144 results on '"Johnson PF"'
Search Results
2. PB.25: Relationship between volumetric breast density, age and hormonal factors
- Author
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Johnson, PF, primary, Sergeant, JC, additional, Bydder, M, additional, Maxwell, A, additional, Whiteside, S, additional, Stavrinos, P, additional, Wilson, M, additional, Evans, DG, additional, Howell, A, additional, and Astley, SM, additional
- Published
- 2013
- Full Text
- View/download PDF
3. An analysis of prosthodontic procedures evaluated by state and regional dental licensing boards
- Author
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Nimmo, A, primary and Johnson, PF, additional
- Published
- 1990
- Full Text
- View/download PDF
4. Sponsoring Institution-Based Fellowships: Health Care Administration, Leadership, and Management.
- Author
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Johnson PF and Weiss KB
- Subjects
- Humans, Leadership, Delivery of Health Care, Fellowships and Scholarships, Internship and Residency
- Published
- 2023
- Full Text
- View/download PDF
5. Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.
- Author
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Meng Q, Chen C, Yang N, Gololobova O, Shi C, Dunn CA, Rossi M, Martindale JL, Basisty N, Ding J, Delannoy M, Basu S, Mazan-Mamczarz K, Shin CH, Yang JH, Johnson PF, Witwer KW, Biragyn A, Sen P, Abdelmohsen K, De S, and Gorospe M
- Subjects
- Humans, Dipeptidyl Peptidase 4 genetics, Dipeptidyl Peptidase 4 metabolism, HeLa Cells, Aging, Cellular Senescence, Extracellular Vesicles metabolism
- Abstract
Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.
- Published
- 2023
- Full Text
- View/download PDF
6. Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest.
- Author
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Rossi M, Anerillas C, Idda ML, Munk R, Shin CH, Donega S, Tsitsipatis D, Herman AB, Martindale JL, Yang X, Piao Y, Mazan-Mamczarz K, Fan J, Ferrucci L, Johnson PF, De S, Abdelmohsen K, and Gorospe M
- Subjects
- Humans, Animals, Mice, Secretome, Aging genetics, Cytokines metabolism, Cellular Senescence genetics, B-Cell Activating Factor genetics, B-Cell Activating Factor metabolism, B-Cell Activating Factor pharmacology
- Abstract
Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-β-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy., Competing Interests: MR, CA, MI, RM, CS, SD, DT, AH, JM, XY, YP, KM, JF, LF, PJ, SD, KA, MG No competing interests declared
- Published
- 2023
- Full Text
- View/download PDF
7. The ACGME Framework for Medically Underserved Areas and Populations and Graduate Medical Education.
- Author
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McDougal L, Johnson PF, Kirk L, and Weiss KB
- Subjects
- Humans, Medically Underserved Area, Education, Medical, Graduate, Accreditation, Internship and Residency
- Published
- 2023
- Full Text
- View/download PDF
8. Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.
- Author
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Liu TT, Kim S, Desai P, Kim DH, Huang X, Ferris ST, Wu R, Ou F, Egawa T, Van Dyken SJ, Diamond MS, Johnson PF, Kubo M, Murphy TL, and Murphy KM
- Subjects
- Animals, Basic-Leucine Zipper Transcription Factors metabolism, CCAAT-Enhancer-Binding Proteins metabolism, Epistasis, Genetic, Inhibitor of Differentiation Protein 2, Lymphocytes cytology, Mice, Myeloid Cells cytology, Nematospiroides dubius immunology, Repressor Proteins, Th2 Cells cytology, Th2 Cells immunology, Cell Differentiation genetics, Dendritic Cells classification, Dendritic Cells cytology, Dendritic Cells pathology, Enhancer Elements, Genetic genetics, Mutation, Zinc Finger E-box Binding Homeobox 2 genetics
- Abstract
The divergence of the common dendritic cell progenitor
1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6 -but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH 2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH 2 responses to helminths9-11 . Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2022
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9. Characterization of Cationic Bolaamphiphile Vesicles for siRNA Delivery into Tumors and Brain.
- Author
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Kim T, Viard M, Afonin KA, Gupta K, Popov M, Salotti J, Johnson PF, Linder C, Heldman E, and Shapiro BA
- Abstract
Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing., (Published by Elsevier Inc.)
- Published
- 2020
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10. Elevating Resident Voices in Health Systems Change: Lessons From the Closure of Hahnemann University Hospital.
- Author
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Nasca TJ, Johnson PF, Weiss KB, and Brigham TP
- Subjects
- Accreditation, Fellowships and Scholarships, Humans, Philadelphia, Education, Medical, Graduate, Health Facility Closure, Hospitals, University, Internship and Residency, Public Policy, Stakeholder Participation
- Abstract
The closure of Philadelphia's Hahnemann University Hospital (HUH) in summer 2019 brought an abrupt end to its status as a sponsor of graduate medical education (GME). The Accreditation Council for Graduate Medical Education (ACGME) provided assistance to ensure that more than 550 residents and fellows in HUH's 35 ACGME-accredited programs were able to transfer to new programs in which they could continue their education. As the ACGME joined other organizations in responding to HUH's closure, it was apparent that the voices of residents and fellows should be emphasized in regulatory processes and policies that address substantial disruptions to GME and affect their education, their daily lives, and their professional futures.
- Published
- 2020
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11. Regulation of senescence and the SASP by the transcription factor C/EBPβ.
- Author
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Salotti J and Johnson PF
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta chemistry, Genes, ras physiology, Humans, MAP Kinase Signaling System physiology, NF-kappa B physiology, Phosphorylation, Protein Multimerization, Signal Transduction physiology, CCAAT-Enhancer-Binding Protein-beta physiology, Cellular Senescence physiology, Oncogenes physiology
- Abstract
Oncogene-induced senescence (OIS) serves as an important barrier to tumor progression in cells that have acquired activating mutations in RAS and other oncogenes. Senescent cells also produce a secretome known as the senescence-associated secretory phenotype (SASP) that includes pro-inflammatory cytokines and chemokines. SASP factors reinforce and propagate the senescence program and identify senescent cells to the immune system for clearance. The OIS program is executed by several transcriptional effectors that include p53, RB, NF-κB and C/EBPβ. In this review, we summarize the critical role of C/EBPβ in regulating OIS and the SASP. Post-translational modifications induced by oncogenic RAS signaling control C/EBPβ activity and dimerization, and these alterations switch C/EBPβ to a pro-senescence form during OIS. In addition, C/EBPβ is regulated by a unique 3'UTR-mediated mechanism that restrains its activity in tumor cells to facilitate senescence bypass and suppression of the SASP., (Published by Elsevier Inc.)
- Published
- 2019
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12. Author Correction: C/EBPβ regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer's disease.
- Author
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Wang ZH, Gong K, Liu X, Zhang Z, Sun X, Wei ZZ, Yu SP, Manfredsson FP, Sandoval IM, Johnson PF, Jia J, Wang JZ, and Ye K
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2019
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13. Localized RAS signaling drives cancer.
- Author
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Basu SK, Basu S, and Johnson PF
- Abstract
Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.
- Published
- 2019
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14. RNA Fibers as Optimized Nanoscaffolds for siRNA Coordination and Reduced Immunological Recognition.
