The Disassociation of the A20/HSP90 Complex via Downregulation of HSP90 Restores the Effect of A20 Enhancing the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeted Agents

NF-κB (nuclear factor κB) is a regulator of hepatocellular cancer (HCC)-related inflammation and enhances HCC cells’ resistance to antitumor therapies by promoting cell survival and anti-apoptosis processes. In the present work, we demonstrate that A20, a dominant-negative regulator of NF-κB, forms a complex with HSP90 (heat-shock protein 90) and causes the disassociation of the A20/HSP90 complex via downregulation of HSP90. This process restores the antitumor activation of A20. In clinical specimens, the expression level of A20 did not relate with the outcome in patients receiving sorafenib; however, high levels of HSP90 were associated with poor outcomes in these patients. A20 interacted with and formed complexes with HSP90. Knockdown of HSP90 and treatment with an HSP90 inhibitor disassociated the A20/HSP90 complex. Overexpression of A20 alone did not affect HCC cells. Downregulation of HSP90 combined with A20 overexpression restored the effect of A20. Overexpression of A20 repressed the expression of pro-survival and anti-apoptosis-related factors and enhanced HCC cells’ sensitivity to sorafenib. These results suggest that interactions with HSP90 could be potential mechanisms of A20 inactivation and disassociation of the A20/HSP90 complex and could serve as a novel strategy for HCC treatment.

Download Full-text

Perturbation of β1-Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0086 ◽

2002 ◽

Vol 13 (10) ◽

pp. 3521-3531 ◽

Cited By ~ 28

Author(s):

Marisa M. Faraldo ◽

Marie-Ange Deugnier ◽

Sylvie Tlouzeau ◽

Jean Paul Thiery ◽

Marina A. Glukhova

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Negative Regulator ◽

Dominant Negative Mutant ◽

Β1 Integrin ◽

Mrna Levels ◽

Pregnancy And Lactation

To study the mechanism of β1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of β1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-β1-cyto). Mammary glands from MMTV-β1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of β1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in β-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-κB, a negative regulator of STAT5 signaling. This is the first study demonstrating in vivo that cell–extracellular matrix interactions involving β1-integrins play an important role in the control of milk gene transcription and in the maintenance of the mammary epithelial cell differentiated state.

Download Full-text

HSP90 Inhibitor 17-AAG Attenuates Nucleus Pulposus Inflammation and Catabolism Induced by M1-Polarized Macrophages

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.796974 ◽

2022 ◽

Vol 9 ◽

Author(s):

Shuo Zhang ◽

Peng Wang ◽

Binwu Hu ◽

Weijian Liu ◽

Xiao Lv ◽

...

Keyword(s):

Nucleus Pulposus ◽

Heat Shock Protein 90 ◽

Nuclear Factor Κb ◽

Hsp90 Inhibitor ◽

Protective Role ◽

Janus Kinase ◽

Mitogen Activated Protein Kinase ◽

Inflammatory Factors ◽

M1 Macrophages

Overactivated inflammation and catabolism induced by proinflammatory macrophages are involved in the pathological processes of intervertebral disc (IVD) degeneration (IVDD). Our previous study suggested the protective role of inhibiting heat shock protein 90 (HSP90) in IVDD, while the underlying mechanisms need advanced research. The current study investigated the effects of HSP90 inhibitor 17-AAG on nucleus pulposus (NP) inflammation and catabolism induced by M1-polarized macrophages. Immunohistochemical staining of degenerated human IVD samples showed massive infiltration of macrophages, especially M1 phenotype, as well as elevated levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)13. The conditioned medium (CM) of inflamed NP cells (NPCs) enhanced M1 polarization of macrophages, while the CM of M1 macrophages but not M2 macrophages promoted the expression of inflammatory factors and matrix proteases in NPCs. Additionally, we found that 17-AAG could represent anti-inflammatory and anti-catabolic effects by modulating both macrophages and NPCs. On the one hand, 17-AAG attenuated the pro-inflammatory activity of M1 macrophages via inhibiting nuclear factor-κB (NF-κB) pathway and mitogen-activated protein kinase (MAPK) pathways. On the other hand, 17-AAG dampened M1-CM-induced inflammation and catabolism in NPCs by upregulating HSP70 and suppressing the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway. Moreover, both in vitro IVD culture models and murine disc puncture models supported that 17-AAG treatment decreased the levels of inflammatory factors and matrix proteases in IVD tissues. In conclusion, HSP90 inhibitor 17-AAG attenuates NP inflammation and catabolism induced by M1 macrophages, suggesting 17-AAG as a promising candidate for IVDD treatment.

