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1.
Virol J ; 12: 77, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25976933

ABSTRACT

BACKGROUND: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways, leading to less protein production and activating genes that encode important proteins in folding processes, including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However, HIV infection's effect on the UPR has scarcely been investigated. METHODS: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered. RESULTS: In in vitro and in vivo analyses, our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile. CONCLUSIONS: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.


Subject(s)
Activating Transcription Factor 6/analysis , Anti-Retroviral Agents/therapeutic use , Antiretroviral Therapy, Highly Active/methods , Endoribonucleases/analysis , HIV Infections/drug therapy , Protein Serine-Threonine Kinases/analysis , Unfolded Protein Response , eIF-2 Kinase/analysis , Adult , Blotting, Western , Female , HIV Infections/pathology , Humans , Male , Middle Aged , Young Adult
2.
Methods Mol Biol ; 1292: 3-18, 2015.
Article in English | MEDLINE | ID: mdl-25804744

ABSTRACT

Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic reticulum, commonly resulting in an accumulation of unfolded proteins in the ER lumen which is sensed by three ER-resident transmembrane proteins, PERK, ATF6, and IRE1. Their activation by such ER stress affects the unfolded protein response, which consists of a shutoff of protein translation and at the same time the switching-on of specific transcription factors that control genes which function to reduce the burden of unfolded proteins to the ER. Here, we describe two sets of methods for monitoring the occurrence of ER stress and UPR signaling in human cells by analyzing markers of activation of all three ER stress sensor proteins. The first set of methods is based on the qualitative and quantitative analysis of UPR-induced transcripts by qPCR. The second set of methods consists of Western blot-based analysis of UPR-induced proteins or protein modifications. Their combined analysis allows assessment of activation of all three ER stress-activated signaling pathways that in combination are characteristic for the UPR.


Subject(s)
Biological Assay/methods , Endoplasmic Reticulum Stress/physiology , Unfolded Protein Response/physiology , Activating Transcription Factor 6/analysis , Activating Transcription Factor 6/metabolism , Endoribonucleases/analysis , Endoribonucleases/metabolism , Humans , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/metabolism , eIF-2 Kinase/analysis , eIF-2 Kinase/metabolism
3.
J Dent Res ; 93(5): 483-9, 2014 May.
Article in English | MEDLINE | ID: mdl-24570149

ABSTRACT

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L ß-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.


Subject(s)
Activating Transcription Factor 6/physiology , Odontoblasts/physiology , Activating Transcription Factor 6/analysis , Adenoviridae/genetics , Alkaline Phosphatase/analysis , Animals , Bone Morphogenetic Protein 2/pharmacology , Calcification, Physiologic/physiology , Cell Culture Techniques , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cell Nucleus/ultrastructure , Cytoplasm/ultrastructure , Dental Pulp/cytology , Extracellular Matrix Proteins/analysis , Gene Expression Regulation/genetics , Genetic Vectors/genetics , Humans , Odontoblasts/drug effects , Phosphoproteins/analysis , Rats , Rats, Sprague-Dawley , Sialoglycoproteins/analysis , Tooth Germ/cytology , Tooth Germ/growth & development , Transcription Factor CHOP/analysis , Unfolded Protein Response/physiology
4.
Cancer Sci ; 102(1): 275-81, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21062387

ABSTRACT

The accumulation of viral proteins in endoplasmic reticulum (ER) may cause ER stress responses and lead to either apoptosis or survival depending on the driving signals. The strong expression of latent membrane protein-1 (LMP1) in Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL) cells raises the question whether LMP1-induced ER stress response is associated with the characteristic tumor biology in HL. In this study, we investigated the expression of ER stress signals (glucose-regulated protein 78 [GRP78], X-box binding protein 1 [XBP1], activating transcription factor 6 [ATF6], CCAAT enhance-binding protein homologous protein [CHOP] and phospho-apoptosis signal-regulating kinase 1 [pASK1]) on 156 cases of HL. Furthermore, LMP1 transfection on EBV-negative HL cell lines was used to explore the regulation of ER stress signals by EBV-LMP1. Interestingly, we demonstrated that the survival signals of ER stress response (GRP78, 62%; XBP1u [unspliced], 55%; XBP1s [spliced], 38%; ATF6, 91%) were dominantly expressed over the ER death signals (CHOP, 10%; pASK1, 7%) in all histological subtypes of HL with a similar level in both EBV-positive and EBV-negative cases. However, expression of ER signals did not bear prognostic significance. In vitro, LMP1 transfection increased the expression of GRP78 and XBP1, but attenuated the expression of death signals, CHOP and pASK1. These data indicate that EBV-LMP1 may play a role in shifting EBV-infected cells towards the survival pathway in the presence of ER stress in EBV-positive HL cases.


Subject(s)
Endoplasmic Reticulum/metabolism , Hodgkin Disease/mortality , Activating Transcription Factor 6/analysis , Adult , Cell Line, Tumor , DNA-Binding Proteins/analysis , Endoplasmic Reticulum Chaperone BiP , Female , Heat-Shock Proteins/analysis , Hodgkin Disease/pathology , Hodgkin Disease/virology , Humans , Male , Regulatory Factor X Transcription Factors , Transcription Factors/analysis , Viral Matrix Proteins/physiology , X-Box Binding Protein 1
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