- Author
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Rackley L, Stewart JM, Salotti J, Krokhotin A, Shah A, Halman JR, Juneja R, Smollett J, Lee L, Roark K, Viard M, Tarannum M, Vivero-Escoto J, Johnson PF, Dobrovolskaia MA, Dokholyan NV, Franco E, and Afonin KA
- Abstract
RNA is a versatile biomaterial that can be used to engineer nanoassemblies for personalized treatment of various diseases. Despite promising advancements, the design of RNA nanoassemblies with minimal recognition by the immune system remains a major challenge. Here, an approach is reported to engineer RNA fibrous structures to operate as a customizable platform for efficient coordination of siRNAs and for maintaining low immunostimulation. Functional RNA fibers are studied in silico and their formation is confirmed by various experimental techniques and visualized by atomic force microscopy (AFM). It is demonstrated that the RNA fibers offer multiple advantages among which are: i) programmability and modular design that allow for simultaneous controlled delivery of multiple siRNAs and fluorophores, ii) reduced immunostimulation when compared to other programmable RNA nanoassemblies, and iii) simple production protocol for endotoxin-free fibers with the option of their cotranscriptional assembly. Furthermore, it is shown that functional RNA fibers can be efficiently delivered with various organic and inorganic carriers while retaining their structural integrity in cells. Specific gene silencing triggered by RNA fibers is assessed in human breast cancer and melanoma cell lines, with the confirmed ability of functional fibers to selectively target single nucleotide mutations., Competing Interests: Conflict of Interest The authors declare no conflict of interest.
- Published
- 2018
- Full Text
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15. A RAS-CaMKKβ-AMPKα2 pathway promotes senescence by licensing post-translational activation of C/EBPβ through a novel 3'UTR mechanism.
- Author
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Basu SK, Gonit M, Salotti J, Chen J, Bhat A, Gorospe M, Viollet B, Claffey KP, and Johnson PF
- Subjects
- A549 Cells, Animals, Cell Line, Tumor, ELAV-Like Protein 1 metabolism, Enzyme Activation, HEK293 Cells, Humans, MCF-7 Cells, Mice, Mice, Knockout, NIH 3T3 Cells, 3' Untranslated Regions genetics, AMP-Activated Protein Kinases metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism, Cellular Senescence physiology, Neoplasms pathology, ras Proteins metabolism
- Abstract
Oncogene-induced senescence (OIS) is an intrinsic tumor suppression mechanism that requires the p53 and RB pathways and post-translational activation of C/EBPβ through the RAS-ERK cascade. We previously reported that in transformed/proliferating cells, C/EBPβ activation is inhibited by G/U-rich elements (GREs) in its 3'UTR. This mechanism, termed "3'UTR regulation of protein activity" (UPA), maintains C/EBPβ in a low-activity state in tumor cells and thus facilitates senescence bypass. Here we show that C/EBPβ UPA is overridden by AMPK signaling. AMPK activators decrease cytoplasmic levels of the GRE binding protein HuR, which is a key UPA component. Reduced cytoplasmic HuR disrupts 3'UTR-mediated trafficking of Cebpb transcripts to the peripheral cytoplasm-a fundamental feature of UPA-thereby stimulating C/EBPβ activation and growth arrest. In primary cells, oncogenic RAS triggers a Ca
++ -CaMKKβ-AMPKα2-HuR pathway, independent of AMPKα1, that is essential for C/EBPβ activation and OIS. This axis is disrupted in cancer cells through down-regulation of AMPKα2 and CaMKKβ. Thus, CaMKKβ-AMPKα2 signaling constitutes a key tumor suppressor pathway that activates a novel UPA-cancelling mechanism to unmask the cytostatic and pro-senescence functions of C/EBPβ.- Published
- 2018
- Full Text
- View/download PDF
16. C/EBPβ regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer's disease.
- Author
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Wang ZH, Gong K, Liu X, Zhang Z, Sun X, Wei ZZ, Yu SP, Manfredsson FP, Sandoval IM, Johnson PF, Jia J, Wang JZ, and Ye K
- Subjects
- Alzheimer Disease enzymology, Animals, Cells, Cultured, Central Nervous System metabolism, Cognition Disorders pathology, Cysteine Endopeptidases genetics, Disease Models, Animal, Female, Glucose metabolism, HEK293 Cells, Humans, Inflammation pathology, Male, Mice, Inbred C57BL, Mice, Knockout, Neurons pathology, Oxygen metabolism, RNA, Messenger genetics, Rats, Transcription, Genetic, Up-Regulation, Alzheimer Disease metabolism, Alzheimer Disease pathology, Amyloid Precursor Protein Secretases metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism
- Abstract
Delta-secretase cleaves both APP and Tau to mediate the formation of amyloid plaques and neurofibrillary tangle in Alzheimer's disease (AD). However, how aging contributes to an increase in delta-secretase expression and AD pathologies remains unclear. Here we show that a CCAAT-enhancer-binding protein (C/EBPβ), an inflammation-regulated transcription factor, acts as a key age-dependent effector elevating both delta-secretase (AEP) and inflammatory cytokines expression in mediating pathogenesis in AD mouse models. We find that C/EBPβ regulates delta-secretase transcription and protein levels in an age-dependent manner. Overexpression of C/EBPβ in young 3xTg mice increases delta-secretase and accelerates the pathological features including cognitive dysfunctions, which is abolished by inactive AEP C189S. Conversely, depletion of C/EBPβ from old 3xTg or 5XFAD mice diminishes delta-secretase and reduces AD pathologies, leading to amelioration of cognitive impairment in these AD mouse models. Thus, our findings support that C/EBPβ plays a pivotal role in AD pathogenesis via increasing delta-secretase expression.
- Published
- 2018
- Full Text
- View/download PDF
17. Oncogenic RAS-Induced Perinuclear Signaling Complexes Requiring KSR1 Regulate Signal Transmission to Downstream Targets.
- Author
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Basu SK, Lee S, Salotti J, Basu S, Sakchaisri K, Xiao Z, Walia V, Westlake CJ, Morrison DK, and Johnson PF
- Subjects
- A549 Cells, Animals, CCAAT-Enhancer-Binding Protein-beta metabolism, Cell Line, Tumor, HEK293 Cells, HeLa Cells, Hep G2 Cells, Humans, MAP Kinase Signaling System, Mice, NIH 3T3 Cells, Phosphorylation, Signal Transduction, Protein Kinases metabolism, ras Proteins metabolism
- Abstract
The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2α colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4-6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBPβ in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBPβ activation is suppressed by 3'UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBPβ. Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBPβ. Cancer Res; 78(4); 891-908. ©2017 AACR ., (©2017 American Association for Cancer Research.)
- Published
- 2018
- Full Text
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18. IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.
- Author
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Song NY, Zhu F, Wang Z, Willette-Brown J, Xi S, Sun Z, Su L, Wu X, Ma B, Nussinov R, Xia X, Schrump DS, Johnson PF, Karin M, and Hu Y
- Subjects
- Acetophenones, Acetylcysteine, Adenocarcinoma metabolism, Animals, Cell Proliferation, Cellular Senescence, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Epigenesis, Genetic, Humans, Lung Neoplasms metabolism, Mice, NADPH Oxidase 2 metabolism, NF-E2-Related Factor 2 metabolism, Proto-Oncogene Proteins p21(ras) metabolism, Reactive Oxygen Species metabolism, Tumor Suppressor Protein p53 metabolism, Adenocarcinoma genetics, I-kappa B Kinase physiology, Lung Neoplasms genetics
- Abstract
Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation ( Ikkα
ΔLu ) induces spontaneous ADCs and promotes KrasG12D -initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D -mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)- Published
- 2018
- Full Text
- View/download PDF
19. Report of the SI2025 Task Force.
- Author
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Duval JF, Opas LM, Nasca TJ, Johnson PF, and Weiss KB
- Subjects
- Humans, United States, Advisory Committees, Education, Medical, Graduate trends, Internship and Residency, Organizational Objectives, Specialty Boards
- Published
- 2017
- Full Text
- View/download PDF
20. Dual negative roles of C/EBPα in the expansion and pro-tumor functions of MDSCs.
- Author
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Mackert JR, Qu P, Min Y, Johnson PF, Yang L, and Lin PC
- Subjects
- Animals, Carcinoma, Lewis Lung genetics, Carcinoma, Lewis Lung metabolism, Cell Differentiation, Melanoma, Experimental genetics, Melanoma, Experimental metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Myeloid-Derived Suppressor Cells metabolism, Neovascularization, Pathologic metabolism, Tumor Cells, Cultured, Tumor Microenvironment, CCAAT-Enhancer-Binding Protein-alpha physiology, Carcinoma, Lewis Lung pathology, Cell Proliferation, Gene Expression Regulation, Neoplastic, Melanoma, Experimental pathology, Myeloid-Derived Suppressor Cells pathology, Neovascularization, Pathologic pathology
- Abstract
Myeloid-derived suppressor cells (MDSCs) are greatly expanded in cancer patients and tumor-bearing mice. They infiltrate into tumors and modulate the tumor microenvironment. In an effort to identify molecular mediators responsible for expansion and the tumor-promoting function of MDSCs, we discovered CCAAT/enhancer binding protein alpha (C/EBPα) expression was significantly reduced in MDSCs from tumor-bearing mice compared to non-tumor-bearing hosts. Tumor-conditioned medium down-regulated C/EBPα expression, suggesting tumor secreted factors inhibiting the gene expression. Consistent with the function of C/EBPα in regulating the balance between proliferation and growth arrest in hematopoietic progenitors, myeloid lineage specific deletion of C/EBPα resulted in significantly enhanced MDSC proliferation and expansion, as well as an increase of myeloid progenitors and a decrease of mature cells. In addition, deletion of C/EBPα in MDSCs enhanced the pro-angiogenic, immune suppressive and pro-tumorigenic behavior of these cells by upregulating the production of iNOS and arginase, as well as MMP-9 and VEGF. Accordingly, tumors growing in C/EBPα conditional null mice displayed greater MDSC infiltration, increased vascularization and accelerated tumor growth. Taken together, this study reveals dual negative roles of C/EBPα in the expansion as well as pro-angiogenic and immune suppressive functions in MDSCs.
- Published
- 2017
- Full Text
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21. Identification of senescent cell surface targetable protein DPP4.
- Author
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Kim KM, Noh JH, Bodogai M, Martindale JL, Yang X, Indig FE, Basu SK, Ohnuma K, Morimoto C, Johnson PF, Biragyn A, Abdelmohsen K, and Gorospe M
- Subjects
- Adult, Aged, Aged, 80 and over, Antibody-Dependent Cell Cytotoxicity, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p16 genetics, Diploidy, Fibroblasts metabolism, Flow Cytometry, Humans, Killer Cells, Natural metabolism, Lymphocyte Subsets enzymology, Mass Spectrometry, RNA, Messenger metabolism, RNA, Ribosomal metabolism, Cellular Senescence physiology, Dipeptidyl Peptidase 4 metabolism, Membrane Proteins metabolism
- Abstract
Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination., (© 2017 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2017
- Full Text
- View/download PDF
22. LPS independent activation of the pro-inflammatory receptor Trem1 by C/EBPε in granulocytes.
- Author
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Suh HC, Benoukraf T, Shyamsunder P, Yin T, Cao Q, Said J, Lee S, Lim R, Yang H, Salotti J, Johnson PF, Madan V, and Koeffler HP
- Subjects
- Animals, Cell Differentiation physiology, Lipopolysaccharides, Mice, Neutrophils metabolism, Transcriptome, Bone Marrow Cells metabolism, CCAAT-Enhancer-Binding Proteins metabolism, Granulocytes metabolism, Triggering Receptor Expressed on Myeloid Cells-1 metabolism
- Abstract
C/EBPε is a critical transcriptional factor for granulocyte differentiation and function. Individuals with germline mutations of C/EBPε fail to develop normal granulocytes and suffer from repeated infections. In order to gain a global view of the transcriptional machinery regulated by C/EBPε, we performed whole-genome ChIP-Seq using mouse bone marrow cells. To complement the C/EBPε DNA binding analyses, RNA-Sequencing was done in parallel using sorted mature and immature granulocytes from WT and C/EBPε KO bone marrow. This approach led to the identification of several direct targets of C/EBPε, which are potential effectors of its role in granulocytic differentiation and function. Interestingly, Trem1, a gene critical to granulocyte function, was identified as a direct C/EBPε target gene. Trem1 expression overlaps very closely with expression signature of C/EBPε during hematopoietic development. Luciferase reporter and EMSA assays revealed that C/EBPε binds to the regulatory elements of Trem1 and regulates its expression during granulocytic differentiation. In addition, we provide evidence that inflammatory stimuli (LPS) can also control the expression of Trem1 independent of C/EBPε. Overall, this study provides comprehensive profiling of the transcriptional network controlled by C/EBPε during granulopoiesis and identifies Trem1 as one of its downstream effectors involved in eliciting an immune response.
- Published
- 2017
- Full Text
- View/download PDF
23. The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation.
- Author
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Ratner D, Orning MP, Proulx MK, Wang D, Gavrilin MA, Wewers MD, Alnemri ES, Johnson PF, Lee B, Mecsas J, Kayagaki N, Goguen JD, and Lien E
- Subjects
- Animals, Disease Models, Animal, Mice, Mice, Knockout, Yersinia pestis immunology, Bacterial Outer Membrane Proteins immunology, Inflammasomes immunology, Plague immunology, Pyrin immunology
- Abstract
Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1β and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1β/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1β/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1β activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1β generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1β/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague., Competing Interests: BL and NK are employees of Genentech, Inc.
- Published
- 2016
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24. C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4.
- Author
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Huggins CJ, Mayekar MK, Martin N, Saylor KL, Gonit M, Jailwala P, Kasoji M, Haines DC, Quiñones OA, and Johnson PF
- Subjects
- Activating Transcription Factor 4 analysis, Activating Transcription Factor 4 genetics, Animals, CCAAT-Enhancer-Binding Proteins analysis, CCAAT-Enhancer-Binding Proteins genetics, Cell Line, Female, Fetus abnormalities, Fetus metabolism, Gene Deletion, Gene Expression Regulation, Glutathione metabolism, Humans, Male, Mice, Inbred C57BL, Neoplasms genetics, Neoplasms metabolism, Protein Multimerization, Response Elements, Transcription Factor CHOP metabolism, Activating Transcription Factor 4 metabolism, CCAAT-Enhancer-Binding Proteins metabolism, Oxidative Stress
- Abstract
The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPβ:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPβ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg(-/-) mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg(-/-) mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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25. An Arf-Egr-C/EBPβ pathway linked to ras-induced senescence and cancer.
