scholarly journals Nox4 Mediates RANKL-induced ER-Phagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

2020 ◽  
Author(s):  
Jing Sun ◽  
wugui chen ◽  
Songtao Li ◽  
Sizhen Yang ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bone Resorption ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Regulatory Effects ◽  
Protein Response

Abstract Background: Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Methods: Osteoclastogenesis was evaluated by number of TRAP-positive multinuclear (≥3) osteoclasts, bone resorption pits and expression levels of related genes. Autophagy activity were evaluated by LC3-II/LC3-I ratio, number of autophagic vacuoles and adenovirus-mRFP-GFP-tagged LC3 reporting system; Inhibitor chloroquine (CQ) was used to verified the role of autophagy in RANKL-induced osteoclastogenesis; Via downregulating Nox4 with inhibitor (DPI) and retrovirus-conveyed shRNA, we further explored the importance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, as well as the regulatory effects of Nox4 on nonmitochondrial reactive oxygen species (ROS) and PERK/eIF-2α/ATF4 pathway. Intracellular ROS scavenger (NAC), mitochondrial-targeted antioxidant (MitoTEMPO) and inhibitor of PERK (GSK2606414) were also employed to investigate the role of ROS and PERK/eIF-2α/ATF4 pathway in RANKL-induced autophagy and osteoclastogenesis. Results: RANKL markedly increased autophagy, while CQ treatment caused reduction of RANKL-induced autophagy and osteoclastogenesis. Consistent with the increased autophagy, the protein levels of Nox4 were significantly increased, and Nox4 was selectively localized within the endoplasmic reticulum (ER) after RANKL stimulation. DPI and shRNA efficiently decreased the protein level and (or) activity of Nox4 in the ER and inhibited RANKL-induced autophagy and osteoclastogenesis. Mechanistically, we found that Nox4 regulates RANKL-induced autophagy activation and osteoclastogenesis by stimulating the production of nonmitochondrial ROS. Additionally, Nox4-derived nonmitochondrial ROS dramatically activate PERK/eIF-2α/ATF4, which is a critical unfolded protein response (UPR)-related signaling pathway during ER stress. Blocking the activation of the PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS antioxidant or PERK inhibitor (GSK2606414) treatment significantly inhibited endoplasmic reticulum autophagy (ER-phagy) during RANKL-induced osteoclastogenesis. Conclusions: Our findings provide new insights into the processes of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesis-related diseases.

scholarly journals Nox4 mediates RANKL-induced ER-phagy and Osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 Pathway

2021 ◽  
Author(s):  
Jing Sun ◽  
Wugui Cheng ◽  
Songtao Li ◽  
Sizhen Yang ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Ros Scavenger ◽  
Regulatory Effects ◽  
Protein Response

Abstract Background: Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Results: Inhibitor chloroquine (CQ) was used to verified the role of autophagy in RANKL-induced osteoclastogenesis; Via downregulating Nox4 with inhibitor (DPI) and retrovirus-conveyed shRNA, we further explored the importance of Nox4 in RANKL-induced autophagy and osteoclastogenesis, as well as the regulatory effects of Nox4 on nonmitochondrial reactive oxygen species (ROS) and PERK/eIF-2α/ATF4 pathway.Intracellular ROS scavenger (NAC), mitochondrial-targeted antioxidant (Mito-TEMPO) and inhibitor of PERK (GSK2606414) were also employed to investigate the role of ROS and PERK/eIF-2α/ATF4 pathway in RANKL-induced autophagy and osteoclastogenesis. RANKL markedly increased autophagy, while CQ treatment caused reduction of RANKL-induced autophagy and osteoclastogenesis. Consistent with the increased autophagy, the protein levels of Nox4 were significantly increased, and Nox4 was selectively localized within the endoplasmic reticulum (ER) after RANKL stimulation. DPI and shRNA efficiently decreased the protein level and (or) activity of Nox4 in the ER and inhibited RANKL-induced autophagy and osteoclastogenesis. Mechanistically, we found that Nox4 regulates RANKL-induced autophagy activation by stimulating the production of nonmitochondrial ROS. Additionally, Nox4-derived nonmitochondrial ROS dramatically activate PERK/eIF-2α/ATF4, which is a critical unfolded protein response (UPR)-related signaling pathway. Blocking the activation of the PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS antioxidant or PERK inhibitor (GSK2606414) treatment significantly inhibited endoplasmic reticulum autophagy (ER-phagy) during RANKL-induced osteoclastogenesis. Conclusions: Our findings provide new insights into the processes of RANKL-induced osteoclastogenesis and will help the development of new therapeutic strategies for osteoclastogenesis-related diseases.

