Molecular analysis of the human cytoglobin mRNA isoforms.

Multiple functions have been proposed for the ubiquitously expressed vertebrate globin cytoglobin (Cygb), including nitric oxide (NO) metabolism, lipid peroxidation/signalling, superoxide dismutase activity, reactive oxygen/nitrogen species (RONS) scavenging, regulation of blood pressure, antifibrosis, and both tumour suppressor and oncogenic effects. Since alternative splicing can expand the biological roles of a gene, we investigated whether this mechanism contributes to the functional diversity of Cygb. By mining of cDNA data and molecular analysis, we identified five alternative mRNA isoforms for the human CYGB gene (V-1 to V-5). Comprehensive RNA-seq analyses of public datasets from human tissues and cells confirmed that the canonical CYGB V-1 isoform is the primary CYGB transcript in the majority of analysed datasets. Interestingly, we revealed that isoform V-3 represented the predominant CYGB variant in hepatoblastoma (HB) cell lines and in the majority of analysed normal and HB liver tissues. CYGB V-3 mRNA is transcribed from an alternate upstream promoter and hypothetically encodes a N-terminally truncated CYGB protein, which is not recognized by some antibodies used in published studies. Little to no transcriptional evidence was found for the other CYGB isoforms. Comparative transcriptomics and flow cytometry on CYGB+/+ and gene-edited CYGB-/- HepG2 HB cells did not unveil a knockout phenotype and, thus, a potential function for CYGB V-3. Our study reveals that the CYGB gene is transcriptionally more complex than previously described as it expresses alternative mRNA isoforms of unknown function. Additional experimental data are needed to clarify the biological meaning of those alternative CYGB transcripts.

Transcriptomics of a cytoglobin knockout mouse: Insights from hepatic stellate cells and brain.

The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.

The Antioxidative Role of Cytoglobin in Podocytes: Implications for a Role in Chronic Kidney Disease.

Aims: Cytoglobin (CYGB) is a member of the mammalian globin family of respiratory proteins. Despite extensive research efforts, its physiological role remains largely unknown, but potential functions include reactive oxygen species (ROS) detoxification and signaling. Accumulating evidence suggests that ROS play a crucial role in podocyte detachment and apoptosis during diabetic kidney disease. This study aimed to explore the potential antioxidative renal role of CYGB both in vivo and in vitro. Results: Using a Cygb-deficient mouse model, we demonstrate a Cygb-dependent reduction in renal function, coinciding with a reduced number of podocytes. To specifically assess the putative antioxidative function of CYGB in podocytes, we first confirmed high endogenous CYGB expression levels in two human podocyte cell lines and subsequently generated short hairpin RNA-mediated stable CYGB knockdown podocyte models. CYGB-deficient podocytes displayed increased cell death and accumulation of ROS as assessed by 2'7'-dichlorodihydrofluorescein diacetate assays and the redox-sensitive probe roGFP2-Orp1. CYGB-deficient cells also exhibited an impaired cellular bioenergetic status. Consistently, analysis of the CYGB-dependent transcriptome identified dysregulation of multiple genes involved in redox balance, apoptosis, as well as in chronic kidney disease (CKD). Finally, genome-wide association studies and expression studies in nephropathy biopsies indicate an association of CYGB with CKD. Innovation: This study demonstrates a podocyte-related renal role of Cygb, confirms abundant CYGB expression in human podocyte cell lines, and describes for the first time an association between CYGB and CKD. Conclusion: Our results provide evidence for an antioxidative role of CYGB in podocytes.

Critical re-evaluation of neuroglobin expression reveals conserved patterns among mammals.

Neuroglobin (Ngb) is a respiratory protein that is almost exclusively expressed in the vertebrate nervous system. Despite many years of research, the exact function and even the expression sites of Ngb are still a matter of debate. However, to investigate hypotheses surrounding the potential roles of Ngb, a detailed knowledge of its major and minor expression sites is indispensable. We have therefore evaluated Ngb expression by extensive bioinformatic analysis using publicly available transcriptome data (RNA-Seq). During mammalian brain development, we observed low embryonic expression of Ngb mRNA and an increase after birth, arguing against a role of Ngb in fetal hypoxia tolerance. In adult mouse brain, we found highest Ngb mRNA levels in the hypothalamus, where expression was up to 100-fold stronger than in cerebral cortex, cerebellum or hippocampus, as confirmed by qRT-PCR and Western blotting. High Ngb expression in the hypothalamus was found conserved in humans and other mammals. Thus, Ngb mRNA is expressed at a basal level in many mammalian brain regions, but shows distinctive regional peaks. RNA-Seq analysis further revealed only low levels of Ngb mRNA in retina and testes and no signal in standard tumor cell lines, thus raising questions concerning previous studies and functional hypotheses. In conclusion, this broad-scale expression study may point to distinct Ngb functions for high- and low-expressing cells and tissues and argues against a single, generic role of Ngb as an oxygen supplier or as an endogenous protectant in all nerve cells.