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1.
Cancers (Basel) ; 13(19)2021 Sep 30.
Article in English | MEDLINE | ID: mdl-34638423

ABSTRACT

Maraviroc (MVC), a CCR5 antagonist, reduces liver fibrosis, injury and tumour burden in mice fed a hepatocarcinogenic diet, suggesting it has potential as a cancer therapeutic. We investigated the effect of MVC on liver progenitor cells (LPCs) and macrophages as both have a role in hepatocarcinogenesis. Mice were fed the hepatocarcinogenic choline-deficient, ethionine-supplemented diet (CDE) ± MVC, and immunohistochemistry, RNA and protein expression were used to determine LPC and macrophage abundance, migration and related molecular mechanisms. MVC reduced LPC numbers in CDE mice by 54%, with a smaller reduction seen in macrophages. Transcript and protein abundance of LPC-associated markers correlated with this reduction. The CDE diet activated phosphorylation of AKT and STAT3 and was inhibited by MVC. LPCs did not express Ccr5 in our model; in contrast, macrophages expressed high levels of this receptor, suggesting the effect of MVC is mediated by targeting macrophages. MVC reduced CD45+ cells and macrophage migration in liver and blocked the CDE-induced transition of liver macrophages from an M1- to M2-tumour-associated macrophage (TAM) phenotype. These findings suggest MVC has potential as a re-purposed therapeutic agent for treating chronic liver diseases where M2-TAM and LPC numbers are increased, and the incidence of HCC is enhanced.

2.
Gene Expr ; 20(1): 39-52, 2020 06 12.
Article in English | MEDLINE | ID: mdl-32317048

ABSTRACT

Liver progenitor cells (LPCs) contribute to liver regeneration during chronic damage and are implicated as cells of origin for liver cancers including hepatocellular carcinoma (HCC). The CDKN2A locus, which encodes the tumor suppressors alternate reading frame protein (ARF) and INK4A, was identified as one of the most frequently altered genes in HCC. This study demonstrates that inactivation of CDKN2A enhances tumorigenic transformation of LPCs. The level of ARF and INK4A expression was determined in a panel of transformed and nontransformed wild-type LPC lines. Moreover, the transforming potential of LPCs with inactivated CDKN2A was shown to be enhanced in LPCs derived from Arf-/- and CDKN2Afl/fl mice and in wild-type LPCs following CRISPR-Cas9 suppression of CDKN2A. ARF and INK4A abundance is consistently reduced or ablated following LPC transformation. Arf-/- and CDKN2A-/- LPCs displayed hallmarks of transformation such as anchorage-independent and more rapid growth than control LPC lines with unaltered CDKN2A. Transformation was not immediate, suggesting that the loss of CDKN2A alone is insufficient. Further analysis revealed decreased p21 expression as well as reduced epithelial markers and increased mesenchymal markers, indicative of epithelial-to-mesenchymal transition, following inactivation of the CDKN2A gene were required for tumorigenic transformation. Loss of ARF and INK4A enhances the propensity of LPCs to undergo a tumorigenic transformation. As LPCs represent a cancer stem cell candidate, identifying CDKN2A as a driver of LPC transformation highlights ARF and INK4A as viable prognostic markers and therapeutic targets for HCC.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p16/physiology , Liver Neoplasms, Experimental/genetics , Stem Cells/pathology , Animals , Azacitidine/pharmacology , CRISPR-Cas Systems , Cell Line, Transformed , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p16/deficiency , DNA Methylation/drug effects , Epithelial-Mesenchymal Transition , Gene Deletion , Gene Expression Regulation, Neoplastic/genetics , Gene Knockout Techniques , Genes, p16 , Liver/cytology , Liver/embryology , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Phenotype , Snail Family Transcription Factors/biosynthesis , Snail Family Transcription Factors/genetics , Tumor Stem Cell Assay , Vimentin/biosynthesis , Vimentin/genetics
3.
FEBS Lett ; 593(3): 253-276, 2019 02.
Article in English | MEDLINE | ID: mdl-30570758

ABSTRACT

The Hippo pathway has emerged as a major eukaryotic signalling pathway and is increasingly the subject of intense interest, as are the key effectors of canonical Hippo signalling, YES-associated protein (YAP) and TAZ. The Hippo pathway has key roles in diverse biological processes, including network signalling regulation, development, organ growth, tissue repair and regeneration, cancer, stem cell regulation and mechanotransduction. YAP and TAZ are multidomain proteins and function as transcriptional coactivators of key genes to evoke their biological effects. YAP and TAZ interact with numerous partners and their activities are controlled by a complex set of processes. This review provides an overview of Hippo signalling and its role in growth. In particular, the functional domains of YAP and TAZ and the complex mechanisms that regulate their protein stability and activity are discussed. Notably, the similarities and key differences are highlighted between the two paralogues including which partner proteins interact with which functional domains to regulate their activity.


Subject(s)
Mechanotransduction, Cellular , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Regeneration , Transcription Factors/metabolism , Acyltransferases , Animals , Cell Cycle Proteins , Hippo Signaling Pathway , Humans , Neoplasms/pathology , Protein Stability , Stem Cells/metabolism , Stem Cells/pathology
4.
Biochem Biophys Rep ; 6: 24-31, 2016 Jul.
Article in English | MEDLINE | ID: mdl-28018981

ABSTRACT

The yes-associated protein (YAP) is a key effector of the mammalian Hippo signaling pathway. YAP has eight known alternately spliced isoforms and these are widely expressed across multiple tissues. Variable effects have been ascribed to different YAP isoforms by inducing their expression in cells, but whether these differences are due to variability in the transcriptional potency of individual YAP isoforms has not been addressed. Indeed a systematic comparison of the transcriptional potencies of YAP isoforms has not been done. To address this, using overexpression and transcriptional reporter analyses we investigated the transcriptional activities of several human YAP isoforms and determined the effects of the splice variant insertions within the transactivation domain on its transcriptional potency. Utilising full-length coding sequence constructs we determined that the number of WW domains and disruption of the leucine zipper motif within YAP's transactivation domain both contribute to transcriptional activity. Notably, disruption of YAP's leucine zipper had a greater effect on transcriptional activity than the absence of the second WW domain. Using GAL4-YAP transcriptional activation domain fusion proteins we found that disruption of the leucine zipper significantly decreased YAP's transcriptional activity in several cell lines. Our data indicates that expression of different YAP isoforms with varying transcriptional potencies may enable fine control of Hippo pathway signaling. Furthermore the specific isoform being utilised should be taken into consideration when interpreting published data or when designing experiments to ascribe YAP's function.

5.
PLoS One ; 11(11): e0164003, 2016.
Article in English | MEDLINE | ID: mdl-27806040

ABSTRACT

Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cell Death/drug effects , Cycloheximide/pharmacology , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Protein Synthesis Inhibitors/pharmacology , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Animals , Apoptosis/drug effects , Cell Death/genetics , Cell Line , Dose-Response Relationship, Drug , Gene Knockout Techniques , Humans , Mice , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/genetics
6.
Stem Cells Int ; 2016: 5702873, 2016.
Article in English | MEDLINE | ID: mdl-27777588

ABSTRACT

Liver progenitor cells (LPCs) can proliferate extensively, are able to differentiate into hepatocytes and cholangiocytes, and contribute to liver regeneration. The presence of LPCs, however, often accompanies liver disease and hepatocellular carcinoma (HCC), indicating that they may be a cancer stem cell. Understanding LPC biology and establishing a sensitive, rapid, and reliable method to detect their presence in the liver will assist diagnosis and facilitate monitoring of treatment outcomes in patients with liver pathologies. A transcriptomic meta-analysis of over 400 microarrays was undertaken to compare LPC lines against datasets of muscle and embryonic stem cell lines, embryonic and developed liver (DL), and HCC. Three gene clusters distinguishing LPCs from other liver cell types were identified. Pathways overrepresented in these clusters denote the proliferative nature of LPCs and their association with HCC. Our analysis also revealed 26 novel markers, LPC markers, including Mcm2 and Ltbp3, and eight known LPC markers, including M2pk and Ncam. These markers specified the presence of LPCs in pathological liver tissue by qPCR and correlated with LPC abundance determined using immunohistochemistry. These results showcase the value of global transcript profiling to identify pathways and markers that may be used to detect LPCs in injured or diseased liver.

