Ultraviolet Radiation Resistance-associated Gene Research Articles

Impaired autophagy mechanisms associated with phospholamban-R14del disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Phospholamban (PLN) is a key modulator of sarcoplasmic reticulum Ca2+ homeostasis and cardiac contractility. The PLN variant c.40_42delAGA (p.R14del) leads to arrhythmogenic cardiomyopathy characterized by life-threatening ventricular arrhythmias and sudden cardiac death. A large number of PLN-R14del carriers have been identified worldwide, although the expanding use of genetic testing and increased awareness is unveiling a higher frequency. PLN-containing aggregates are emerging as a hallmark of this disease, and are increasingly believed to be associated with disease pathogenesis. However, the mechanism leading to the formation of aggregates is unknown. We performed an in depth molecular and cellular analysis utilizing in vitro systems to determine if these aggregates result from aberrations in autophagy, and the underlying mechanisms. Utilizing cell culture systems we studied the expression level and subcellular distribution of fundamental autophagy-related proteins and evaluated the impact of autophagy inducers and inhibitors. Expression of PLN-R14del in different cell culture systems (HEK293 and H9c2 cells) was found to result in PLN aggregate formation, which were determined to contain the autophagy marker proteins sequestosome-1 (p62) and microtubule-associated protein light chain 3 (LC3), indicating inefficient degradation by the autophagy pathway. In support of this, PLN-R14del expressing cells were shown to exhibit increased p62 and LC3-II protein levels. This was associated with significantly reduced autophagic flux, as demonstrated by examination of LC3-II protein levels after autophagy inhibition. Moreover, by detailed immunofluorescence analysis, we determined reduced co-localization of the autophagosomal protein LC3 and the lysosomal associated membrane protein 1 (LAMP1) in PLN-R14del cells, suggesting impaired autophagosome-lysosome fusion. We next examined the distribution of key proteins mediating autophagosome-lysosome membrane fusion including the Ras-related protein Rab7, the ultraviolet radiation resistance-associated gene (UVRAG) and membrane tether proteins. In PLN-R14del transfected cells, significant alterations in their co-localization with LC3 were observed, indicating their impaired recruitment to the autophagosomes. Importantly, in PLN-WT cells changes in cytosolic Ca2+ levels by thapsigargin treatment were found to recapitulate the autophagic defects of PLN-R14del, suggesting that Ca2+ alterations are a contributing factor of PLN-R14del mediated aberrant autophagy. In conclusion, our findings reveal a causal relationship between PLN-R14del and autophagy dysregulation, through inhibition of autophagosome-lysosome fusion and autophagic flux reduction. This ultimately leads to aggregate formation, an early pathological sign in human patients.

UVRAG Promotes Tumor Progression through Regulating SP1 in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common type of cancer. The ultraviolet radiation resistance-associated gene (UVRAG) plays a role in autophagy and has been implicated in tumor progression and prognosis. However, the role of UVRAG expression in CRC has remained elusive. In this study, the prognosis was analyzed via immunohistochemistry, and the genetic changes were compared between the high UVRAG expression group and the low UVRAG expression group using RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) data, and genetic changes were then identified by in vitro experiments. It was found that UVRAG could enhance tumor migration, drug resistance, and CC motif chemokine ligand 2 (CCL2) expression to recruit macrophages by upregulating SP1 expression, resulting in poor prognosis of CRC patients. In addition, UVRAG could upregulate the expression of programmed death-ligand 1 (PD-L1). In summary, the relationship between UVRAG expression and the prognosis of CRC patients as well as the potential mechanisms in CRC were explored, providing evidence for the treatment of CRC.

Dual effect of ultraviolet B on cholesterol efflux and regulated by ultraviolet radiation resistance-associated gene-mediated autophagy.

In addition to diet and metabolism, the occurrence of foam cells and atherosclerosis are also related to environmental factors. Individual studies have shown that ultraviolet B (UVB) can regulate the progression of atherosclerosis, but with different results. Whether or not UVB has a dual effect on atherosclerosis and what mechanism is involved has not been reported. After THP-1-derived foam cells were treated with UVB in different ways, the effects of UVB on foam cells were investigated by western blotting, cholesterol efflux experiment, oil red O staining and other methods. UVB plays a dual role on foam cell formation, and this effect is related to cholesterol efflux. UVB of 50mJ/cm2 can promote cholesterol efflux in foam cells, while UVB of 200mJ/cm2 can inhibit cholesterol efflux. UVB induces cholesterol efflux from foam cells in an autophagy-dependent manner, as the beneficial effect of UVB at 50mJ/cm2 can be reversed by the autophagy inhibitor 3-Methyladenine (3-MA). In addition, silencing the expression of ultraviolet radiation resistance-associated gene (UVRAG) can inhibit autophagy and reduce cholesterol efflux, and overexpressing UVRAG yields the opposite result. In conclusion, our research proves that UVB exhibits a dual role in foam cell formation by regulating cholesterol efflux. Further more, we also reveal that UVRAG-mediated autophagy is the underlying mechanism of UVB-induced cholesterol efflux.

MiR-183 inhibits autophagy and apoptosis in gastric cancer cells by targeting ultraviolet radiation resistance-associated gene.

Ultraviolet radiation resistance‑associated gene (UVRAG) regulates autophagy by promoting the formation and maturation of autophagosomes. The aim of the present study was to investigate the effects of UVRAG and UVRAG‑targeting miRNA on the regulation of autophagy and apoptosis in gastric cancer (GC). TargetScan was used to predict that miR‑183 targets the 3'‑untranslated region (UTR) of UVRAG, while the interaction between miR‑183 and the 3'‑UTR of UVRAG was assessed using a dual luciferase reporter assay. Autophagy was induced in the GC cell line MKN28 by serum starvation. miR‑NC mimics, miR‑183 mimics, miR‑NC inhibitors and miR‑183 inhibitors were transfected into MKN28 cells, followed by assessment of the UVRAG expression, cell viability and cell apoptosis by western blotting, reverse transcription‑quantitative polymerase chain reaction, Cell Counting Kit‑8 and flow cytometry, respectively. It was demonstrated that autophagy resulted in miR‑183 downregulation and overexpression of UVRAG mRNA and protein in MKN28 cells. Transfection with miR‑183 mimic or inhibitor affected the expression of miR‑183 and, consequently, UVRAG. miR‑183 overexpression reversed the starvation‑induced inhibition of cell proliferation, while inhibition of miR‑183 reduced the proliferation of GC cells. miR‑183 overexpression reversed starvation‑induced apoptosis, while miR‑183 inhibition promoted starvation‑induced apoptosis in GC cells. Finally, miR‑183 overexpression attenuated starvation‑induced autophagy (LC‑3), apoptosis (Bax/Bcl‑2) and UVRAG expression, while miR‑183 inhibition exerted the opposite effects. In conclusion, the results of the present study indicated that miR‑183 inhibits starvation‑induced autophagy and apoptosis by targeting UVRAG in human GC cells.

Role of autophagy in the occurrence and development of vitiligo

Autophagy is a central cellular process of delivering organelles and cytoplasm to lysosomes for degradation and recycling of intracellular molecular components. Vitiligo is an acquired depigmented skin disease, characterized by loss or dysfunction of melanocytes in lesions. Vitiligo has a great impact on the quality of life of patients, and its pathogenesis is not yet clear. Studies have found that several mechanisms affect the occurrence and development of vitiligo, including the presence of ultraviolet radiation resistance-associated gene polymorphisms, autophagic degradation of melanosomes in keratinocytes, synergistic effects of autophagy pathway and oxidative stress, and regulation of the expression of microphthalmia transcription factor by microtubule-associated protein light chain 3. Recent studies have indicated that vitamin D could induce autophagy in a variety of cell types. The vitamin D derivative calcipotriol, which has been widely used in the topical treatment of vitiligo, can also induce autophagy of melanocytes. Therefore, researches on the relationship between autophagy and vitiligo are expected to provide new ideas for treating vitiligo. Key words: Vitiligo; Autophagy; Melanocytes; Genes; Vitamin D

Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats

Autophagy disruption leads to neuronal damage in hypoxic–ischemic brain injury. Rab7, a member of the Rab GTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPC rats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.

UVRAG Deficiency Exacerbates Doxorubicin-Induced Cardiotoxicity

Doxorubicin (DOX) is an effective chemotherapeutic drug in the treatment of various types of cancers. However, its clinical application has been largely limited by potential development of cardiotoxicity. Previously we have shown that ultra-violet radiation resistance-associated gene (UVRAG), an autophagy-related protein, is essential for the maintenance of autophagic flux in the heart under physiological conditions. Here, we sought to determine the role of UVRAG-mediated autophagy in DOX-induced cardiotoxicity. Mouse models of acute or chronic DOX-induced cardiotoxicity were established. UVRAG deficiency exacerbated DOX-induced mortality and cardiotoxicity manifested by increased cytoplasmic vacuolization, enhanced collagen accumulation, elevated serum activities of lactate dehydrogenase and myocardial muscle creatine kinase, higher ROS levels, aggravated apoptosis and more depressed cardiac function. Autophagic flux was impaired in DOX-induced cardiotoxicity. UVRAG deficiency aggravated impaired autophagic flux in DOX-induced cardiotoxicity. Intermittent fasting restored autophagy and ameliorated pathological alterations of DOX-induced cardiotoxicity. Collectively, our data suggest that UVRAG deficiency exacerbates DOX-induced cardiotoxicity, at least in part, through aggravation of DOX-induced impaired autophagic flux. Intermittent fasting, which restores blunted autophagic flux and ameliorates pathology in the mouse models of DOX-induced cardiotoxicity, may be used as a potential preventive or therapeutic approach for DOX cardiotoxicity.

Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions.

Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling.

Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K—a master regulator of endocytosis, endosomal sorting and autophagy—provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance.

An expanded role for mTORC1 in autophagy

Mechanistic target of rapamycin complex 1 (mTORC1) negatively regulates autophagy at early stages by phosphorylating Unc51-like kinase 1 (ULK1). Our recent study expanded the roles of mTORC1 in autophagy by identifying ultraviolet radiation resistance-associated gene product (UVRAG) as a substrate of mTORC1. This finding has provided new insight into the roles of mTORC1 in cellular membrane processes and cancer.

UVRAG: A Multifunctional Protein with Essential Roles in the Heart

The ultraviolet (UV) radiation resistance-associated gene (UVRAG) was initially identified for its capability to partially complement UV sensitivity in xeroderma pigmentosum (XP) cells. Subsequently, UVRAG is recognized as a tumor suppressor and autophagy-related protein. In recent years, substantial bodies of work provide new insight into UVRAG structure and multiple biological functions. UVRAG consists of a proline-rich domain, a calcium-dependent phospholipid binding C2 domain, a coiled- coil domain, a DNA-dependent protein kinase binding domain and a CEP-63 binding domain. In addition to its tumor suppression activity, UVRAG regulates autophagy and endocytic trafficking, participates in apoptosis, maintains genomic stability, gets involved in organ rotation during development. In the heart, UVRAG is required to maintain cardiac structure and function. UVRAG deficiency in the heart leads to age- related dilated cardiomyopathy. In this review, we summarize these recent findings of UVRAG that are shedding new light on its crucial roles in many biological processes and implications for the pathophysiology of various diseases including heart disease.

Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer's disease-like scenario.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2-amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer's disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD-Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

Association of ultraviolet radiation resistance-associated gene polymorphisms with rheumatoid arthritis

The ultraviolet radiation resistance-associated gene (UVRAG) protein binds to the Beclin 1/PI3-kinase III complex and promotes autophagy. Autophagy may be upregulated by endoplasmic reticulum (ER) stress. Persistent and excessive ER stress may alter synovial fibroblast apoptosis and this alteration may affect the pathogenesis of rheumatoid arthritis (RA). In this study, we investigated whether UVRAG genetic polymorphisms are associated with RA. To determine the association between UVRAG polymorphisms and RA, we genotyped five UVRAG single-nucleotide polymorphisms (SNPs; rs7111334, intron C/T; rs7933235, intron A/G; rs1380075, intron T/A; rs1458836, near the 5' gene terminal G/A; and rs636420, exon 15 C/T) using a direct sequencing method in 243 RA patients and 417 control subjects. Among these, one SNP (rs7111334) exhibited significant genotypic/allelic differences between RA patients and the control group. Therefore, this study suggested a possible association between UVRAG polymorphisms and RA susceptibility.

High expression of the autophagy gene Beclin‐1 is associated with favorable prognosis for salivary gland adenoid cystic carcinoma

Although autophagy is universally involved in tumorigenesis and tumor progression, the roles of autophagy and autophagy-regulating genes in salivary gland adenoid cystic carcinoma (ACC) remain unknown. In this study, we investigated the expression of the autophagy-regulating genes Beclin-1, death-associated protein kinase-1, ultraviolet radiation resistance-associated gene, and phosphatase and tensin homolog in salivary gland ACC samples. Immunohistochemistry and real-time polymerase chain reaction were used to analyze the expression of these genes in 89 ACC samples and normal salivary gland tissue samples. The relationship of their expression with clinicopathological features was analyzed. The data showed significantly lower expression of these genes in the tumor samples than in normal salivary gland tissue samples. Furthermore, Beclin-1 expression was significantly correlated with histological pattern of ACC (P<0.05), and high expression of ultraviolet radiation resistance-associated gene was associated with distant metastasis (P<0.05). Most importantly, univariate and multivariate survival analyses suggested that Beclin-1 protein and mRNA expression in cancer cells were independent prognostic indicators for ACC. Our results suggest that autophagy-regulating genes may participate in the pathogenesis of salivary gland ACC. Further research will be required to gain a better understanding of autophagy in ACC.

Association of UVRAG polymorphisms with susceptibility to non‐segmental vitiligo in a Korean sample

Autoimmune, self-destructive, oxidative stress and genetic theories have been proposed for the pathogenesis of vitiligo. Autophagy is essential for cellular homeostasis and is implicated in many pathophysiological conditions such as cancer, response to oxidative stress and autoimmunity. The ultraviolet (UV) radiation resistance-associated gene (UVRAG) activates the Beclin1-PI(3)KC3 complex, promoting autophagy. To evaluate whether UVRAG polymorphisms are associated with non-segmental vitiligo (NSV) patients in a Korean sample, we conducted a case-control association study of 225 NSV patients and 439 matched healthy controls. A total of five single nucleotide polymorphisms (SNPs) of UVRAG were selected for analysis. Among these, two SNPs (rs1458836, rs7933235) showed significant genotypic differences between the NSV patient group and the control group. These two SNPs were located within a strong linkage disequilibrium (LD) block. In addition, the haplotype of the UVRAG polymorphism was associated with NSV. This study suggests a possible association between UVRAG and NSV susceptibility.

Frameshift mutation of UVRAG, an autophagy-related gene, in gastric carcinomas with microsatellite instability

Alteration of autophagy is involved in tumor development. Beclin1, an important regulator of autophagy, acts as a tumor suppressor. Ultraviolet (UV) radiation resistance-associated gene (UVRAG) binds with Beclin1 and induces autophagy. There is a polyadenine tract in UVRAG gene (A10 in exon 8) that is a target for frameshift mutations in colorectal carcinomas with microsatellite instability (MSI). Functionally, colon cancer cells with the frameshift mutation of UVRAG show reduced autophagy formation and increased tumorigenicity. The aim of this study was to determine whether the frameshift mutations of UVRAG are also present in gastric carcinomas with MSI. For this, we analyzed human UVRAG exon 8 in 45 gastric carcinomas with MSI and 92 gastric carcinomas without MSI by a single-strand conformation polymorphism analysis. Overall, we detected 3 frameshift mutations of UVRAG in the polyadenine tract (3/45; 6.7%), and all of them were found in MSH-high (H) subtypes (3/32; 9.4%). The 3 mutations consisted of 2 c.708_709delA and 1 c.709delA which would result in premature stops of the UVRAG protein synthesis. The present data indicate that frameshift mutations in the polyadenine tract in UVRAG gene are present in gastric carcinomas as well and suggest that the affected gastric cancer cells with the mutations may have a reduced autophagy activity.