Effects of JUN and NFE2L2 knockdown on oxidative status and NFE2L2/AP-1 targets expression in HeLa cells in basal conditions and upon sub-lethal hydrogen peroxide treatment.

Although NFE2L2 transcription factor is considered to make the most significant contribution to the NFE2L2/AP-1-pathway-dependent antioxidants regulation in the human cell, AP-1 has the potential to provide significant backup and even play an equal role in the cell. Considering this, the present study is focused on revealing how JUN, an AP-1 component, and NFE2L2 contribute to regulation of four target genes containing AREs with embedded TREs-SQSTM1, FTH1, HMOX1 and CBR3 and to cellular oxidative status in general in basal conditions and under pro-oxidative influence. NFE2L2 and JUN were down-regulated in HeLa cells using siRNA-mediated knockdown approach. These cells were subsequently exposed to 400 µM hydrogen peroxide in the medium or equal volume of sterile water. They revealed some evidence of both backup functioning and competing between the two factors. Importantly, JUN demonstrated a high level of participation (inc. as a negative regulator) in functioning of the classic NFE2L2 targets and in cellular oxidative status establishment in general. One of the key findings was a dramatic increase in JUN expression following NFE2L2 knockdown in basal conditions. The both AP-1 and NFE2L2 sub-pathways equally determine the outcome of the NFE2L2/AP-1 pathway activation induced by various stimuli, and the outcome is stimulus type- and stimulus-intensity-specific and results from either of the two eventually dominating sub-pathways.

Individual expression features of GPX2, NQO1 and SQSTM1 transcript variants induced by hydrogen peroxide treatment in HeLa cells

Abstract Pathway activity assessment-based approaches are becoming highly influential in various fields of biology and medicine. However, these approaches mostly rely on analysis of mRNA expression, and total mRNA from a given locus is measured in the majority of cases. Notably, a significant portion of protein-coding genes produces more than one transcript. This biological fact is responsible for significant noise when changes in total mRNA transcription of a single gene are analyzed. The NFE2L2/AP-1 pathway is an attractive target for biomedical applications. To date, there is a lack of data regarding the agreement in expression of even classical target genes of this pathway. In the present paper we analyzed whether transcript variants of GPX2, NQO1 and SQSTM1 were characterized by individual features of expression when HeLa cells were exposed to pro-oxidative stimulation with hydrogen peroxide. We found that all the transcripts (10 in total) appeared to be significantly individually regulated under the conditions tested. We conclude that individual transcripts, rather than total mRNA, are best markers of pathway activation. We also discuss here some biological roles of individual transcript regulation.

Individual expression features of GPX2, NQO1 and SQSTM1 transcript variants induced by hydrogen peroxide treatment in HeLa cells.

Pathway activity assessment-based approaches are becoming highly influential in various fields of biology and medicine. However, these approaches mostly rely on analysis of mRNA expression, and total mRNA from a given locus is measured in the majority of cases. Notably, a significant portion of protein-coding genes produces more than one transcript. This biological fact is responsible for significant noise when changes in total mRNA transcription of a single gene are analyzed. The NFE2L2/AP-1 pathway is an attractive target for biomedical applications. To date, there is a lack of data regarding the agreement in expression of even classical target genes of this pathway. In the present paper we analyzed whether transcript variants of GPX2, NQO1 and SQSTM1 were characterized by individual features of expression when HeLa cells were exposed to pro-oxidative stimulation with hydrogen peroxide. We found that all the transcripts (10 in total) appeared to be significantly individually regulated under the conditions tested. We conclude that individual transcripts, rather than total mRNA, are best markers of pathway activation. We also discuss here some biological roles of individual transcript regulation.

Testing the concept of the interatomic status of the NFE2L2/AP1 pathway as a systemic biomarker for examination stress.

BACKGROUND AND OBJECTIVES: Oxidative status-based interactomic profiling is a promising approach for fundamental integrative cell biology, diagnostics, and therapy. However, this approach has been neither utilized as a method nor tested as a tool. Thus, we aimed (1) to develop an oxidative status pathway state assessment-based analytical procedure relying on NFE2L2/AP1 pathway evaluation, and (2) to preliminarily assess its responsiveness, performance and diagnostic properties when applied to deciphering stress conditions of the academic examination period and academic term. These conditions were chosen as those representing a common model of mild, everyday-life stressors causing shifts in oxidative status. METHODS: To meet the aim of the study, we performed a repetitive-measurements study collating gene expression of NFE2L2/AP1 pathway targets and controllers under the two stress conditions using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Surprisingly, even with some sensitivity limitations of the methods employed, a pathway state analysis approach based on a multiple target-to-controller ratio calculation was highly responsive and yielded very high receiver operating characteristics in deciphering the model stress conditions. CONCLUSION: Although further testing of the approach is required, the interactomic pathway activation assaying concept was preliminarily experimentally proven to be a highly promising clinical diagnostic tool that may easily be adapted for current tasks.