The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates.

Petter Angell Olsen,Stefan Krauss

doi:10.3390/genes12121920

Abstract

HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/β-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/β-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/β-catenin signaling components and on transcriptional regulation of WNT/β-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

Highlights

IntroductionThe WNT/β-catenin signaling pathway plays a pivotal role during development, organogenesis, stem cell maintenance and self-renewal of many tissues, and aberrant
We show that while β-catenin is predominantly localized at the cell membrane, AXIN1, APC, DVL2 and TNKS all co-localize in large cytoplasmic aggregates that are enclosed by a narrow layer of the adenoviral E1B-55k protein present in HEK293 cells
We showed that in HEK293 cells central components of the WNT/βcatenin signaling pathway display atypical cellular localizations by being sequestered in large cytoplasmic E1B-55k aggregates

Summary

Introduction

The WNT/β-catenin signaling pathway plays a pivotal role during development, organogenesis, stem cell maintenance and self-renewal of many tissues, and aberrant. WNT/β-catenin signaling activity is associated with the development of several disease conditions including cancer [1,2]. The main determinant of WNT/β-catenin signaling activity is the level of free cytoplasmic β-catenin that can translocate to the nucleus and activate target genes through interactions of β-catenin with transcription factors of the TCF/LEF family [3]. In cells with low WNT/β-catenin signaling activity, the level of cytoplasmic βcatenin is kept low by the constant action of a degradosome that targets cytosolic β-catenin (by phosphorylation and ubiquitination) for proteolytic degradation. The degradosome comprises in its core of the structural protein APC, the rate limiting structural proteins

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