- Author
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Salotti J, Sakchaisri K, Tourtellotte WG, and Johnson PF
- Subjects
- 3T3 Cells, Animals, Cell Line, Tumor, Cell Proliferation, Cellular Senescence, Fibroblasts metabolism, Genotype, Humans, Mice, Mice, Inbred C57BL, NIH 3T3 Cells, Promoter Regions, Genetic, Cell Transformation, Neoplastic, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Early Growth Response Protein 1 metabolism, Neoplasms metabolism, ras Proteins metabolism
- Abstract
Oncogene-induced senescence (OIS) protects normal cells from transformation by Ras, whereas cells lacking p14/p19(Arf) or other tumor suppressors can be transformed. The transcription factor C/EBPβ is required for OIS in primary fibroblasts but is downregulated by H-Ras(V12) in immortalized NIH 3T3 cells through a mechanism involving p19(Arf) loss. Here, we report that members of the serum-induced early growth response (Egr) protein family are also downregulated in 3T3(Ras) cells and directly and redundantly control Cebpb gene transcription. Egr1, Egr2, and Egr3 recognize three sites in the Cebpb promoter and associate transiently with this region after serum stimulation, coincident with Cebpb induction. Codepletion of all three Egrs prevented Cebpb expression, and serum induction of Egrs was significantly blunted in 3T3(Ras) cells. Egr2 and Egr3 levels were also reduced in Ras(V12)-expressing p19(Arf) null mouse embryonic fibroblasts (MEFs), and overall Egr DNA-binding activity was suppressed in Arf-deficient but not wild-type (WT) MEFs, leading to Cebpb downregulation. Analysis of human cancers revealed a strong correlation between EGR levels and CEBPB expression, regardless of whether CEBPB was increased or decreased in tumors. Moreover, overexpression of Egrs in tumor cell lines induced CEBPB and inhibited proliferation. Thus, our findings identify the Arf-Egr-C/EBPβ axis as an important determinant of cellular responses (senescence or transformation) to oncogenic Ras signaling., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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26. Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300.
- Author
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Bhaumik P, Davis J, Tropea JE, Cherry S, Johnson PF, and Miller M
- Subjects
- Amino Acid Sequence, CCAAT-Enhancer-Binding Proteins chemistry, Crystallography, X-Ray, Molecular Sequence Data, Phosphorylation, Protein Conformation, Sequence Homology, Amino Acid, CCAAT-Enhancer-Binding Proteins metabolism, p300-CBP Transcription Factors chemistry
- Abstract
Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723-1818 of p300 Taz2 and residues 37-61 of C/EBPℇ. The segment corresponding to the C/EBPℇ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBPℇ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP-p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPβ N-terminus, which associates with the C/EBPβ-p300 complex.
- Published
- 2014
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27. C/EBPγ suppresses senescence and inflammatory gene expression by heterodimerizing with C/EBPβ.
- Author
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Huggins CJ, Malik R, Lee S, Salotti J, Thomas S, Martin N, Quiñones OA, Alvord WG, Olanich ME, Keller JR, and Johnson PF
- Subjects
- Animals, CCAAT-Enhancer-Binding Proteins genetics, Cell Cycle, Cell Line, Cell Line, Tumor, Cell Proliferation, Cells, Cultured, Fibroblasts cytology, Fibroblasts metabolism, Gene Deletion, Gene Expression Regulation, Neoplastic, Hematopoiesis, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Humans, Inflammation genetics, Inflammation metabolism, Mice, Mice, Inbred C57BL, NIH 3T3 Cells, Neoplasms metabolism, Protein Multimerization, CCAAT-Enhancer-Binding Protein-beta metabolism, CCAAT-Enhancer-Binding Proteins metabolism, Cellular Senescence, Gene Expression Regulation, Neoplasms genetics
- Abstract
C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β∼β) inhibited the proliferation and tumorigenicity of Ras(V12)-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γ∼β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg(-/-) MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg(-/-) MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg(-/-) bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.
- Published
- 2013
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28. Authors' reply.
- Author
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Yan C, Tang H, Gao H, Johnson PF, Ye Y, and Wu M
- Subjects
- Animals, Acute Lung Injury metabolism, Acute Lung Injury pathology, CCAAT-Enhancer-Binding Protein-delta metabolism
- Published
- 2013
29. Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction by modulating glutamate receptor recycling in Down's syndrome.
- Author
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Wang X, Zhao Y, Zhang X, Badie H, Zhou Y, Mu Y, Loo LS, Cai L, Thompson RC, Yang B, Chen Y, Johnson PF, Wu C, Bu G, Mobley WC, Zhang D, Gage FH, Ranscht B, Zhang YW, Lipton SA, Hong W, and Xu H
- Subjects
- Animals, Antineoplastic Combined Chemotherapy Protocols, C-Reactive Protein physiology, CCAAT-Enhancer-Binding Protein-beta, CCAAT-Enhancer-Binding Proteins physiology, Cerebral Cortex cytology, Cerebral Cortex physiopathology, Cisplatin, Down-Regulation, Female, Hippocampus cytology, Hippocampus physiopathology, Humans, Ifosfamide, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, MicroRNAs physiology, Mitomycin, Nerve Tissue Proteins physiology, Transcription Factors physiology, Down Syndrome physiopathology, Receptors, Glutamate metabolism, Sorting Nexins physiology, Synapses physiology
- Abstract
Sorting nexin 27 (SNX27), a brain-enriched PDZ domain protein, regulates endocytic sorting and trafficking. Here we show that Snx27(-/-) mice have severe neuronal deficits in the hippocampus and cortex. Although Snx27(+/-) mice have grossly normal neuroanatomy, we found defects in synaptic function, learning and memory and a reduction in the amounts of ionotropic glutamate receptors (NMDA and AMPA receptors) in these mice. SNX27 interacts with these receptors through its PDZ domain, regulating their recycling to the plasma membrane. We demonstrate a concomitant reduced expression of SNX27 and CCAAT/enhancer binding protein β (C/EBPβ) in Down's syndrome brains and identify C/EBPβ as a transcription factor for SNX27. Down's syndrome causes overexpression of miR-155, a chromosome 21-encoded microRNA that negatively regulates C/EBPβ, thereby reducing SNX27 expression and resulting in synaptic dysfunction. Upregulating SNX27 in the hippocampus of Down's syndrome mice rescues synaptic and cognitive deficits. Our identification of the role of SNX27 in synaptic function establishes a new molecular mechanism of Down's syndrome pathogenesis.
- Published
- 2013
- Full Text
- View/download PDF
30. CCAAT/enhancer-binding protein δ is a critical mediator of lipopolysaccharide-induced acute lung injury.
- Author
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Yan C, Johnson PF, Tang H, Ye Y, Wu M, and Gao H
- Subjects
- Acute Lung Injury chemically induced, Acute Lung Injury enzymology, Animals, Bronchoalveolar Lavage Fluid, CCAAT-Enhancer-Binding Protein-delta deficiency, Cell Line, Chemokines biosynthesis, Extracellular Signal-Regulated MAP Kinases metabolism, Gene Knockdown Techniques, Interleukin-6 metabolism, Lipopolysaccharides, Luciferases metabolism, Lung enzymology, Lung pathology, Macrophages, Alveolar enzymology, Macrophages, Alveolar pathology, Mice, Mitogen-Activated Protein Kinase 3 metabolism, Tumor Necrosis Factor-alpha metabolism, p38 Mitogen-Activated Protein Kinases metabolism, Acute Lung Injury metabolism, Acute Lung Injury pathology, CCAAT-Enhancer-Binding Protein-delta metabolism
- Abstract
Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA-mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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31. Critical role for CCAAT/enhancer-binding protein β in immune complex-induced acute lung injury.