7.
Eur J Med Chem ; 120: 275-83, 2016 Sep 14.
Article in English | MEDLINE | ID: mdl-27208658

ABSTRACT

BACKGROUND & AIMS: The availability of non-tumorigenic and tumorigenic liver progenitor cell (LPC) lines affords a method to screen putative anti-liver cancer agents to identify those that are selectively effective. To prove this principle we tested thalidomide and a range of its derivatives and compared them to lenalidomide and sorafenib, to assess their growth-inhibitory effects. METHODS: Cell growth, the mitotic and apoptotic index of cell cultures were measured using the Cellavista instrument (SynenTec) using commercially available reagents. RESULTS: Neither lenalidomide nor thalidomide (100 µM) affected tumorigenic LPCs but killed their non-tumorigenic counterparts. Sorafenib arrested growth in both cell types. All but two derivatives of thalidomide were ineffective; of the two effective derivatives, one (thalidomide C1) specifically affected the tumorigenic cell line (10 µM). Mitotic and apoptotic analyses revealed that thalidomide C1 induced apoptotic cell death and not mitotic arrest. CONCLUSIONS: This study shows that screens incorporating non-tumorigenic and tumorigenic liver cell lines are a sound approach to identify agents that are effective and selective. A high throughput instrument such as the Cellavista affords robust and reproducible objective measurements with a large number of replicates that are reliable. These experiments show that neither lenalidomide nor thalidomide are potentially useful for anti-liver cancer therapy as they kill non-tumorigenic liver cells and not their tumorigenic counterparts. Sorafenib in contrast, is highly effective, but not selective. One tested thalidomide derivative has potential as an anti-tumor drug since it induced growth arrest; and importantly, it selectively induced apoptotic cell death only in tumorigenic liver progenitor cells.


Subject(s)
Liver Neoplasms/drug therapy , Stem Cells/drug effects , Thalidomide/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Lenalidomide , Liver Neoplasms/pathology , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Phenylurea Compounds/pharmacology , Sorafenib , Stem Cells/pathology , Thalidomide/analogs & derivatives
8.
J Biol Chem ; 290(46): 27928-38, 2015 Nov 13.
Article in English | MEDLINE | ID: mdl-26432639

ABSTRACT

The mammalian Hippo signaling pathway regulates cell growth and survival and is frequently dysregulated in cancer. YAP and TAZ are transcriptional coactivators that function as effectors of this signaling pathway. Aberrant YAP and TAZ activity is reported in several human cancers, and normally the expression and nuclear localization of these proteins is tightly regulated. We sought to establish whether a direct relationship exists between YAP and TAZ. Using knockdown and overexpression experiments we show YAP inversely regulates the abundance of TAZ protein by proteasomal degradation. Interestingly this phenomenon was uni-directional since TAZ expression did not affect YAP abundance. Structure/function analyses suggest that YAP-induced TAZ degradation is a consequence of YAP-targeted gene transcription involving TEAD factors. Subsequent investigation of known regulators of TAZ degradation using specific inhibitors revealed a role for heat shock protein 90 and glycogen synthase kinase 3 but not casein kinase 1 nor LATS in YAP-mediated TAZ loss. Importantly, this phenomenon is conserved from mouse to human; however, interestingly, different YAP isoforms varied in their ability to degrade TAZ. Since shRNA-mediated TAZ depletion in HeLa and D645 cells caused apoptotic cell death, we propose that isoform-specific YAP-mediated TAZ degradation may contribute to the contradicting roles reported for YAP overexpression. This study identifies a novel mechanism of TAZ regulation by YAP, which has significant implications for our understanding of Hippo pathway regulation, YAP-isoform specific signaling, and the role of these proteins in cell proliferation, apoptosis, and tumorigenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Cycle Proteins , Cell Proliferation , Gene Knockdown Techniques , Glycogen Synthase Kinase 3/metabolism , HSP90 Heat-Shock Proteins/metabolism , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Phosphoproteins/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling Proteins
9.
Dis Model Mech ; 8(12): 1635-41, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26496771