scholarly journals Nox4 Promotes RANKL-Induced Autophagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Sun ◽  
Wugui Chen ◽  
Songtao Li ◽  
Sizhen Yang ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Nuclear Factor Κb ◽  
Potential Therapeutic Target ◽  
Unfolded Protein ◽  
Nadph Oxidase 4 ◽  
Receptor Activator ◽  
Ros Scavenger ◽  
Protein Response

Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Via conducting a series of biochemical experiments with in vitro cell lines, this study investigated the role and mechanism of NADPH oxidase 4 (Nox4) in RANKL-induced autophagy and osteoclastogenesis. In the current study, we found that RANKL dramatically induced autophagy and osteoclastogenesis, inhibition of autophagy with chloroquine (CQ) markedly attenuates RANKL-induced osteoclastogenesis. Interestingly, we found that the protein level of Nox4 was remarkably upregulated by RANKL treatment. Inhibition of Nox4 by 5-O-methyl quercetin or knockdown of Nox4 with specific shRNA markedly attenuated RANKL-induced autophagy and osteoclastogenesis. Furthermore, we found that Nox4 stimulated the production of nonmitochondrial reactive oxygen species (ROS), activating the critical unfolded protein response (UPR)-related signaling pathway PERK/eIF-2α/ATF4, leading to RANKL-induced autophagy and osteoclastogenesis. Blocking the activation of PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS scavenger (NAC) or PERK inhibitor (GSK2606414) significantly inhibited autophagy during RANKL-induced osteoclastogenesis. Collectively, this study reveals that Nox4 promotes RANKL-induced autophagy and osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 pathway, suggesting that the pathway may be a novel potential therapeutic target for osteoclastogenesis-related disease.

scholarly journals Dual role of Endoplasmic Reticulum Stress-Mediated Unfolded Protein Response Signaling Pathway in Carcinogenesis

2019 ◽  
Vol 20 (18) ◽  
pp. 4354 ◽  
Author(s):  
Natalia Siwecka ◽  
Wioletta Rozpędek ◽  
Dariusz Pytel ◽  
Adam Wawrzynkiewicz ◽  
Adam Dziki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Signaling Pathway ◽  
Cancer Progression ◽  
Cellular Response ◽  
Current Knowledge ◽  
Clinical Prognosis ◽  
Unfolded Protein ◽  
Protein Response

Cancer constitutes a grave problem nowadays in view of the fact that it has become one of the main causes of death worldwide. Poor clinical prognosis is presumably due to cancer cells metabolism as tumor microenvironment is affected by oxidative stress. This event triggers adequate cellular response and thereby creates appropriate conditions for further cancer progression. Endoplasmic reticulum (ER) stress occurs when the balance between an ability of the ER to fold and transfer proteins and the degradation of the misfolded ones become distorted. Since ER is an organelle relatively sensitive to oxidative damage, aforementioned conditions swiftly cause the activation of the unfolded protein response (UPR) signaling pathway. The output of the UPR, depending on numerous factors, may vary and switch between the pro-survival and the pro-apoptotic branch, and hence it displays opposing effects in deciding the fate of the cancer cell. The role of UPR-related proteins in tumorigenesis, such as binding the immunoglobulin protein (BiP) and inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6) or the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), has already been specifically described so far. Nevertheless, due to the paradoxical outcomes of the UPR activation as well as gaps in current knowledge, it still needs to be further investigated. Herein we would like to elicit the actual link between neoplastic diseases and the UPR signaling pathway, considering its major branches and discussing its potential use in the development of a novel, anti-cancer, targeted therapy.

scholarly journals Potential Roles of Endoplasmic Reticulum Stress and Cellular Proteins Implicated in Diabesity