- Author
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Yan C, Wu M, Cao J, Tang H, Zhu M, Johnson PF, and Gao H
- Subjects
- Acute Lung Injury genetics, Acute Lung Injury pathology, Animals, Antigen-Antibody Complex adverse effects, CCAAT-Enhancer-Binding Protein-beta biosynthesis, CCAAT-Enhancer-Binding Protein-beta deficiency, CCAAT-Enhancer-Binding Protein-delta deficiency, CCAAT-Enhancer-Binding Protein-delta genetics, CCAAT-Enhancer-Binding Protein-delta physiology, Cell Line, Disease Models, Animal, Immunoglobulin G administration & dosage, Immunoglobulin G adverse effects, Inflammation Mediators administration & dosage, Inflammation Mediators adverse effects, Inflammation Mediators physiology, Macrophages, Alveolar immunology, Macrophages, Alveolar metabolism, Macrophages, Alveolar pathology, Male, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Acute Lung Injury immunology, Antigen-Antibody Complex administration & dosage, CCAAT-Enhancer-Binding Protein-beta physiology
- Abstract
C/EBPs, particularly C/EBPβ and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPβ and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPβ and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPβ gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPβ-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPβ in a murine alveolar macrophage cell line. These findings implicate C/EBPβ as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.
- Published
- 2012
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32. CIKS/Act1-mediated signaling by IL-17 cytokines in context: implications for how a CIKS gene variant may predispose to psoriasis.
- Author
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Sønder SU, Paun A, Ha HL, Johnson PF, and Siebenlist U
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Blotting, Western, Cytokines immunology, Cytokines metabolism, Flow Cytometry, Humans, Interleukin-17 metabolism, Mice, Mice, Knockout, Polymorphism, Single Nucleotide, Psoriasis immunology, Psoriasis metabolism, Real-Time Polymerase Chain Reaction, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins immunology, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins metabolism, Genetic Predisposition to Disease genetics, Interleukin-17 immunology, Psoriasis genetics, Signal Transduction immunology, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins genetics
- Abstract
Psoriasis is a relapsing skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory immune cells. Recently, IL-17 cytokines have been strongly implicated as critical for the pathogenesis of this disease. IL-17A (also known as IL-17) and IL-17F are the signature cytokines of Th17 cells, but are also produced by innate cells, including γδ T cells present in skin, whereas epithelial cells, including keratinocytes, may produce IL-17C. IL-17 cytokines signal via the adaptor protein connection to IκB kinase and stress-activated protein kinases (CIKS)/Act1. Psoriasis is a disease with a strong genetic predisposition, and the gene encoding CIKS has recently been identified as a susceptibility locus. Unexpectedly, one predisposing gene variant features a mutation that impairs rather than enhances CIKS-mediated IL-17 cytokine signaling, counter to the predicted role for IL-17 cytokines in psoriatic inflammation. In this study, we demonstrate, however, that this mutant adaptor does not impair the IL-17-specific contributions to the genetic response when combined with TNF-α, a cytokine also prominent in psoriatic inflammation. Interestingly, TNF-α signals compensate IL-17 signaling defects imposed by this mutant adaptor even for genes that are not induced by TNF-α alone, including the transcription factors CCAAT/enhancer binding protein δ and IκBζ, which help regulate secondary gene expression in response to IL-17. Based on these findings we discuss a scenario in which the mutant adaptor may interfere with homeostatic maintenance of epithelial barriers, thereby potentially enabling the initiation of inflammatory responses to insults, whereas this same mutant adaptor would still be able to mediate IL-17-specific contributions to inflammation once TNF-α is present.
- Published
- 2012
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33. C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages.
- Author
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Yan C, Zhu M, Staiger J, Johnson PF, and Gao H
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta genetics, CCAAT-Enhancer-Binding Protein-delta genetics, Cell Line, Complement C5a genetics, Inflammation genetics, Inflammation metabolism, MAP Kinase Signaling System physiology, Mice, Mitogen-Activated Protein Kinase 3 genetics, Mitogen-Activated Protein Kinase 3 metabolism, Receptors, IgG genetics, p38 Mitogen-Activated Protein Kinases genetics, p38 Mitogen-Activated Protein Kinases metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism, CCAAT-Enhancer-Binding Protein-delta metabolism, Chemokines biosynthesis, Complement C5a metabolism, Receptors, IgG metabolism
- Abstract
CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ are known to participate in the regulation of many genes associated with inflammation. However, little is known about the activation and function of C/EBPβ and -δ in inflammatory responses elicited by Fcγ receptor (FcγR) activation. Here we show that C/EBPβ and -δ activation are induced in IgG immune complex (IC)-treated macrophages. The increased expression of C/EBPβ and -δ occurred at both mRNA and protein levels. Furthermore, induction of C/EBPβ and -δ was mediated, to a large extent, by activating FcγRs. Using siRNA-mediated knockdown as well as macrophages deficient for C/EBPβ and/or -δ, we demonstrate that C/EBPβ and -δ play a critical role in the production of TNF-α, MIP-2, and MIP-1α in IgG IC-stimulated macrophages. Moreover, both ERK1/2 and p38 MAPK are involved in C/EBP induction and TNF-α, MIP-2, and MIP-1α production induced by IgG IC. We provide the evidence that C5a regulates IgG IC-induced inflammatory responses by enhancing ERK1/2 and p38 MAPK activities as well as C/EBPβ and -δ activities. Collectively, these data suggest that C/EBPβ and -δ are key regulators for FcγR-mediated induction of cytokines and chemokines in macrophages. Furthermore, C/EBPs may play an important regulatory role in IC-associated inflammatory responses.
- Published
- 2012
- Full Text
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34. Identification of a Src tyrosine kinase/SIAH2 E3 ubiquitin ligase pathway that regulates C/EBPδ expression and contributes to transformation of breast tumor cells.
- Author
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Sarkar TR, Sharan S, Wang J, Pawar SA, Cantwell CA, Johnson PF, Morrison DK, Wang JM, and Sterneck E
- Subjects
- Breast metabolism, Breast pathology, Breast Neoplasms metabolism, Breast Neoplasms pathology, CCAAT-Enhancer-Binding Protein-delta metabolism, Cell Line, Tumor, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, Cyclin D1 genetics, Female, Humans, RNA, Messenger genetics, Signal Transduction, Ubiquitination, Breast Neoplasms genetics, CCAAT-Enhancer-Binding Protein-delta genetics, Cell Transformation, Neoplastic genetics, Gene Expression Regulation, Neoplastic, Nuclear Proteins metabolism, Ubiquitin-Protein Ligases metabolism, src-Family Kinases metabolism
- Abstract
The transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBPδ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBPδ through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBPδ, but not C/EBPβ, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBPδ expression supports elevated cyclin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBPδ expression in an SIAH2-dependent manner, which is necessary for "therapeutic" responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBPδ, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBPδ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.
- Published
- 2012
- Full Text
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35. 3'UTR elements inhibit Ras-induced C/EBPβ post-translational activation and senescence in tumour cells.
- Author
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Basu SK, Malik R, Huggins CJ, Lee S, Sebastian T, Sakchaisri K, Quiñones OA, Alvord WG, and Johnson PF
- Subjects
- Cell Line, Humans, Models, Biological, RNA, Messenger metabolism, 3' Untranslated Regions, Aging, CCAAT-Enhancer-Binding Protein-beta metabolism, Gene Expression Regulation, Oncogene Protein p21(ras) metabolism
- Abstract
C/EBPβ is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPβ is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPβ activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPβ, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPβ target genes, while promoting expression of genes linked to cancers and TGFβ signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPβ translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPβ activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPβ activity and suppress its anti-oncogenic functions.
- Published
- 2011
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36. A distal enhancer in Il12b is the target of transcriptional repression by the STAT3 pathway and requires the basic leucine zipper (B-ZIP) protein NFIL3.