ABSTRACT

The choline-deficient, ethionine-supplemented (CDE) dietary model induces chronic liver damage, and stimulates liver progenitor cell (LPC)-mediated repair. Long-term CDE administration leads to hepatocellular carcinoma in rodents and lineage-tracing studies show that LPCs differentiate into functional hepatocytes in this model. The CDE diet was first modified for mice by our laboratory by separately administering choline-deficient chow and ethionine in the drinking water (CD+E diet). Although this CD+E diet is widely used, concerns with variability in weight loss, morbidity, mortality and LPC response have been raised by researchers who have adopted this model. We propose that these inconsistencies are due to differential consumption of chow and ethionine in the drinking water, and that incorporating ethionine in the choline-deficient chow, and altering the strength, will achieve better outcomes. Therefore, C57Bl/6 mice, 5 and 6 weeks of age, were fed an all-inclusive CDE diet of various strengths (67% to 100%) for 3 weeks. The LPC response was quantitated and cell lines were derived. We found that animal survival, LPC response and liver damage are correlated with CDE diet strength. The 67% and 75% CDE diet administered to mice older than 5 weeks and greater than 18 g provides a consistent and acceptable level of animal welfare and induces a substantial LPC response, permitting their isolation and establishment of cell lines. This study shows that an all-inclusive CDE diet for mice reproducibly induces an LPC response conducive to in vivo studies and isolation, whilst minimizing morbidity and mortality.


Subject(s)
Choline/pharmacology , Diet , Ethionine/pharmacology , Liver/cytology , Morbidity , Stem Cells/cytology , Aging , Alanine Transaminase/blood , Animals , Bile Ducts/cytology , Biomarkers/metabolism , Body Weight , Cell Differentiation , Cell Line , Cell Lineage , Hepatocytes/cytology , Inflammation/pathology , Liver/pathology , Male , Mice, Inbred C57BL , Survival Analysis
10.
PLoS One ; 10(2): e0114813, 2015.
Article in English | MEDLINE | ID: mdl-25658431

ABSTRACT

The Yes-associated protein (YAP) is a potent transcriptional co-activator that functions as a nuclear effector of the Hippo signaling pathway. YAP is oncogenic and its activity is linked to its cellular abundance and nuclear localisation. Activation of the Hippo pathway restricts YAP nuclear entry via its phosphorylation by Lats kinases and consequent cytoplasmic retention bound to 14-3-3 proteins. We examined YAP expression in liver progenitor cells (LPCs) and surprisingly found that transformed LPCs did not show an increase in YAP abundance compared to the non-transformed LPCs from which they were derived. We then sought to ascertain whether nuclear YAP was more abundant in transformed LPCs. We used an antibody that we confirmed was specific for YAP by immunoblotting to determine YAP's sub-cellular localisation by immunofluorescence. This antibody showed diffuse staining for YAP within the cytosol and nuclei, but, noticeably, it showed intense staining of the nucleoli of LPCs. This staining was non-specific, as shRNA treatment of cells abolished YAP expression to undetectable levels by Western blot yet the nucleolar staining remained. Similar spurious YAP nucleolar staining was also seen in mouse embryonic fibroblasts and mouse liver tissue, indicating that this antibody is unsuitable for immunological applications to determine YAP sub-cellular localisation in mouse cells or tissues. Interestingly nucleolar staining was not evident in D645 cells suggesting the antibody may be suitable for use in human cells. Given the large body of published work on YAP in recent years, many of which utilise this antibody, this study raises concerns regarding its use for determining sub-cellular localisation. From a broader perspective, it serves as a timely reminder of the need to perform appropriate controls to ensure the validity of published data.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Nucleolus/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Cell Line , Cell Nucleolus/genetics , Humans , Mice , Phosphoproteins/genetics , Protein Transport/physiology , Transcription Factors , YAP-Signaling Proteins
11.
Int J Biochem Cell Biol ; 54: 288-303, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24731940

ABSTRACT

Since their discovery more than a decade ago microRNAs have been demonstrated to have profound effects on almost every aspect of biology. Numerous studies in recent years have shown that microRNAs have important roles in development and in the etiology and progression of disease. This review is focused on microRNAs and the roles they play in liver development, regeneration and liver disease; particularly chronic liver diseases such as alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, viral hepatitis and primary liver cancer. The key microRNAs identified in liver development and chronic liver disease will be discussed together with, where possible, the target messenger RNAs that these microRNAs regulate to profoundly alter these processes. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.


Subject(s)
Gene Expression Regulation , Liver Diseases/genetics , Liver Regeneration , Liver/pathology , MicroRNAs/genetics , Animals , Humans , Liver/metabolism
12.
Free Radic Biol Med ; 50(6): 689-99, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21172426

ABSTRACT

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.


