Wild-type Overexpression Research Articles

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida.

BackgroundPseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.ResultsA genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.ConclusionProduction of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.

Erythritol production on wheat straw using Trichoderma reesei.

We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native β-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw.

Mitochondrial calcium uniporter activity is dispensable for MDA-MB-231 breast carcinoma cell survival.

Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.

Dub3 controls DNA damage signalling by direct deubiquitination of H2AX

A crucial event in the DNA damage response is the phosphorylation and subsequent ubiquitination of H2AX, required for the recruitment of proteins involved in DNA repair. Here we identify a novel regulator of this process, the ubiquitin hydrolase Dub3. Overexpression of wild type, but not catalytic inactive, Dub3 decreases the DNA damage-induced mono-ubiquitination of H2A(X) whereas downregulation of Dub3 has the opposite effect. Dub3 overexpression abrogates focus formation of 53BP1 and BRCA1 in response to genotoxic stress. However, focus formation of MDC1 and γH2AX, earlier events in this response, are unaffected by Dub3 overexpression. We show that Dub3 counteracts H2AX E3 ligases RNF8 and RNF168. Moreover, Dub3 and H2AX interact and Dub3 deubiquitinates H2AX in vitro. Importantly, overexpression of Dub3 delays H2AX dephosphorylation and recovery of MDC1 focus formation at later time points after DNA damage, whereas H2AX dephosphorylation at later time points is faster after Dub3 depletion. Altogether these results show that Dub3 regulates a correct DNA damage response by controlling H2AX ubiquitination.

Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides)

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.

Mechanisms of resistance to azacitidine in human leukemia cell lines

The DNA methylation inhibitor azacitidine (5-azacytidine) is used against myelodysplastic syndrome and acute myeloid leukemia, but drug resistance is an ongoing, intractable problem. To investigate resistance mechanisms, we generated two azacitidine-resistant cell lines, THP-1/AR and HL60/AR, and studied genetic disparities between them and their corresponding parental lines. In cells treated with azacitidine, significant mitotic variations were noted in parental cells which were absent in resistant cells, suggesting that resistance arises from negating azacitidine-mediated activation of apoptosis signaling and reestablishing G2/M checkpoint. Importantly, both resistant cell lines have common point mutations in the uridine-cytidine kinase 2 (UCK2) gene, which encodes the rate-limiting enzyme of the azacitidine activation pathway. Forced expression of mutated UCK2 in parental THP-1 cells abrogated azacitidine-induced apoptosis, whereas overexpression of wild type UCK2 in resistant THP-1/AR cells restored sensitivity to azacitidine, implying that UCK2 gene mutations perturb azacitidine activation and advance azacitidine resistance. Our study provides new insights into azacitidine resistance and establishes models useful in developing effective strategies to overcome it.

Mechanism Of Anti-Myeloma Effects Due To Silencing Of Deptor

DEPTOR is a recently described 48 kDa protein that binds to mTOR and inhibits this kinase within TORC1 and TORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma genetic sub-class that contains IgH translocations. Most interestingly, DEPTOR knock-down in over-expressing MM cell lines induces growth arrest and apoptosis, indicating DEPTOR could serve as a therapeutic target in selected patients. To investigate the mechanism of MM cytoreduction induced by DEPTOR silencing we generated a doxycycline-inducible DEPTOR shRNA. Induced DEPTOR knockdown in 8226 and OPM-2 cell lines resulted in phosphorylation of p70S6K and 4E-BP1 (due to release of TORC1 inhibition) as well as apoptosis, cell cycle arrest and senescence. RAPTOR knockdown in these MM cells prevented TORC1 stimulation and the anti-MM effects resulting from DEPTOR silencing. Thus, the acute activation of TORC1 mediated the apoptosis and growth arrest. Although DEPTOR silencing and mTORC1 activation resulted in depressed AKT activity, probably due to the TORC1-PI3K/AKT negative feedback loop, this was not relevant to anti-MM effects as over-expression of wild type or a phosphomimetic version of AKT did not prevent the adverse effects. Similarly, induction of ER stress was also not relevant to the anti-MM effects as markers for UPR activation did not occur during DEPTOR silencing. Although western blot did not show alteration of p53 expression, p21 and p27 were induced subsequent to DEPTOR knockdown. Associated RAPTOR knockdown prevented the upregulation of p21 but did not affect p27 levels. In addition, over-expression of AKT did not prevent the upregulation of p21. To test whether p21 was integral to DEPTOR knockdown effects, we silenced p21. P21-silenced MM cells were protected against apoptosis, cell cycle arrest and senescence induced by DEPTOR knockdown. Furthermore, DEPTOR knockdown in a murine xenograft model, achieved by ingestion of doxycyline, resulted in tumor regression, TORC1 activation, apoptosis and p21 induction. These data indicate a novel pathway to explain the lethal effect of DEPTOR knockdown in MM cells. The pathway is initiated by acute activation of mTORC1, with subsequent p21 induction. The data also suggest that DEPTOR over-expression in some MM clones is pro-tumoral due to its ability to suppress expression of CDK inhibitors like p21 and p27. Disclosures: No relevant conflicts of interest to declare.

Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Molecular determinants of desensitization in an ENaC/degenerin channel

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma CellsIn VitroandIn Vivo

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

Autophagic Degradation of Caspase-8 Protects U87MG Cells Against H2O2-induced Oxidative Stress

Oxidative stress induces apoptosis in many cellular systems including glioblastoma cells, with caspase-8 activation was regarded as a major contribution to H2O2-induced cell death. This study focused on the role of the autophagic protein p62 in H2O2-induced apoptosis in U87MG cells. Oxidative stress was applied with H2O2, and cell apoptosis and viability were measured with use of caspase inhibitors or autophagic mediators or siRNA p62, GFP-p62 and GFP-p62-UBA (del) transfection. We found that H2O2 -induced U87MG cell death was correlated with caspase-8. To understand the role of p62 in MG132-induced cell death, the levels of p62/SQSTM1 or autophagy in U87MG cells were modulated with biochemical or genetic methods. The results showed that the over-expression of wild type p62/SQSTM1 significantly reduced H2O2 induced cell death, but knockdown of p62 aggravated the process. In addition, inhibition of autophagy promoted p62 and active caspase-8 increasing H2O2 -induced apoptosis while induction of autophagy manifested the opposite effect. We further demonstrated that the function of p62/SQSTM1 required its C-terminus UBA domain to attenuate H2O2 cytotoxity by inhibition of caspase-8 activity. Our results indicated that p62/SQSTM1 was a potential contributor to mediate caspase-8 activation by autophagy in oxidative stress process.

Abstract 3712: p21-activated kinase-1 in the prevention of inflammation-driven colon cancer.

Abstract Chronic gut inflammation in inflammatory bowel diseases (IBD) predisposes to the development of colorectal cancer, causing increased mortality. Use of mesalamine in the treatment and remittance of ulcerative colitis (UC) modulates the risk of neoplastic progression. We have previously reported (Khare V, et al. Biochem Pharm 2012) that p-21 activated kinase-1 (PAK1) mediates mesalamine activity by orchestrating MAPK signaling; Wnt-β catenin pathway and cell adhesion; all implicated in the colon carcinogenesis. Since mesalamine has primarily an anti-inflammatory function, we evaluated the role of PAK1, its target, in IBD and in colitis-associated cancer (CAC). PAK1 expression was scored by immunohistochemistry in human samples from IBD, CAC and normal mucosa. Compared to normal mucosa and Crohn's disease, a higher PAK1 expression was detected in UC which further increased in CAC. PAK1 expression was also elevated in the inflamed mucosa and carcinomas in IL-10−/− mouse model of colitis. The efficacy of mesalamine in reducing inflammation and tumor incidence in these mice has been previously reported (Brown JB et al, Gastroenetrology 2010). The consequence of PAK1 overexpression was further investigated in vitro. Normal diploid colon epithelial cells (HCEC-1CT) showed higher proliferation (46 ± 3.1% increase) upon overexpression of wildtype (wt) PAK1 while dominant negative (dn) PAK1 overexpression reduced proliferation (25% ± 2.5 decrease) compared to control. Apoptosis (Annexin V staining) was reduced in HCEC-1CT overexpressing wtPAK1 (1.2% ± 0.40) compared to dnPAK1 (11.6% ± 0.3) and control (16% ± 0.83). Mesalamine treatment (20mM) was effective in counteracting the effect of PAK1 overexpression by reducing cell proliferation and inducing apoptosis. Treatment of cells with specific inhibitors (PD98059/LY294002/Rapamycin) of signaling pathways (MEK/PI3K/mTOR) demonstrated that mesalamine regulated PAK1 expression by the MEK, PI3K and mTOR signaling pathways. Our data indicate that (1) PAK1 is upregulated in UC and in CAC, (2) PAK1 may be utilized as a predictive marker for the development of CAC, and (3) PAK1 is a viable target for chemoprevention in the setting of colitis. Mesalamine counteracts PAK1 expression and its effect on cell proliferation and apoptosis. In the longterm management of UC mesalamine may protect from CAC through inhibition of PAK1. Citation Format: Vineeta Khare, Kyle Dammann, Manuela Jambrich, Andres Roig, Jerry Shay, Christoph Gasche. p21-activated kinase-1 in the prevention of inflammation-driven colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3712. doi:10.1158/1538-7445.AM2013-3712