- Author
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Smith AM, Qualls JE, O'Brien K, Balouzian L, Johnson PF, Schultz-Cherry S, Smale ST, and Murray PJ
- Subjects
- Animals, Basic-Leucine Zipper Transcription Factors genetics, Cells, Cultured, Interleukin-10 genetics, Interleukin-10 metabolism, Interleukin-12 Subunit p40 genetics, Mice, Mice, Knockout, Myeloid Cells cytology, Myeloid Cells metabolism, STAT3 Transcription Factor genetics, Basic-Leucine Zipper Transcription Factors metabolism, Enhancer Elements, Genetic physiology, Gene Expression Regulation physiology, Interleukin-12 Subunit p40 biosynthesis, STAT3 Transcription Factor metabolism, Transcription, Genetic physiology
- Abstract
Deregulated IL-12 and IL-23 production from activated myeloid lineage cells is a key driver of numerous T cell-dependent autoimmune and inflammatory diseases. IL-12 and IL-23 share a common p40 subunit encoded by Il12b, which is negatively regulated at the transcriptional level by the STAT3 (signal transducer and activator of transcription 3)-activating anti-inflammatory cytokine IL-10. We found that IL-10 targets an enhancer 10 kb upstream of the Il12b transcriptional start site. Within the enhancer, a single 10-bp site is required for the inhibitory effects of IL-10 and is bound by NFIL3 (nuclear factor, interleukin 3-regulated), a B-ZIP transcription factor. Myeloid cells lacking NFIL3 produce excessive IL-12p40 and increased IL-12p70. Thus, the STAT3-dependent expression of NFIL3 is a key component of a negative feedback pathway in myeloid cells that suppresses proinflammatory responses.
- Published
- 2011
- Full Text
- View/download PDF
37. CCAAT/enhancer-binding proteins (C/EBP)-α and -β are essential for ovulation, luteinization, and the expression of key target genes.
- Author
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Fan HY, Liu Z, Johnson PF, and Richards JS
- Subjects
- Animals, Blood Vessels embryology, Blotting, Western, CCAAT-Enhancer-Binding Protein-alpha genetics, CCAAT-Enhancer-Binding Protein-beta genetics, Chorionic Gonadotropin genetics, Corpus Luteum blood supply, Cyclic AMP-Dependent Protein Kinases metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Female, Granulosa Cells cytology, In Situ Hybridization, In Situ Nick-End Labeling, Luteinizing Hormone metabolism, Mice, Mice, Inbred BALB C, Mice, Knockout, Microarray Analysis, CCAAT-Enhancer-Binding Protein-alpha metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism, Gene Expression Regulation, Granulosa Cells metabolism, Luteinization, Ovulation genetics
- Abstract
LH activation of the epidermal growth factor receptor/RAS/ERK1/2 pathway is essential for ovulation and luteinization because granulosa cell (GC) depletion of ERK1/2 (ERK1/2(gc)(-/-) mice) renders mice infertile. As mediators of ERK1/2-dependent GC differentiation, the CCAAT/enhancer-binding proteins, (C/EBP)α and C/EBPβ, were also disrupted. Female Cebpb(gc)(-/-) mutant mice, but not Cebpa(gc)(-/-) mice, were subfertile whereas Cebpa/b(gc)(-/-) double-mutant females were sterile. Follicles failed to ovulate, ovaries were devoid of corpora lutea, luteal cell marker genes (Lhcgr, Prlr, Ptgfr, Cyp11a1, and Star) were absent, and serum progesterone levels were low. Microarray analyses identified numerous C/EBPα/β target genes in equine chorionic gonadotropin (eCG)-human (h)CG-treated mice. At 4 h post-hCG, a subset (19%) of genes altered in the Cebpa/b-depleted cells was also altered in ERK1/2-depleted cells; hence they are common effectors of ERK1/2. Additional genes down-regulated in the Cebpa/b-depleted cells at 8 and 24 h post-hCG include known (Akr1b7, Runx2, Star, Saa3) and novel (Abcb1b, Apln, Igfbp4, Prlr, Ptgfr Timp4) C/EBP targets and effectors of luteal and vascular cell development. Bhmt, a gene controlling methionine metabolism and thought to be expressed exclusively in liver and kidney, was high in wild-type luteal cells but totally absent in Cebpa/b mutant cells. Because numerous genes potentially associated with vascular development were suppressed in the mutant cells, C/EBPα/β appear to dictate the luteinization process by also controlling genes that regulate the formation of the extensive vascular network required to sustain luteal cells. Thus, C/EBPα/β mediate the terminal differentiation of GCs during the complex process of luteinization.
- Published
- 2011
- Full Text
- View/download PDF
38. CCAAT/Enhancer-binding protein beta DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP).
- Author
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Lee S, Miller M, Shuman JD, and Johnson PF
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Humans, Molecular Sequence Data, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Rats, Signal Transduction, Transcription, Genetic, CCAAT-Enhancer-Binding Protein-beta metabolism, DNA chemistry, Enhancer Elements, Genetic, p300-CBP Transcription Factors metabolism
- Abstract
Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPbeta), which regulates oncogenic H-Ras(V12)-induced senescence and growth arrest. Here we report that C/EBPbeta (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (phiXXphiphi)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPbeta binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPbeta auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third phiXXphiphi motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPbeta N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPbeta derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPbeta with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.
- Published
- 2010
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39. RSK-mediated phosphorylation in the C/EBP{beta} leucine zipper regulates DNA binding, dimerization, and growth arrest activity.
- Author
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Lee S, Shuman JD, Guszczynski T, Sakchaisri K, Sebastian T, Copeland TD, Miller M, Cohen MS, Taunton J, Smart RC, Xiao Z, Yu LR, Veenstra TD, and Johnson PF
- Subjects
- Amino Acid Sequence, Animals, CCAAT-Enhancer-Binding Protein-beta chemistry, CCAAT-Enhancer-Binding Protein-beta genetics, Cell Line, DNA genetics, Growth Substances metabolism, Humans, MAP Kinase Signaling System physiology, Mice, Mitogen-Activated Protein Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinases genetics, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Phosphorylation, Protein Multimerization, Protein Processing, Post-Translational, Rats, Ribosomal Protein S6 Kinases antagonists & inhibitors, Ribosomal Protein S6 Kinases genetics, Sequence Alignment, Transcriptional Activation, ras Proteins genetics, ras Proteins metabolism, CCAAT-Enhancer-Binding Protein-beta metabolism, Cell Cycle physiology, DNA metabolism, Leucine Zippers, Ribosomal Protein S6 Kinases metabolism
- Abstract
The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPbeta activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g<-->e' salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta in Ras(V12)-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPbeta-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.
- Published
- 2010
- Full Text
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40. A central role for transcription factor C/EBP-beta in regulating CD1d gene expression in human keratinocytes.
- Author
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Sikder H, Zhao Y, Balato A, Chapoval A, Fishelevich R, Gade P, Singh IS, Kalvakolanu DV, Johnson PF, and Gaspari AA
- Subjects
- Binding Sites, Cell Line, Cloning, Molecular, Epithelial Cells, Humans, Natural Killer T-Cells, Promoter Regions, Genetic, Skin Diseases etiology, Skin Diseases immunology, Antigens, CD1d genetics, CCAAT-Enhancer-Binding Protein-beta physiology, Gene Expression Regulation, Keratinocytes metabolism, Transcription, Genetic
- Abstract
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-beta binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-beta caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-beta in regulating CD1d transcription.