Subject(s)
Enzyme Inhibitors/pharmacology , Thioredoxin Reductase 1/antagonists & inhibitors , Thioredoxin Reductase 1/metabolism , Thioredoxin Reductase 2/antagonists & inhibitors , Thioredoxin Reductase 2/metabolism , Thioredoxins/metabolism , Animals , Antioxidants/metabolism , Cisplatin/metabolism , Cisplatin/pharmacology , Cytosol/enzymology , Dithionitrobenzoic Acid/metabolism , Humans , Mice , Mitochondria/enzymology , Naphthoquinones/metabolism , Organogold Compounds/pharmacology , Oxidation-Reduction , Recombinant Proteins/metabolism , Substrate Specificity , Thioctic Acid/analogs & derivatives , Thioctic Acid/metabolism , Thioredoxin Reductase 1/chemistry , Thioredoxin Reductase 1/genetics , Thioredoxin Reductase 2/chemistry , Thioredoxin Reductase 2/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism
13.
PLoS One ; 4(11): e8078, 2009 Nov 30.
Article in English | MEDLINE | ID: mdl-19956646

ABSTRACT

The most thoroughly characterized mammalian IAP is XIAP/BIRC4, which can inhibit caspases 9, 3 and 7, but may also regulate apoptosis through interactions with other proteins such as Smac/DIABLO, HtrA2/Omi, XAF1, TAK1, cIAP1, and cIAP2.High throughput sequencing of the mouse genome revealed the existence of a gene resembling Xiap/Birc4 on mouse chromosome 7. To confirm the existence of this gene, and to determine its functional significance, we performed Southern and Northern blot analysis. This showed the presence of the Xiap-like gene in both wild-type and Xiap gene knock-out mice, but the corresponding mRNA was not detected in any tissues examined by Northern blot. Analysis of the gene sequence in all three possible reading frames predicts that expression of this gene would not give rise to a full-length protein, but only non-functional truncated polypeptides. Because its nucleotide sequence is 92% identical to Xiap, but it has no introns corresponding to those of Xiap, we conclude that Xiap-ps1 is a pseudogene generated by retro-transposition of a spliced Xiap message to chromosome 7.


Subject(s)
Chromosome Mapping , Chromosomes , Pseudogenes , X-Linked Inhibitor of Apoptosis Protein/genetics , Amino Acid Sequence , Animals , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Genetic , Molecular Sequence Data , Peptides/chemistry , Retroelements , Sequence Homology, Amino Acid
14.
J Biol Chem ; 284(51): 35906-15, 2009 Dec 18.
Article in English | MEDLINE | ID: mdl-19815541

ABSTRACT

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.


Subject(s)
Apoptosis/physiology , Inhibitor of Apoptosis Proteins/metabolism , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , TNF Receptor-Associated Factor 2/metabolism , Tumor Necrosis Factors/metabolism , Amino Acid Motifs/physiology , Animals , Cell Line , Inhibitor of Apoptosis Proteins/genetics , Mice , Mice, Knockout , NF-kappa B/genetics , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Receptors, Tumor Necrosis Factor, Type I/genetics , TNF Receptor-Associated Factor 2/genetics
15.
J Mol Biol ; 384(2): 313-23, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-18835564

ABSTRACT

Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF(3) cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF(3) cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cell Line , Cell Survival/drug effects , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Mice , Myeloid Cell Leukemia Sequence 1 Protein , Proteasome Inhibitors , Protein Binding/drug effects , bcl-2 Homologous Antagonist-Killer Protein/deficiency , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/deficiency , bcl-2-Associated X Protein/metabolism
16.
Carcinogenesis ; 29(6): 1124-33, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18413365

ABSTRACT

A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here, we compare two matched liver progenitor cell (LPC) lines, one non-tumorigenic [p53-immortalized liver (PIL) 4] and the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild-type non-tumorigenic cell line [bipotential murine oval liver (BMOL)] revealed an increase in expression of genes encoding the antiapoptotic proteins cellular inhibitor of apoptosis protein (cIAP) 1 and yes associate protein in the PIL2 cells, which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (Deltapsi(m)) compared with PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide disumutase, was elevated at transcript and protein levels. Taken together, these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared with PIL4. We tested a new gold compound to show that hyperpolarized Deltapsi(m) led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 or BMOL. The gold compound depolarized the Deltapsi(m), depleted the adenosine triphosphate pool and activated caspase-3 and caspase-9, suggesting that apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPCs are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria-targeted chemotherapeutics that selectively target tumor cells.