Download Full-text

Synergism of Heat Shock Protein 90 and Histone Deacetylase Inhibitors in Synovial Sarcoma

Sarcoma ◽

10.1155/2009/794901 ◽

2009 ◽

Vol 2009 ◽

pp. 1-10 ◽

Cited By ~ 15

Author(s):

Anne Nguyen ◽

Le Su ◽

Belinda Campbell ◽

Neal M. Poulin ◽

Torsten O. Nielsen

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Synovial Sarcoma ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitors ◽

Hdac Inhibitors ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Therapeutic Benefit ◽

Multiple Time

Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of 17-AAG with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only induce cell death but also activate the survival pathway NF-κB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-κB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-κB activation. In our assays, adding 17-AAG blocks NF-κB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-κB inhibitor BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-κB synergize with HDAC inhibitors against synovial sarcoma.

Download Full-text

Identification of heat-shock protein 90 beta in Japanese encephalitis virus-induced secretion proteins

Journal of General Virology ◽

10.1099/vir.0.033993-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2803-2809 ◽

Cited By ~ 23

Author(s):

Chun-Yu Hung ◽

Meng-Chieh Tsai ◽

Yi-Ping Wu ◽

Robert Y. L. Wang

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Molecular Chaperones ◽

Heat Shock Protein 90 ◽

Rna Replication ◽

Hsp90 Inhibitor ◽

Encephalitis Virus ◽

Virus Infectivity ◽

Density Fractionation ◽

Virus Particles

Five host cellular proteins were identified in the secretion medium from Japanese encephalitis virus (JEV)-infected baby hamster kidney-21 (BHK-21) cells, including three molecular chaperones: Hsp70, GRP78 and Hsp90. Hsp90 isoforms were characterized further. Hsp90α was observed to be retained inside the nuclei, whereas Hsp90β associated with virus particles during assembly and was released into the secretion medium upon JEV infection. The association of Hsp90β and viral E protein was demonstrated by using sucrose-density fractionation and Western blot analysis. Moreover, JEV viral RNA replication was not affected by treatment with geldanamycin, an Hsp90 inhibitor, but impaired virus infectivity that was determined by a plaque-forming assay. Our results show that Hsp90β, not Hsp90α, is present in the JEV-induced secretion medium and is required for JEV infectivity in BHK-21 cells.

Download Full-text

Activation of Nuclear Factor κb and bcl-x Survival Gene Expression by Nerve Growth Factor Requires Tyrosine Phosphorylation of IκBα

The Journal of Cell Biology ◽

10.1083/jcb.152.4.753 ◽

2001 ◽

Vol 152 (4) ◽

pp. 753-764 ◽

Cited By ~ 100

Author(s):

Nguyen Truc Bui ◽

Antonia Livolsi ◽

Jean-Francois Peyron ◽

Jochen H.M. Prehn

Keyword(s):

Gene Expression ◽

Tyrosine Phosphorylation ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Nuclear Factor ◽

Human Neuroblastoma ◽

Tnf Α

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-κB (NFκB). We investigated the effect of NGF on the expression of Bcl-xL, an anti–apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1–10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFκB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFκB p65 subunit. NGF-induced NFκB activity and Bcl-xL expression were inhibited in cells overexpressing the NFκB inhibitor, IκBα. Unlike tumor necrosis factor-α (TNF-α), however, NGF-induced NFκB activation occurred without significant degradation of IκBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein–tagged IκBα. Moreover, in contrast to TNF-α, NGF failed to phosphorylate IκBα at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IκBα potently suppressed NFG-, but not TNF-α–induced NFκB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-α–, but not NGF-induced NFκB activation. We conclude that NGF and TNF-α induce different signaling pathways in neurons to activate NFκB and bcl-x gene expression.

Download Full-text

Heat shock protein 90 inhibitors attenuate LPS-induced endothelial hyperpermeability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00350.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. L755-L763 ◽

Cited By ~ 53

Author(s):

Anuran Chatterjee ◽

Connie Snead ◽

Gunay Yetik-Anacak ◽

Galina Antonova ◽

Jingmin Zeng ◽

...

Keyword(s):

Endothelial Cells ◽

Heat Shock ◽

Lung Injury ◽

Heat Shock Protein ◽

Adherens Junction ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Barrier Dysfunction ◽

Focal Adhesion Protein ◽

Endothelial Hyperpermeability

Endothelial hyperperme ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60Src, phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and β-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and β-catenin expression, and inhibited activation of pp60Src and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of β-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.