- Published
- 2009
- Full Text
- View/download PDF
41. MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility.
- Author
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Fan HY, Liu Z, Shimada M, Sterneck E, Johnson PF, Hedrick SM, and Richards JS
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta genetics, Enzyme Activation, Female, Gene Expression Profiling, Granulosa Cells enzymology, Luteinizing Hormone metabolism, MAP Kinase Signaling System, Meiosis, Mice, Mice, Inbred C57BL, Mice, Transgenic, Oocytes physiology, Ovarian Follicle physiology, Phosphorylation, CCAAT-Enhancer-Binding Protein-beta metabolism, Fertility, Granulosa Cells metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Ovulation
- Abstract
A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Because the signaling molecules RAS and ERK1/2 (extracellular signal-regulated kinases 1 and 2) are activated by an LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBPbeta (CCAAT/Enhancer-binding protein-beta) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/EBPbeta constitute an in vivo LH-regulated signaling pathway that controls ovulation- and luteinization-related events.
- Published
- 2009
- Full Text
- View/download PDF
42. C/EBPbeta regulates body composition, energy balance-related hormones and tumor growth.
- Author
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Staiger J, Lueben MJ, Berrigan D, Malik R, Perkins SN, Hursting SD, and Johnson PF
- Subjects
- Adenocarcinoma pathology, Adipose Tissue physiology, Animals, Body Weight, Bone Density, CCAAT-Enhancer-Binding Protein-beta deficiency, CCAAT-Enhancer-Binding Protein-beta genetics, CCAAT-Enhancer-Binding Protein-delta deficiency, CCAAT-Enhancer-Binding Protein-delta genetics, CCAAT-Enhancer-Binding Protein-delta physiology, Cell Division genetics, Colonic Neoplasms pathology, Female, Insulin physiology, Insulin-Like Growth Factor I physiology, Leptin physiology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Obesity complications, Obesity genetics, Body Composition physiology, CCAAT-Enhancer-Binding Protein-beta physiology, Neoplasms epidemiology, Obesity physiopathology
- Abstract
The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPdelta in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPbeta, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBPdelta was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPbeta(-/-) mice, and C/EBPbeta was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPbeta-deficient animals, especially males. Thus, C/EBPbeta contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.
- Published
- 2009
- Full Text
- View/download PDF
43. RasV12-mediated down-regulation of CCAAT/enhancer binding protein beta in immortalized fibroblasts requires loss of p19Arf and facilitates bypass of oncogene-induced senescence.
- Author
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Sebastian T and Johnson PF
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta biosynthesis, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, Cellular Senescence genetics, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Down-Regulation, Fibroblasts metabolism, Fibroblasts physiology, Genes, ras, Humans, Mice, NIH 3T3 Cells, Tumor Suppressor Protein p53 genetics, fas Receptor genetics, ras Proteins metabolism, CCAAT-Enhancer-Binding Protein-beta genetics, Cyclin-Dependent Kinase Inhibitor p16 genetics, ras Proteins genetics
- Abstract
The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) is involved in cellular responses to oncogenic and physiologic Ras signals. C/EBPbeta is required for premature senescence of primary mouse fibroblasts induced by expression of H-Ras(V12), demonstrating its role in oncogene-induced senescence. Here, we have investigated the mechanisms by which Ras inhibits proliferation of normal cells but transforms immortalized cells. We show that oncogenic Ras down-regulates C/EBPbeta expression in NIH 3T3 cells, which are immortalized by a deletion of the CDKN2A locus and, therefore, lack the p16(Ink4a) and p19(Arf) tumor suppressors. Ras(V12)-induced silencing of C/EBPbeta occurred at the mRNA level and involved both the Raf-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-ERK and phosphatidylinositol 3-kinase signaling pathways. Oncogenic Ras decreased C/EBPbeta expression in Ink4a/Arf(-/-) mouse embryo fibroblasts (MEF) but increased C/EBPbeta levels in wild-type MEFs. C/EBPbeta down-regulation in NIH 3T3 cells was reversed by expression of p19(Arf), but not of p53 or p16(Ink4a), highlighting a critical role for p19(Arf) in sustaining C/EBPbeta levels. Ectopic expression of p34 C/EBPbeta (LAP) inhibited Ras(V12)-mediated transformation of NIH 3T3 cells, suppressed their tumorigenicity in nude mice, and reactivated expression of the proapoptotic Fas receptor, which is also down-regulated by Ras. Our findings indicate that Cebpb gene silencing eliminates a growth inhibitory transcription factor that would otherwise restrain oncogenesis. We propose that C/EBPbeta is part of a p53-independent, p19(Arf)-mediated network that enforces Ras-induced cell cycle arrest and tumor suppression in primary fibroblasts.
- Published
- 2009
- Full Text
- View/download PDF
44. The lamin B receptor under transcriptional control of C/EBPepsilon is required for morphological but not functional maturation of neutrophils.
- Author
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Cohen TV, Klarmann KD, Sakchaisri K, Cooper JP, Kuhns D, Anver M, Johnson PF, Williams SC, Keller JR, and Stewart CL
- Subjects
- Animals, CCAAT-Enhancer-Binding Proteins genetics, Cell Differentiation, Cell Nucleus Shape, Cells, Cultured, Disease Models, Animal, Gene Expression Regulation, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutagenesis, Insertional, Neutrophil Activation, Neutrophils physiology, Pelger-Huet Anomaly embryology, Pelger-Huet Anomaly metabolism, Promoter Regions, Genetic, Receptors, Cytoplasmic and Nuclear metabolism, Staphylococcus aureus physiology, Lamin B Receptor, CCAAT-Enhancer-Binding Proteins metabolism, Neutrophils cytology, Pelger-Huet Anomaly genetics, Pelger-Huet Anomaly physiopathology, Receptors, Cytoplasmic and Nuclear genetics, Transcription, Genetic
- Abstract
The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.
- Published
- 2008
- Full Text
- View/download PDF
45. Differential control of the CCAAT/enhancer-binding protein beta (C/EBPbeta) products liver-enriched transcriptional activating protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP) and the regulation of gene expression during the response to endoplasmic reticulum stress.
- Author
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Li Y, Bevilacqua E, Chiribau CB, Majumder M, Wang C, Croniger CM, Snider MD, Johnson PF, and Hatzoglou M
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta deficiency, CCAAT-Enhancer-Binding Protein-beta genetics, Cell Line, Tumor, Genes, Reporter, Glioma genetics, Luciferases genetics, Plasmids, RNA, Neoplasm genetics, RNA, Neoplasm isolation & purification, Rats, CCAAT-Enhancer-Binding Protein-beta physiology, Endoplasmic Reticulum physiology, Liver physiology, Transcription, Genetic
- Abstract
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.