Subject(s)
Antineoplastic Agents/pharmacology , Energy Metabolism , Gold Compounds/pharmacology , Hepatocytes/metabolism , Mitochondria/drug effects , Stem Cells/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Cycle Proteins , Cell Line, Tumor , Drug Resistance, Neoplasm/physiology , Gene Expression , Gene Expression Regulation, Neoplastic , Hepatocytes/cytology , Immunoblotting , Inhibitor of Apoptosis Proteins/metabolism , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Mice , Microarray Analysis , Mitochondria/metabolism , Phosphoproteins/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/drug effects , YAP-Signaling Proteins
17.
Cell ; 131(4): 682-93, 2007 Nov 16.
Article in English | MEDLINE | ID: mdl-18022363

ABSTRACT

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.


Subject(s)
Apoptosis/physiology , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis Regulatory Proteins , Autocrine Communication , Benzoquinones/metabolism , Brefeldin A/metabolism , Caspase 8/metabolism , Caspase Inhibitors , Cell Line , Enzyme Inhibitors/metabolism , Humans , Inhibitor of Apoptosis Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lactams, Macrocyclic/metabolism , Mice , Mitochondrial Proteins/metabolism , Molecular Mimicry , NF-kappa B/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Synthesis Inhibitors/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Serpins/metabolism , Signal Transduction/physiology , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , Viral Proteins/metabolism
18.
FEBS J ; 273(18): 4264-76, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16930133

ABSTRACT

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.


Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Cycle Proteins/isolation & purification , Cell Line , Dimerization , Gene Deletion , Humans , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transfection
19.
Biochem J ; 388(Pt 2): 445-54, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15683364

ABSTRACT

Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.


Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Repressor Proteins/chemistry , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Animals , Cell Line , Dimerization , Enzyme Activation , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins/physiology , Membrane Proteins , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Molecular Sequence Data , Monomeric GTP-Binding Proteins/antagonists & inhibitors , Phosphorylation , Protein Structure, Tertiary , Repressor Proteins/physiology , Signal Transduction
20.
Oncogene ; 21(31): 4812-21, 2002 Jul 18.
Article in English | MEDLINE | ID: mdl-12101419

ABSTRACT

We have cloned a novel SOCS gene from Drosophila, socs36E, which is most homologous to the mammalian socs-5 gene. Socs36E is expressed zygotically, predominantly during embryogenesis, in a highly dynamic pattern. In vivo expression of SOCS36E in transgenic flies results in several adult phenotypes. Engrailed-GAL4 directed expression causes loss of the wing anterior cross vein, humeral outgrowths, absence of halteres and eye pigmentation defects. Expression of SOCS36E under apterous-GAL4 control resulted in outstretched wings. Full penetrance of these phenotypes required the presence of the SH2 and SOCS-box domains of SOCS36E. The observed phenotypes were consistent with defects in JAK/STAT or EGF-R signalling and were exacerbated in flies heterozygous for either the d-jak (hopscotch), d-stat (stat92E) or d-egf-r (der) genes. Conversely, inactivating one copy of the d-cbl gene, a negative regulator of the d-EGF-R, partially rescued the wing phenotypes. These genetic interactions imply that SOCS36E can suppress activities of the JAK/STAT and EGF-R signalling pathways in the wing disc and suggest that SOCS36E interacts with multiple pathways in vivo.


Subject(s)
Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/physiology , Drosophila/embryology , Drosophila/metabolism , Protein Kinases , Repressor Proteins/physiology , Signal Transduction , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cell Line , Cloning, Molecular , DNA-Binding Proteins/antagonists & inhibitors , Drosophila/genetics , Drosophila Proteins/genetics , ErbB Receptors/antagonists & inhibitors , Humans , Janus Kinases , Mice , Molecular Sequence Data , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Messenger/biosynthesis , Receptors, Invertebrate Peptide/antagonists & inhibitors , Repressor Proteins/genetics , STAT Transcription Factors , Sequence Alignment , Suppressor of Cytokine Signaling Proteins , Trans-Activators/antagonists & inhibitors , Transcription Factors , Wings, Animal/anatomy & histology , Wings, Animal/embryology , Wings, Animal/metabolism
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