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Sagir Mustapha ◽  
Mustapha Mohammed ◽  
Ahmad Khusairi Azemi ◽  
Ismaeel Yunusa ◽  
Aishatu Shehu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Unfolded Protein ◽  
Metabolic Functions ◽  
Chronic Hyperglycemia ◽  
Obesity And Diabetes ◽  
Activating Transcription Factor ◽  
Protein Response

The role of the endoplasmic reticulum (ER) has evolved from protein synthesis, processing, and other secretory pathways to forming a foundation for lipid biosynthesis and other metabolic functions. Maintaining ER homeostasis is essential for normal cellular function and survival. An imbalance in the ER implied stressful conditions such as metabolic distress, which activates a protective process called unfolded protein response (UPR). This response is activated through some canonical branches of ER stress, i.e., the protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). Therefore, chronic hyperglycemia, hyperinsulinemia, increased proinflammatory cytokines, and free fatty acids (FFAs) found in diabesity (a pathophysiological link between obesity and diabetes) could lead to ER stress. However, limited data exist regarding ER stress and its association with diabesity, particularly the implicated proteins and molecular mechanisms. Thus, this review highlights the role of ER stress in relation to some proteins involved in diabesity pathogenesis and provides insight into possible pathways that could serve as novel targets for therapeutic intervention.

scholarly journals A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum

1998 ◽  
Vol 143 (4) ◽  
pp. 921-933 ◽  
Author(s):  
Susana Silberstein ◽  
Gabriel Schlenstedt ◽  
Pam A. Silver ◽  
Reid Gilmore
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Physiological Role ◽  
Carboxypeptidase Y ◽  
Reporter Protein ◽  
Unfolded Protein ◽  
A Cell ◽  
The Unfolded Protein Response ◽  
Protein Response

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.

scholarly journals Albendazole reduces hepatic inflammation and endoplasmic reticulum-stress in a mouse model of chronic Echinococcus multilocularis infection

2022 ◽  
Vol 16 (1) ◽  
pp. e0009192
Author(s):  
Michael Weingartner ◽  
Simon Stücheli ◽  
Fadi Jebbawi ◽  
Bruno Gottstein ◽  
Guido Beldi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Echinococcus Multilocularis ◽  
Molecular Mechanisms ◽  
Disease Recurrence ◽  
Unfolded Protein ◽  
Programmed Death ◽  
Protein Response ◽  
Host Parasite ◽  
Related Proteins

Background Echinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice. Methods E. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes. Results E. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels. Conclusions and significance AE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE.

scholarly journals Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

1999 ◽  
Vol 10 (12) ◽  
pp. 4059-4073 ◽  
Author(s):  
Maddalena de Virgilio ◽  
Claudia Kitzmüller ◽  
Eva Schwaiger ◽  
Michael Klein ◽  
Gert Kreibich ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
The Other ◽  
Slow Phase ◽  
Type I ◽  
Unfolded Protein ◽  
Other Hand ◽  
The Unfolded Protein Response ◽  
Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

scholarly journals ScreeningMycobacterium tuberculosisSecreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Chelsea E. Stamm ◽  
Breanna L. Pasko ◽  
Sujittra Chaisavaneeyakorn ◽  
Luis H. Franco ◽  
Vidhya R. Nair ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mycobacterium Tuberculosis ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Membrane Binding ◽  
Host Cells ◽  
Secreted Proteins ◽  
Content Type ◽  
Unfolded Protein ◽  
Protein Response

ABSTRACTMycobacterium tuberculosis(Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCEAdvances have been made to identify secreted proteins ofMycobacterium tuberculosisduring animal infections. These data, combined with transposon screens identifying genes important forM. tuberculosisvirulence, have generated a vast resource of potentialM. tuberculosisvirulence proteins. However, the function of many of these proteins inM. tuberculosispathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200M. tuberculosissecreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) duringM. tuberculosisinfection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

scholarly journals Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2167 ◽  
Author(s):  
Lavinia Raimondi ◽  
Angela De Luca ◽  
Simona Fontana ◽  
Nicola Amodio ◽  
Viviana Costa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Endoplasmic Reticulum ◽  
Extracellular Vesicle ◽  
Scientific Data ◽  
Daily Experience ◽  
Unfolded Protein ◽  
Xbp1 Mrna ◽  
Protein Response ◽  
Upr Pathway

Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption.

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