- Published
- 2008
- Full Text
- View/download PDF
46. Transcriptional regulation of fatty acid translocase/CD36 expression by CCAAT/enhancer-binding protein alpha.
- Author
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Qiao L, Zou C, Shao P, Schaack J, Johnson PF, and Shao J
- Subjects
- 3T3-L1 Cells, Adipocytes pathology, Animals, CCAAT-Enhancer-Binding Protein-alpha genetics, CCAAT-Enhancer-Binding Protein-beta genetics, CCAAT-Enhancer-Binding Protein-beta metabolism, CD36 Antigens genetics, Cell Differentiation genetics, Diet, Embryo, Mammalian pathology, Fibroblasts pathology, Gene Expression Regulation genetics, Humans, Mice, Mice, Knockout, Obesity genetics, Obesity metabolism, Obesity pathology, Point Mutation, RNA, Messenger biosynthesis, RNA, Messenger genetics, Response Elements genetics, Adipocytes metabolism, CCAAT-Enhancer-Binding Protein-alpha metabolism, CD36 Antigens biosynthesis, Embryo, Mammalian metabolism, Fibroblasts metabolism, Transcription, Genetic genetics
- Abstract
Fatty acid translocase (FAT/CD36) plays an important role in facilitating long chain fatty acid transport. FAT/CD36 gene deletion protects mice from high fat diet-induced obesity. In this study we have investigated the regulatory mechanism of FAT/CD36 expression at the transcription level. FAT/CD36 expression was activated during 3T3-L1 adipocyte differentiation, and FAT/CD36 protein levels were positively correlated with CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma. However, a negative correlation was detected between FAT/CD36 and C/EBPbeta. Overexpression of C/EBPalpha or C/EBPbeta increased FAT/CD36 mRNA and protein levels in several types of cells. Restoration of C/EBPalpha or C/EBPbeta expression in C/EBPalpha- or C/EBPbeta-deficient mouse embryonic fibroblasts increased FAT/CD36 expression. However, in mouse embryonic fibroblasts C/EBPalpha was a more potent activator of FAT/CD36 expression than was C/EBPbeta. Expression of C/EBPalpha robustly increased FAT/CD36 proximal promoter-directed luciferase expression in human embryonic kidney 293 cells. A C/EBP-responsive element was identified in the FAT/CD36 promoter by using 5' and specific site mutations. The binding of C/EBPalpha in the FAT/CD36 promoter was detected by chromatin immunoprecipitation in 3T3-L1 adipocytes. These results demonstrated that C/EBPalpha regulates FAT/CD36 gene expression at the transcriptional level.
- Published
- 2008
- Full Text
- View/download PDF
47. Genetic ablation of CCAAT/enhancer binding protein alpha in epidermis reveals its role in suppression of epithelial tumorigenesis.
- Author
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Loomis KD, Zhu S, Yoon K, Johnson PF, and Smart RC
- Subjects
- Animals, Apoptosis, Cell Differentiation, Cell Proliferation, Keratinocytes cytology, Mice, Mice, Inbred BALB C, Mice, Knockout, Mutation, NIH 3T3 Cells, ras Proteins metabolism, CCAAT-Enhancer-Binding Protein-alpha genetics, Epidermis metabolism, Epithelial Cells metabolism, Gene Expression Regulation, Neoplastic
- Abstract
CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPalpha is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPalpha mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPalpha occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPalpha as an epithelial tumor suppressor is lacking due to the absence of C/EBPalpha mutations in epithelial tumors and the lethal effect of C/EBPalpha deletion in mouse model systems. To examine the function of C/EBPalpha in epithelial tumor development, an epidermal-specific C/EBPalpha knockout mouse was generated. The epidermal-specific C/EBPalpha knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPalpha is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPalpha knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPalpha displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPalpha contributes to deregulation of tumor cell proliferation. C/EBPalpha had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPalpha blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPalpha is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPalpha is a suppressor of epithelial tumorigenesis.
- Published
- 2007
- Full Text
- View/download PDF
48. A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation.
- Author
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Lopez AB, Wang C, Huang CC, Yaman I, Li Y, Chakravarty K, Johnson PF, Chiang CM, Snider MD, Wek RC, and Hatzoglou M
- Subjects
- Activating Transcription Factor 3 metabolism, Activating Transcription Factor 4 metabolism, Animals, CCAAT-Enhancer-Binding Protein-beta metabolism, Cationic Amino Acid Transporter 1 metabolism, Dimerization, Eukaryotic Initiation Factor-2 metabolism, Feedback, Physiological, Phosphorylation, Promoter Regions, Genetic, RNA, Messenger metabolism, Rats, Transcription, Genetic, Transfection, Tumor Cells, Cultured, Amino Acids metabolism, Arginine metabolism, Cationic Amino Acid Transporter 1 genetics, Gene Expression Regulation, Lysine metabolism
- Abstract
The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-beta and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPbeta activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPbeta. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.
- Published
- 2007
- Full Text
- View/download PDF
49. C/EBPbeta serine 64, a phosphoacceptor site, has a critical role in LPS-induced IL-6 and MCP-1 transcription.
- Author
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Spooner CJ, Sebastian T, Shuman JD, Durairaj S, Guo X, Johnson PF, and Schwartz RC
- Subjects
- Animals, CCAAT-Enhancer-Binding Protein-beta genetics, Cell Line, Chemokine CCL2 genetics, Genes, Reporter, Interleukin-6 genetics, Mice, Phosphorylation, Promoter Regions, Genetic, CCAAT-Enhancer-Binding Protein-beta metabolism, Chemokine CCL2 metabolism, Interleukin-6 metabolism, Lipopolysaccharides immunology, Serine metabolism, Transcription, Genetic
- Abstract
C/EBPbeta is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. Serine 64 in the transactivation domain of C/EBPbeta has recently been identified as a Ras-induced phosphoacceptor site. The integrity of serine 64 along with threonine 189 is important for the Ha-ras(V12)-induced transformation of NIH3T3 cells, however no target genes dependent upon serine 64 for their expression have been reported. In order to evaluate a potential role of serine 64 in C/EBPbeta-regulated cytokine expression, we expressed a form of C/EBPbeta with an alanine substitution at serine 64 (C/EBPbeta(S64A)) in P388 murine B lymphoblasts, which lack endogenous C/EBPbeta expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. In comparison to wild type C/EBPbeta, which robustly supports the LPS-induced expression of IL-6 and MCP-1, C/EBPbeta(S64A) was severely impaired in its ability to support the LPS-induced transcription of IL-6 and MCP-1. Furthermore, LPS stimulation increased the level of phosphorylation detected at serine 64. Thus, serine 64, probably through its phosphorylation, is a critical determinant of C/EBPbeta activity in the transcription of IL-6 and MCP-1.
- Published
- 2007
- Full Text
- View/download PDF
50. Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription.
- Author
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Spooner CJ, Guo X, Johnson PF, and Schwartz RC
- Subjects
- Animals, Cell Line, Tumor, Chemokine CCL2 biosynthesis, Chemokine CCL2 physiology, Interleukin-6 biosynthesis, Leukemia P388, Mice, Peptide Fragments biosynthesis, Peptide Fragments genetics, Protein Structure, Tertiary physiology, CCAAT-Enhancer-Binding Protein-beta physiology, Chemokine CCL2 genetics, Gene Expression Regulation immunology, Interleukin-6 genetics, Peptide Fragments physiology, Transcription, Genetic immunology
- Abstract
C/EBPbeta is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. To examine the roles of the C/EBPbeta transactivation and regulatory domains in LPS-induced MCP-1 and IL-6 expression, we expressed various N-terminal truncations and deletions of C/EBPbeta in P388 murine B lymphoblasts, which lack endogenous C/EBPbeta expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. Unexpectedly, a region between amino acids 105 and 212 of C/EBPbeta that includes regulatory domains 1 and 2 facilitates C/EBPbeta activation of IL-6 expression, while having an inhibitory effect on MCP-1 expression. Thus, this region can mediate promoter-specific effects on cytokine and chemokine gene transcription. LIP, the naturally occurring truncated form of C/EBPbeta, largely retains these regulatory domains and stimulates IL-6 but not MCP-1 transcription.
- Published
- 2007
- Full Text
- View/download PDF
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