Ágnes Donkó

Peroxidasin is secreted and incorporated into the extracellular matrix of myofibroblasts and fibrotic kidney.

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

Spatial and Temporal Analysis of NADPH Oxidase-Generated Hydrogen Peroxide Signals by Novel Fluorescent Reporter Proteins

AIMS Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2. RESULTS We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci. INNOVATION Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells. CONCLUSION Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.

Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models

Nox/Duox NADPH oxidases are now considered the primary, regulated sources of reactive oxygen species (ROS). These enzymes are expressed in diverse cells and tissues, and their products are essential in several physiological settings. Knockout mouse models are instrumental in identifying the physiological functions of Nox/Duox enzymes as well as in exploring the impact of their pharmacological targeting on disease progression. The currently available data from experiments on knockout animals suggest that the lack of non-phagocytic Nox/Duox enzymes often modifies the course and phenotype in many disease models. Nevertheless, as illustrated by studies on Nox4-deficient animals, the absence of Nox-derived ROS can also lead to aggravated disease manifestation, reinforcing the need for a more balanced view on the role of ROS in health and disease.

Peroxidasin-like protein: A novel peroxidase homologue in the human heart

AIMS Peroxidases serve diverse biological functions including well-characterized activities in host defence and hormone biosynthesis. More recently, peroxidasin (PXDN) was found to be involved in collagen IV cross-linking in the extracellular matrix (ECM). The aim of this study was to characterize the expression and function of peroxidasin-like protein (PXDNL), a previously unknown peroxidase homologue. METHODS AND RESULTS We cloned the PXDNL cDNA from the human heart and identified its expression pattern by northern blot, in situ hybridization, and immunohistochemistry. PXDNL is expressed exclusively in the heart and it has evolved to lose its peroxidase activity. The protein is produced by cardiomyocytes and localizes to cell-cell junctions. We also demonstrate that PXDNL can form a complex with PXDN and antagonizes its peroxidase activity. Furthermore, we show an increased expression of PXDNL in the failing myocardium. CONCLUSION PXDNL is a unique component of the heart with a recently evolved inactivation of peroxidase function. The elevation of PXDNL levels in the failing heart may contribute to ECM dysregulation due to its antagonism of PXDN function.

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2.

In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2(thyd)) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H2O2, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.

Deficiency in Duox2 activity alleviates ileitis in GPx1- and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium

Mice deficient in glutathione peroxidase (GPx)-1 and -2 (GPx1-/-GPx2-/- double knockout or DKO mice) develop very-early-onset (VEO) ileocolitis, suggesting that lack of defense against reactive oxygen species (ROS) renders susceptibility to intestinal inflammation. Two members of ROS-generating NADPH oxidase family, NOX1 and DUOX2, are highly inducible in the intestinal epithelium. Previously, we reported that Nox1 deficiency ameliorated the pathology in DKO mice (Nox1-TKO). The role of Duox2 in ileocolitis of the DKO mice is evaluated here in Duoxa-TKO mice by breeding DKO mice with Duoxa-/- mice (Duoxa-TKO), which do not have Duox2 activity. Similar to Nox1-TKO mice, Duoxa-TKO mice no longer have growth retardation, shortened intestine, exfoliation of crypt epithelium, crypt abscesses and depletion of goblet cells manifested in DKO mice by 35 days of age. Unlike Nox1-TKO mice, Duoxa-TKO mice still have rampant crypt apoptosis, elevated proliferation, partial loss of Paneth cells and diminished crypt density. Treating DKO mice with NOX inhibitors (di-2-thienyliodonium/DTI and thioridazine/THZ) and an antioxidant (mitoquinone/MitoQ) significantly reduced gut pathology. Furthermore, in the inflamed human colon, DUOX protein expression is highly elevated in the apical, lateral and perinuclear membrane along the whole length of gland. Taken together, we conclude that exfoliation of crypt epithelium, but not crypt apoptosis, is a major contributor to inflammation. Both Nox1 and Duox2 induce exfoliation of crypt epithelium, but only Nox1 induces apoptosis. NOX1 and DUOX2 may be potential therapeutic targets for treating ileocolitis in human patients suffering inflammatory bowel disease (IBD).

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1.

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.

Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation.

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H2O2). It was observed that LPO was able to form dityrosine at low H2O2 concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H2O2 production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H2O2 measurement.