Regulation of peroxiredoxin-3 gene expression under basal and hyperglycemic conditions: Key roles for transcription factors Sp1, CREB and NF-κB

Silpa Arkat; Sundar Poovitha; Anupama Vijayakumar; Rohini Dhat; Sandhya L Sitasawad; Nitish R Mahapatra

doi:10.1016/j.bbadis.2023.166691

Regulation of peroxiredoxin-3 gene expression under basal and hyperglycemic conditions: Key roles for transcription factors Sp1, CREB and NF-κB

Biochim Biophys Acta Mol Basis Dis. 2023 Jun;1869(5):166691. doi: 10.1016/j.bbadis.2023.166691. Epub 2023 Mar 16.

Authors

Silpa Arkat¹, Sundar Poovitha¹, Anupama Vijayakumar¹, Rohini Dhat², Sandhya L Sitasawad², Nitish R Mahapatra³

Affiliations

¹ Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
² National Centre for Cell Science, NCCS Complex, S.P. Pune University, Ganeshkhind, Pune 411007, Maharashtra, India.
³ Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India. Electronic address: nmahapatra@iitm.ac.in.

PMID: 36933848
DOI: 10.1016/j.bbadis.2023.166691

Abstract

Peroxiredoxin-3 (Prx-3), a thioredoxin-dependent peroxidase located exclusively in the mitochondrial matrix, catalyses peroxides/peroxinitrites. Altered levels of Prx-3 is associated with diabetic cardiomyopathy (DCM). However, molecular mechanisms of Prx-3 gene regulation remain partially understood. We undertook a systemic analysis of the Prx-3 gene to identify the key motifs and transcriptional regulatory molecules. Transfection of promoter-reporter constructs in the cultured cells identified -191/+20 bp domain as the core promoter region. Stringent in silico analysis of this core promoter revealed putative binding sites for specificity protein 1 (Sp1), cAMP response element-binding protein (CREB) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Interestingly, while co-transfection of the -191/+20 bp construct with Sp1/CREB plasmid diminished Prx3 promoter-reporter activity, mRNA and protein levels, co-transfection with NF-κB expression plasmid augmented the same. Consistently, inhibition of Sp1/CREB/NF-κB expression reversed the promoter-reporter activity, mRNA and protein levels of Prx-3, thereby confirming their regulatory effects. ChIP assays provided evidence for interactions of Sp1/CREB/NF-κB with the Prx-3 promoter. H9c2 cells treated with high glucose as well as streptozotocin (STZ)-treated diabetic rats showed time-dependent reduction in promoter activity, endogenous transcript and protein levels of Prx-3. Augmentation of Sp1/CREB protein levels and their strong binding with Prx-3 promoter are responsible for diminished Prx-3 levels under hyperglycemia. The activation/increase in the NF-κB expression under hyperglycemia was not sufficient to restore the reduction of endogenous Prx-3 levels owing to its weak binding affinity. Taken together, this study elucidates the previously unknown roles of Sp1/CREB/NF-κB in regulating Prx-3 gene expression under hyperglycemic condition.

Keywords: Diabetic cardiomyopathy; Gene regulation; Heart; Mitochondria; Peroxiredoxin-3; Streptozotocin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cyclic AMP Response Element-Binding Protein / genetics
Cyclic AMP Response Element-Binding Protein / metabolism
Diabetes Mellitus, Experimental* / genetics
Gene Expression
NF-kappa B* / genetics
NF-kappa B* / metabolism
Peroxiredoxin III / genetics
RNA, Messenger / metabolism
Rats
Sp1 Transcription Factor

Substances

Cyclic AMP Response Element-Binding Protein
NF-kappa B
Peroxiredoxin III
RNA, Messenger
Creb1 protein, rat
Sp1 Transcription Factor