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

Year 2024, Volume: 48 Issue: 3, 949 - 961, 10.09.2024

Yalçın Erzurumlu , Hatice Kübra Doğan , Deniz Çataklı

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

Abstract

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.

Keywords

ER stres, katlanmamış protein yanıtı, sirkadiyen saat

Ethical Statement

Bu çalışma herhangi bir etik izin gerektirmemektedir

Project Number

TSG-2021-8302, TAB-2020-8253

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

Abstract

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.

Keywords

Circadian clock, ER stress, unfolded protein response

Ethical Statement

This study does not require any ethical permission

Supporting Institution

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

Project Number

TSG-2021-8302, TAB-2020-8253

Thanks

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.

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