A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation
2020; Elsevier BV; Volume: 19; Issue: 12 Linguagem: Inglês
10.1074/mcp.ra120.002316
ISSN1535-9484
AutoresYi Liu, Michael J. Trnka, Shenheng Guan, Doyoung Kwon, Do‐Hyung Kim, J.-J. Chen, Peter A. Greer, Alma L. Burlingame, Maria Almira Correia,
Tópico(s)Autophagy in Disease and Therapy
ResumoMallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria. Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria. Protein aggregates and inclusion bodies are linked to various neurodegenerative, muscular and hepatic diseases [Alzheimer's, Parkinson's, Desmin-related myopathies, and Mallory-Denk-bodies (MDBs) (1Strnad P. Zatloukal K. Stumptner C. Kulaksiz H. Denk H. Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies.Biochim. Biophys. Acta. 2008; 1782: 764-774Crossref PubMed Scopus (82) Google Scholar)]. Although different aggregates vary with each tissue source and in predominant protein composition, they contain common components such as p62/Sequestosome-1, ubiquitin, chaperones and proteasome constituents, which are often misfolded, highly insoluble, and cross-linked (1Strnad P. Zatloukal K. Stumptner C. Kulaksiz H. Denk H. Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies.Biochim. Biophys. Acta. 2008; 1782: 764-774Crossref PubMed Scopus (82) Google Scholar, 2Zatloukal K. Stumptner C. Fuchsbichler A. Heid H. Schnoelzer M. Kenner L. 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To our knowledge, this is the first evidence that NF-κB is also activated through IκB-sequestration into insoluble aggregates. We believe such a novel mechanism accounts for the persistent hepatic NF-κB activation that may directly contribute to the severe inflammatory responses and liver injury observed not only in various MDB-featuring liver diseases and experimental MDB-models but also in acute erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLPP) (28Cox T.M. Protoporphyria.in: Karl M. Kadish K.M.S. Roger Guilard The Porphyrin Handbook: Medical aspects of porphyrins. Elsevier Science, U. S. A2003Crossref Scopus (9) Google Scholar, 29Thapar M. Bonkovsky H.L. The diagnosis and management of erythropoietic protoporphyria.Gastroenterol. Hepatol. (N Y). 2008; 4: 561-566PubMed Google Scholar, 30Balwani M. Doheny D. Bishop D.F. Nazarenko I. Yasuda M. Dailey H.A. Anderson K.E. Bissell D.M. Bloomer J. Bonkovsky H.L. Phillips J.D. Liu L. Desnick R.J. 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Fresh primary mouse hepatocytes were cultured on Type I collagen-coated 60 mm Permanox plates (Thermo Scientific, Grand Island, NY) in William's E Medium (Invitrogen, Grand Island, NY) supplemented with 2 mM l-glutamine, insulin-transferrin-selenium (Invitrogen), 0.1% bovine albumin Fraction V (Invitrogen), Penicillin-Streptomycin (Invitrogen) and 0.1 μM dexamethasone. Cells were allowed to attach for 4 to 6 h and then overlaid with Matrigel (Corning, Oneonta, NY). From the 2nd day after plating, the medium was replaced daily, and cells were further cultured for 4–5 days with daily light microscopic examination for any signs of cell death and/or cytotoxicity. On day 5, some cells were treated with 30 μM NMPP (Frontier Scientific, Logan, UT; dissolved in DMSO), 10 μM PPIX (Sigma-Aldrich, St. Louis, MO; dissolved in DMSO with sonication) or 10 μM zinc(II)-PPIX (ZnPP, Sigma-Aldrich, St. Louis, MO; dissolved in DMSO and complexed with BSA (BSA) at a molar ratio of 4:1 to keep it solubilized in the medium) for various times as indicated (Results). In some cases, hepatocytes were pretreated with inhibitors of various cellular processes as indicated (Results) for 1 h before treatment with NMPP, PPIX or ZnPP. HepG2 and HeLa cells were cultured in minimal Eagle's medium (MEM) containing 10% v/v fetal bovine serum (FBS) and supplemented with nonessential amino acids and 1 mM sodium pyruvate. HEK293T and MEF cells were cultured with Dulbecco's Modified