Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Nitric Oxide Derived from Cytoglobin-Deficient Hepatic Stellate Cells Causes Suppression of Cytochrome c Oxidase Activity in Hepatocytes.

Aims: Cell-cell interactions between hepatocytes (Hep) and other liver cells are key to maintaining liver homeostasis. Cytoglobin (CYGB), expressed exclusively by hepatic stellate cells (HSC), is essential in mitigating mitochondrial oxidative stress. CYGB absence causes Hep dysfunction and evokes hepatocarcinogenesis through an elusive mechanism. CYGB deficiency is speculated to hinder nitric oxide dioxygenase (NOD) activity, resulting in the elevated formation and release of nitric oxide (NO). Hence, we hypothesized that NO accumulation induced by the loss of NOD activity in CYGB-deficient HSC could adversely affect mitochondrial function in Hep, leading to disease progression. Results: NO, a membrane-permeable gas metabolite overproduced by CYGB-deficient HSC, diffuses into the neighboring Hep to reversibly inhibit cytochrome c oxidase (CcO), resulting in the suppression of respiratory function in an electron transport chain (ETC). The binding of NO to CcO is proved using purified CcO fractions from Cygb knockout (Cygb-/-) mouse liver mitochondria. Its inhibitory action toward CcO-specific activity is fully reversed by the external administration of oxyhemoglobin chasing away the bound NO. Thus, these findings indicate that the attenuation of respiratory function in ETC causes liver damage through the formation of excessive reactive oxygen species. Treating Cygb-/- mice with an NO synthase inhibitor successfully relieved NO-induced inhibition of CcO activity in vivo. Innovation and Conclusion: Our findings provide a biochemical link between CYGB-absence in HSC and neighboring Hep dysfunction; mechanistically the absence of CYGB in HSC causes mitochondrial dysfunction of Hep via the inhibition of CcO activity by HSC-derived NO. Antioxid. Redox Signal. 38, 463-479.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

Hexa Histidine-Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species.

BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.

Autophagy in the Central Nervous System and Effects of Chloroquine in Mucopolysaccharidosis Type II Mice.

Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.

Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice.

Cytoglobin (CYGB), discovered in hepatic stellate cells (HSCs), is known to possess a radical scavenger function, but its pathophysiological roles remain unclear. Here, for the first time, we generated a new transgenic (TG) mouse line in which both Cygb and mCherry reporter gene expression were under the control of the native Cygb gene promoter. We demonstrated that the expression of Cygb-mCherry was related to endogenous Cygb in adult tissues by tracing mCherry fluorescence together with DNA, mRNA, and protein analyses. Administration of a single dose (50 mg/kg) of thioacetamide (TAA) in Cygb-TG mice resulted in lower levels of alanine transaminase and oxidative stress than those in WT mice. After 10 weeks of TAA administration, Cygb-TG livers exhibited reduced neutrophil accumulation, cytokine expression and fibrosis but high levels of quiescent HSCs. Primary HSCs isolated from Cygb-TG mice (HSCCygb-TG) exhibited significantly decreased mRNA levels of α-smooth muscle actin (αSMA), collagen 1α1, and transforming growth factor ß-3 after 4 days in culture relative to WT cells. HSCsCygb-TG were resistant to H2O2-induced αSMA expression. Thus, cell-specific overexpression of Cygb attenuates HSC activation and protects mice against TAA-induced liver fibrosis presumably by maintaining HSC quiescence. Cygb is a potential new target for antifibrotic approaches.

Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system.

The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.

The p53-independent nuclear translocation of cyclin G1 in degenerating neurons by ischemic and traumatic insults.

Cyclin G1 (CG1) was identified as a p53-transactivated target gene, and yet its physiological and pathological roles have been unclear. Here, we demonstrate that CG1 is translocated from cytoplasm to the nuclei of neurons in response to variety of injuries. In the normal matured rodent brain, CG1 immunoreactivity was hardly observed; however, some brain injuries exhibited intense CG1 immunoreactivity in the nuclei of the damaged neurons. Transient common carotid artery occlusion (CCAO) in the gerbil showed strong CG1-like immunoreactivity in the hippocampal CA1 neurons, and permanent middle cerebral artery occlusion (MCAO) in the mouse showed strong CG1-like immunoreactivity in the nuclei of neurons located in the ischemic brain regions. TUNEL staining did not exactly overlap with the CG1-positive cells, but overlapped highly with Fluoro-Jade B staining, a degeneration marker. Brain trauma caused by knife cut, cold injury, and kinate injection also showed CG1 accumulation in the neuronal nuclei located near the injury site. These observations were obtained in p53-deficient mice as well, suggesting that the accumulation of CG1 in the injured neurons is p53-independent. A similar nuclear translocation of endogenous CG1 was confirmed in a primary culture of cortical neurons when a toxic level of N-methyl-D-aspartate (NMDA) was applied. These results demonstrate that nuclear translocation of CG1 from cytoplasmic region occurs in damaged and degenerating neurons in a p53-independent manner, and the CG1 nuclear staining could be a good marker for the neurons received fatal damages.