Klaus Steger

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47 phox and p67 phox subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47 phox and p67 phox are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47 phox homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67 phox homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47 phox was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.

Transcriptional and translational regulation of gene expression in haploid spermatids

 During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications resulting in the formation of mature spermatozoa. While in round spermatids histones and non-histone proteins are replaced by transition proteins, in elongating spermatids, transition proteins are removed from the condensing chromatin and are replaced by protamines, which are the principal basic nuclear proteins of mature spermatozoa. The tightly packed DNA-protamine complexes cease transcription several days before the completion of spermiogenesis. Thus, major modifications in both nuclear and cytoplasmic structures continue throughout spermiogenesis, stringent temporal and stage-specific gene expression is a prerequisite for the correct differentiation of round spermatids into mature spermatozoa. The genes for transition proteins and protamines are transcribed in round and elongating spermatids. Transcription is regulated via methylation and trans-acting factors that bind to the TATA-box, the CRE-box, or other specific DNA sequences in the promoter region. The transcripts are stored as ribonucleoprotein particles in a translationally repressed state for several days and are translated in elongating and elongated spermatids. It has been demonstrated that, in haploid spermatids, essentially every mRNA exhibits evidence of translational repression. Translational regulation involves protein repressors that bind to the poly-A tail or specific RNA sequences located in the 3′-UTR.

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease

Reactive oxygen species (ROS) are at the centre of many physiological and pathological processes. NOX1, a ROS‐producing NADPH oxidase, is highly expressed in the colon but its function in colonic physiology or pathology is still poorly understood. It has been suggested to play a role in host defence, but also in cell growth and possibly malignant transformation. In this study we characterized NOX1 expression in human colon samples derived from healthy control subjects and patients with colon cancer or inflammatory bowel disease (IBD). NOX1 mRNA expression was assessed by dot‐blot hybridization, real‐time PCR and in situ hybridization, using samples derived from surgical specimens from patients undergoing colon resection. In normal tissues, NOX1 expression was low in the ileum, intermediate in the right colon, and high in the left colon (p = 0.0056 right vs. left colon). NOX1 mRNA levels were not influenced by factors linked to colon tumourigenesis, such as age or sex. Moreover, there was no statistical difference in NOX1 expression between samples derived from adenomas, well differentiated or poorly differentiated colon adenocarcinomas. At a cellular level, NOX1 was highly expressed in colon epithelial cells, both within the crypts and on the luminal surface. In addition, a population of lymphocytes, particularly in the appendix, showed NOX1 expression. Lymphocytes in lesions of Crohns disease and ulcerative colitis were also strongly positive for NOX1. In conclusion, NOX1 is an enzyme that is constitutively expressed in colon epithelium and is not associated with tumourigenesis. Its distribution in crypts and on the luminal surface, as well as its left‐to‐right gradient in the colon, suggests a role in host defence function. In addition to the known epithelial localization, we define lymphocytes as a novel site of NOX1 expression, where it may potentially be involved in the pathogenesis of inflammatory bowel diseases. Copyright

Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma.

PIK3CA, encoding the catalytic subunit p110α of phosphatidylinositol 3‐kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well‐defined regions of formalin‐fixed tissue sections and to compare these data with protein and mRNA expression of p110α. The mRNA and protein levels of p110α were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low‐to‐moderate dysplasias (17%) and in seven of nine high‐grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high‐grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110α mRNA and protein expression, whereas only two cases (18%) (one high‐grade dysplasia and one carcinoma) with no gain or low‐level gain displayed increased p110α protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110α mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events. Copyright

Haploid spermatids exhibit translationally repressed mRNAs.

During spermiogenesis, DNA-binding histones are replaced by protamines. DNA-protamine-interactions result in chromatin condensation causing cessation of transcription in elongating spermatids. Thus, in haploid spermatids, processes of transcription and translation need to be temporally uncoupled in order to ensure protein synthesis in transcriptionally silent germ cells. Post-transcriptional events, such as processing, transport, and storage of mRNAs, therefore, play important roles in determining when transcripts become functionally available for translation. This review focuses on mechanisms of translational regulation in haploid spermatids and male infertility caused by defects of translational control processes.

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis

Summary.  Histone‐to‐protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA–histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 ± 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10–8, 93.1 ± 6.6%) and impaired spermatogenesis (scores 7–1, 74.9 ± 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 ± 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone‐to‐protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone‐to‐protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine‐1 results in precocious chromatin condensation followed by sterility.

Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors

BackgroundMost testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency.ResultsIn the present study, we show that BLIMP1 and PRMT5 were expressed and arginine dimethylation of histones H2A and H4 was detected in human male gonocytes at weeks 12–19 of gestation, indicating a role of this mechanism in human fetal germ cell development as well. Moreover, BLIMP1/PRMT5 and histone H2A and H4 arginine 3 dimethylation was present in IGCNU and most seminomas, while downregulated in embryonal carcinoma (EC) and other nonseminomatous tumors.ConclusionThese data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors.

Expression of connexin 43 in human testis.

Abstract In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency.