Project Details
Projekt
Analysis of the transcriptional and post-transcriptional regulation of human iNOS expression
Subject Area
Pharmacology
Biochemistry
Immunology
Biochemistry
Immunology
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 273680753
The high amounts of NO produced by the inducible nitric oxide synthase (iNOS) have beneficial antiviral, antiparasital and antitumoral effects. However, aberrant iNOS induction is involved in the pathophysiology of human autoimmune diseases (rheumatoid arthritis), cancer and septic shock. Therefore it is important to understand the mechanisms by which cells regulate iNOS-related NO production. NO production by iNOS is mainly regulated by regulation of iNOS expression. Beside transcription human iNOS expression is regulated by different post-transcriptional mechanisms. In the 5-untranslated region (5-UTR) upstream of the bona fide iNOS coding sequence (cds) there is a small upstream open reading frame (µORF). Our recent data show that this µORF seems to be translatable and therefore should block the translation of the bona fide iNOS cds. However iNOS protein expression can be observed after induction of cells. Our data indicate that a binding site of the poly-A binding protein (PABP) and an overlapping putative internal ribosomal entry site (IRES) sequence located behind the µORF may be involved in a cap-independent translation of the human iNOS mRNA. In the present application we want to analyze the mechanisms enabling the translation of the iNOS cds in the presence of a µORF. The human iNOS 5-UTR is encoded by exon 1 and in part by exon 2. The stop codon of the µORF is located 50 bp in the front of the intron 1. This hints for a regulation of iNOS expression by the nonsense mediated mRNA decay (NMD). Our data show that siRNA-mediated downregulation of the expression of Upf1 (a central mediator of NMD) enhances human iNOS mRNA- and protein expression. In the present application we want to analyze the involvement of the NMD in human iNOS expression in detail. Korneev et al. describe the expression of a long non coding RNA (lncRNA) with a partial anti-sense homology to the bona fide iNOS gene (as-iNOS-lncRNA) in human cells. Our data show that the expression of the as-iNOS-lncRNA is upregulated by cytokine incubation. In addition down-regulation of as-iNOS-lncRNA significantly reduced cytokine-induced iNOS mRNA- and protein expression. This hints for an regulation of the human iNOS gene by the as-iNOS-lncRNA. In the present application we want to analyze the mechanisms of this regulation. In human C-28/I2 chondrocytes iNOS expression depends on cell differentiation. The mechanisms important for differentiation-dependent iNOS regulation are unknown. Beside changes in the post-transcriptional mechanisms this may be explained by changes in the inducibility of the human iNOS promoter. Our data show, that in contrast to most cells the inducibility of the human iNOS promoter resembles the inducibility of the iNOS mRNA expression (> 100 fold) in differentiated C-28/I2 cells. In the application we want to analyze the differentiation-dependent mechanisms regulating human iNOS expression in C-28/I2 chondrocytes.
DFG Programme
Research Grants