Eagle high glucose medium (DMEM) containing 10% v/v FBS. For transfection experiments, cells were seeded on 6-well plates, when cells were 60% confluent, each cell well was transfected with 3 μg plasmid DNA complexed with TurboFect transfection reagent (ThermoFisher, Grand Island, NY) for HEK293T cells and X-tremeGENE HP transfection reagent (Roche, Indianapolis, IN) for HepG2 cells according to the manufacturers' instructions. At 40-72 h after transfection, cells were either treated as indicated or directly harvested for assays. HRI-knockout mice: Were generated as described (33Han A.P. Yu C. Lu L. Fujiwara Y. Browne C. Chin G. Fleming M. Leboulch P. Orkin S.H. Chen J.J. Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency.EMBO J. 2001; 20: 6909-6918Crossref PubMed Scopus (262) Google Scholar). p62 knockout mice: Originally generated as detailed (34Okada K. Yanagawa T. Warabi E. Yamastu K. Uwayama J. Takeda K. Utsunomiya H. Yoshida H. Shoda J. Ishii T. 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ATG5 MEF cells: Were generated by Prof. Noboru Mizushima's laboratory (36Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1120) Google Scholar), University of Tokyo, and provided by Prof. Randy Schekman's laboratory (UC Berkeley). CAPN4 MEF cells: Were generated as described (37Tan Y. Dourdin N. Wu C. De Veyra T. Elce J.S. Greer P.A. Conditional disruption of ubiquitous calpains in the mouse.Genesis. 2006; 44: 297-303Crossref PubMed Scopus (53) Google Scholar). PentaKO (p62/NBR1/OPTN/NDP52/TAX1BP1) HeLa cells: Were generated and provided by Dr. Richard J. Youle's laboratory NIH (38Lazarou M. Sliter D.A. Kane L.A. Sarraf S.A. Wang C. Burman J.L. Sideris D.P. Fogel A.I. Youle R.J. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.Nature. 2015; 524: 309-314Crossref PubMed Scopus (1309) Google Scholar). PPIX content was determined using the intrinsic PPIX fluorescence as described (39Chisolm Jr., J. Brown D.H. Micro-scale photofluorometric determination of “free erythrocyte pophyrin” (protoporphyrin IX).Clin. Chem. 1975; 21: 1669-1682Crossref PubMed Scopus (115) Google Scholar). Briefly, 50 μl of cell lysates were first extracted with 400 μl of an ethyl acetate and acetic acid mixture (3:1, EtOAc-HAc) and then re-extracted with another 400 μl of EtOAc-HAc. The extracts were pooled and re-extracted with 400 μl of 3 M HCl. After centrifugation, the aqueous phase was recovered for fluorescent determination in a SpectrumMax M5 plate reader at an excitation of 405 nm. The intensity at an emission of 610 nm was quantified relative to a standard curve prepared with known concentrations of pure PPIX. pCMV-3HA-IκBα and pCMV-3HA-IκBα-S32A/S36A were gifts from Dr. Warner Greene (plasmids #21985, #24143; Addgene, Cambridge, MA). C1-Emerald was a gift from Dr. Michael Davidson (Addgene plasmid #54734). pcDNA6 and pcDNA3 vectors were from Invitrogen (Grand Island, NY). pcDNA6-HA-rHRI, C1-Emerald-IκBα, pcDNA6-p62-myc and pcDNA3-NBR1 were constructed by us. The primers, templates, vectors and restriction enzymes (RE) used are summarized below (Table I). Nuclear fractions of cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher, Grand Island, NY). EMSA were performed using LightShift Chemiluminescent EMSA Kit (ThermoFisher). Briefly, 2–5 μg of nuclear extracts were incubated with 1 μl of biotin-labeled NF-κB DNA probes (Panomics, Cleveland, OH) in binding buffer [10 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 5 mM MgCl2, 0.2 mM EDTA, 5% glycerol, 1 μg poly dI-dC]. The final volume of the mixture was adjusted to 20 μl and incubated at room temperature for 30 min, and then mixed with 5 μl loading buffer for loading onto 5% TBE Polyacrylamide Gel (Bio-Rad, Hercules, CA). The gel was run at 120 V until the dye reached the gel bottom, and then transferred to Hybond-N+ positively charged nylon membrane (GE Life Sciences, Marlborough, MA). The NF-κB complex shifted probes were detected by blotting with HRP-coupled streptavidin. For super-shift EMSA, the binding mixture (in the absence of the biotin-labeled probes) was first incubated with p65 antibody for 20 min on ice, and then the biotin-labeled probes were added and further incubated at room temperature for another 30 min before gel loading. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturers' instructions. Total RNA (2 μg) was used to perform reverse transcription using SuperScript VILO Master Mix (Invitrogen, Grand Island, NY) in a 20 μl-reaction. Reverse transcribed first strand cDNA (1 μl) was used in PCRs. GAPDH was used as the internal control. PCR primers were as follows: IL6-RT forward: ACAACCACGGCCTTCCCTACTT; IL6-RT reverse: CACGATTTCCCAGAGAACATGT; GAPDH-RT forward: ACCACAGTCCATGCCATCAC; GAPDH-RT reverse: CACCACCCTGTTGCTGTAGCC. For Western IB analyses, whole-cell extracts were prepared with Cell Lysis buffer (Cell Signaling Technology, Danvers, MA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and supplemented with 10% glycerol and protease/phosphatase inhibitor mixture (Pierce, Grand Island, NY). Cell lysates were sonicated for 10 s and then cleared by centrifugation at 4 °C in a tabletop centrifuge at 14,000 × g for 10 min. Protein concentrations were determined by BCA assay and equal amounts of proteins were separated on 4–15% Tris-Glycine eXtended (TGX) polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) for IB analyses. The following primary antibodies were employed: c-Myc (9E10, Santa Cruz Technology, Dallas, TX), HA (C29F4, Cell Signaling Technology), IκBα (N terminus, E130, Abcam, Cambridge, MA), IκBα (C terminus, 44D4, Cell Signaling Technology), p62 (2C11, Abnova, Taipei City, Taiwan), phospho-eIF2α (Ser52) (polyclonal, Invitrogen), eIF2α (Invitrogen), IκBα (Mouse mAb for IP, 112B2, Cell Signaling Technology), NF-κB p65 (D14E12, Cell Signaling Technology), LC3B (Novus, Littleton, CO), β-Actin (Sigma, St. Louis, MO), Histone H3 (Abcam), Nup153 (Bethyl, Montgomery, TX), Nup153 (QE5, BioLegend, San Diego, CA). The following secondary antibodies were also used: Rabbit anti-mouse IgG H&L (HRP) (Abcam) and Goat anti-rabbit IgG H&L (HRP) (Abcam). The immunoblots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Grand Island, NY). Whole-cell extracts were prepared as described above. Cell lysates (1 mg) were then incubated with indicated antibodies (2 µg) or control IgGs at 4 °C overnight. Antibody-antigen complexes were then captured by protein G Dynabeads (Invitrogen, Grand Island, NY) at room temperature for 1 h, and then eluted by heating at 95 °C for 10 min in 2 × SDS-loading buffer. Eluates were subjected to IB analyses as described above. Cells were harvested in cell lysis buffer (≈ 300 µl) as described above and the cell lysates were cleared by centrifugation at 14,000 × g. The pellet was then solubilized in RIPA buffer (Cell Signaling Technology, Danvers, MA) supplemented with 0.1% SDS, 10% glycerol and protease/phosphatase inhibitor mixture with sonication followed by centrifugation at 14,000 × g for 10 min. The resulting pellet was then solubilized in half the volume (≈150 µl) of cell lysis buffer with sonication in urea/CHAPS buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, and 30 mM DTT and supplemented with protease/phosphatase inhibitor mixture. High salt buffer (HSB)-extraction (Fig. 3C) was carried out as described previously (40Ku N.O. Toivola D.M. Zhou Q. Tao G.Z. Zhong B. Omary M.B. Studying simple epithelial keratins in cells and tissues.Methods Cell Biol. 2004; 78: 489-517Crossref PubMed Scopus (0) Google Scholar). Briefly, cells were first harvested in cell lysis buffer containing 1% Triton as described above, the resulting pellet was then suspended in a cell lysis buffer supplemented with 1.5 M KCl (HSB) with sonication. Upon sedimentation, the resulting pellet was solubilized in Laemmli buffer c
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