Download Full-text

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5725-5737.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5725-5737

Author(s):

M Au-Fliegner ◽

E Helmer ◽

J Casanova ◽

B M Raaka ◽

H H Samuels

Keyword(s):

Retinoic Acid ◽

Thyroid Hormone ◽

Dominant Negative ◽

Heptad Repeat ◽

Specific Response ◽

Wild Type ◽

Negative Effects ◽

Related Factors ◽

Response Element ◽

Response Elements

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

Download Full-text

MicroRNA-146a-5p enhances radiosensitivity in hepatocellular carcinoma through replication protein A3-induced activation of the DNA repair pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00189.2018 ◽

2019 ◽

Vol 316 (3) ◽

pp. C299-C311 ◽

Cited By ~ 12

Author(s):

Jing Luo ◽

Zhong-Zhou Si ◽

Ting Li ◽

Jie-Qun Li ◽

Zhong-Qiang Zhang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Dna Damage ◽

Dna Repair ◽

Dna Damage Repair ◽

Replication Protein ◽

Repair Pathway ◽

Related Factors ◽

Damage Repair ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is known for its high mortality rate worldwide. Based on intensive studies, microRNA (miRNA) expression functions in tumor suppression. Therefore, we aimed to evaluate the contribution of miR-146a-5p to radiosensitivity in HCC through the activation of the DNA damage repair pathway by binding to replication protein A3 (RPA3). First, the limma package of R was performed to differentially analyze HCC expression chip, and regulative miRNA of RPA3 was predicted. Expression of miR-146a-5p, RPA3, and DNA damage repair pathway-related factors in tissues and cells was determined. The effects of radiotherapy on the expression of miR-146a-5p and RPA3 as well as on cell radiosensitivity, proliferation, cell cycle, and apoptosis were also assessed. The results showed that there exists a close correlation between miR-146a and the radiotherapy effect on HCC progression through regulation of RPA3 and the DNA repair pathway. The positive rate of ATM, pCHK2, and Rad51 in HCC tissues was higher when compared with that of the paracancerous tissues. SMMC-7721 and HepG2 cell proliferation were significantly inhibited following 8 Gy 6Mv dose. MiR-146a-5p restrained the expression of RPA3 and promoted the expression of relative genes associated with the DNA repair pathway. In addition, miR-146a-5p overexpression suppresses cell proliferation and enhances radiosensitivity and cell apoptosis in HCC cells. In conclusion, the present study revealed that miR-146a-5p could lead to the restriction of proliferation and the promotion of radiosensitivity and apoptosis in HCC cells through activation of DNA repair pathway and inhibition of RPA3.

Download Full-text

Decreased tetrahydrobiopterin and disrupted association of Hsp90 with eNOS by hyperglycemia impair myocardial ischemic preconditioning

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01078.2010 ◽

2011 ◽

Vol 301 (5) ◽

pp. H2130-H2139 ◽

Cited By ~ 28

Author(s):

Nikolina Vladic ◽

Zhi-Dong Ge ◽

Thorsten Leucker ◽

Anna K. Brzezinska ◽

Jian-Hai Du ◽

...

Keyword(s):

Nitric Oxide ◽

Infarct Size ◽

Ischemic Preconditioning ◽

Myocardial Infarct ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Myocardial Infarct Size ◽

Human Coronary Artery ◽

Area At Risk ◽

Synthesis Inhibitor

Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH4) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly ( P < 0.05) decreased myocardial infarct size (46 ± 1 to 19 ± 2% of the area at risk in control and IPC rabbits, respectively) and increased BH4 concentrations (HPLC; 7.6 ± 0.2 to 10.2 ± 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 ± 0.3 to 5.4 ± 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH4, Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH4 synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH4 precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH4 concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 ± 0.06 to 0.59 ± 0.3) and nitric oxide production (184 ± 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH4 concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia.

Download Full-text

Effect of Heldanamycin Binding on the Thr90 Phosphorylation Site of Hsp90

Biomedical Chemistry Research and Methods ◽

10.18097/bmcrm00145 ◽

2021 ◽

Vol 4 (3) ◽

pp. e00145

Author(s):

K.A. Shcherbakov ◽

D.S. Shcherbinin ◽

A.V. Veselovsky

Keyword(s):

Prostate Cancer ◽

Transcription Factor ◽

Androgen Receptor ◽

Molecular Modeling ◽

Phosphorylation Site ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Inhibitor Binding ◽

Atp Binding Site ◽

Modeling Methods

Prostate cancer is hormone-dependent and the androgen receptor (AR) is involved in its development. AR is a transcription factor that is activated by ligand binding, result in its translocation into the nucleus, where it initiates gene transcription. In an inactive state in cytoplasm AR exists as a complex with heat shock protein 90 (HSP90) and some other proteins. When the agonist binds, a conformational change in AR occurs, resulting in HSP90 and other chaperones dissociating. Recently it has been shown that for the dissociation of the HSP90-AR complex and the translocation of the latter into the nucleus, phosphorylation of the Thr-90 residue of the N-terminal domain of HSP90 is necessary. In this work, the effect of the HSP90 inhibitor, heldanamycin, interacting with the ATP-binding site, on the Thr90 phosphorylation site was investigated by molecular modeling methods. It has been shown that inhibitor binding slightly affects the size and mobility of cavity around Thr90. It is suggested that inhibitor binding to HSP90 does not result in changing the protein structure and does not influence on protein phosphorylation, and partially explains low effectiveness of such type of drugs in the therapy of prostate cancer.

Download Full-text