday, M., Qadri, O., Bashir, A., Hilal, N., Nida-I-Fatima, Rader, S., Fazili, K.M. (2021). The molecular mechanism and functional diversity of UPR signaling sensor IRE1. Life sciences, 265, 118740. [CrossRef]
• 37. Maillo, C., Martín, J., Sebastián, D., Hernández-Alvarez, M., García-Rocha, M., Reina, O., Zorzano, A., Fernandez, M., Méndez, R. (2017). Circadian- and UPR-dependent control of CPEB4 mediates a translational response to counteract hepatic steatosis under ER stress. Nature Cell Biology, 19(2), 94-105. [CrossRef]
• 38. Bhattarai, K.R., Riaz, T.A., Kim, H.R., Chae, H.J. (2021). The aftermath of the interplay between the endoplasmic reticulum stress response and redox signaling. Experimental & Molecular Medicine, 53(2), 151-167. [CrossRef]
• 39. Erzurumlu, Y., Dogan, H.K., Catakli, D., Aydogdu, E., Muhammed, M.T. (2023). Estrogens drive the endoplasmic reticulum-associated degradation and promote proto-oncogene c-Myc expression in prostate cancer cells by androgen receptor/estrogen receptor signaling. Journal of Cell Communication and Signaling, 17(3), 793-811. [CrossRef]
• 40. Guo, D., Zhu, Y., Wang, H., Wang, G., Wang, C., Ren, H. (2020). E3 ubiquitin ligase HRD1 modulates the circadian clock through regulation of BMAL1 stability. Experimental and Therapeutic Medicine, 20(3), 2639-2648. [CrossRef]
• 41. Kim, H., Wei, J., Song, Z., Mottillo, E., Samavati, L., Zhang, R., Li, L., Chen, X., Jena, B.P., Lin, J.D., Fang, D., Zhang, K. (2021). Regulation of hepatic circadian metabolism by the E3 ubiquitin ligase HRD1-controlled CREBH/PPARα transcriptional program. Molecular Metabolism, 49, 101192. [CrossRef]
• 42. Cretenet, G., Le Clech, M., Gachon, F. (2010). Circadian clock-coordinated 12 Hr period rhythmic activation of the IRE1alpha pathway controls lipid metabolism in mouse liver. Cell Metabolism, 11(1), 47-57. [CrossRef]
• 43. Bu, Y., Yoshida, A., Chitnis, N., Altman, B.J., Tameire, F., Oran, A., Gennaro, V., Armeson, K.E., McMahon, S.B., Wertheim, G.B., Dang, C.V., Ruggero, D., Koumenis, C., Fuchs, S.Y., Diehl, J.A. (2018). A PERK-miR-211 axis suppresses circadian regulators and protein synthesis to promote cancer cell survival. Nature Cell Biology, 20(1), 104-115. [CrossRef]