The E3 ubiquitin ligase Itch regulates tumor suppressor protein RASSF5/NORE1 stability in an acetylation-dependent manner

Ras association (RalGDS/AF-6) domain family member RASSF5 is a non-enzymatic RAS effector super family protein, known to be involved in cell growth regulation. Expression of RASSF5 is found to be extinguished by promoter hypermethylation in different human cancers, and its ectopic expression suppresses cell proliferation and tumorigenicity. Interestingly, this role in tumorigenesis has been confounded by the fact that regulation at molecular level remains unclear and many transformed cells actually display elevated RASSF5 expression. Here, we demonstrate that E3 ubiquitin ligase Itch is a unique binding partner of RASSF5. Itch can interact with PPxY motif in RASSF5 both in vivo and in vitro through its WW domains. Importantly, the overexpression of Itch induces RASSF5 degradation by poly-ubiquitination via 26S proteasome pathway. In addition, our results indicate that the elevated levels of RASSF5 found in tumor cells due to acetylation, which restricts its binding to Itch and results in a more stable inert protein. Inhibition of RASSF5 acetylation permits its interaction with Itch and provokes proteasomal degradation. These data suggest that apart from promoter methylation, hyperacetylation could also be downregulating RASSF5 function in different human cancer. Finally, results from functional assays suggest that the overexpression of wild type, not the ligase activity defective Itch negatively regulate RASSF5-mediated G1 phase transition of cell cycle as well as apoptosis, suggesting that Itch alone is sufficient to alter RASSF5 function. Collectively, the present investigation identifies a HECT class E3 ubiquitin ligase Itch as a unique negative regulator of RASSF5, and suggests the possibility that acetylation as a potential therapeutic target for human cancer.

Abstract C33: Akt-EphA2 signaling axis promotes diffuse infiltrative invasion of glioma in vivo and regulates glioma stem cell properties

Abstract Glioblastoma multiforme (GBM) is among the most lethal types of human cancer. Diffuse infiltrative invasion is a major cause for the poor prognosis; however the underlying mechanisms remain to be fully elucidated. Using human glioblastoma stem-like cells (GSCs) that recapitulate the highly invasive propensity of primary glioblastomas, we find that ephrin receptor A2 (EphA2) critically regulates glioma invasion in vivo. ShRNA knockdown of EphA2 markedly diminished intracranial invasion. Conversely overexpression of EphA2 enhanced the invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro, the Akt-EphA2 signaling axis in GSCs was maintained in the absence of ephrin-A ligands and disrupted upon ligand stimulation. In accordance, GSCs invasion was significantly increased in brain slices from the Efna1;Efna3;Efna4 triple knockout mice compared with those from the wild type littermates. Mechanistically overexpression of wild type but not S897A mutant EphA2 promoted GSC self-renewal, which was correlated with the increased expression of stem cell markers. Taken together, the results show that EphA2 stimulates glioma invasiveness in vivo in cooperation with Akt, which is negatively regulated by ephrin-As in tumor microenvironment. Citation Format: Hui Miao, Nickolas W. Gale, Bingcheng Wang. Akt-EphA2 signaling axis promotes diffuse infiltrative invasion of glioma in vivo and regulates glioma stem cell properties. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C33.

NHERF2 is crucial in ERM phosphorylation in pulmonary endothelial cells

BackgroundEBP50 and NHERF2 adaptor proteins are incriminated in various signaling pathways of the cell. They can bind ERM proteins and mediate ERM-membrane protein interactions.ResultsBinding of ERM to EBP50 and NHERF2 was compared in pulmonary artery endothelial cells by immunoprecipitation. NHERF2 associates with all three ERM, but EBP50 appeared to be a weak binding partner if at all. Furthermore, we detected co-localization of NHERF2 and phospho-ERM at the cell membrane and in the filopodia of dividing cells. Silencing of NHERF2 prevented agonist or angiogenesis induced phosphorylation of ERM, while overexpression of the adaptor elevated the phosphorylation level of ERM, likely catalyzed by Rho kinase 2, which co-immunoprecipitated with NHERF2/ERM in control EC, but did not bind to ERM in NHERF2 depleted cells. Dependence of ERM phosphorylation on NHERF2 was also shown in Matrigel tube formation assay, and NHERF2 was proved to be important in angiogenesis as well. Furthermore, when NHERF2 was depleted or cells were overexpressing a mutant form of NHERF2 unable to bind ERM, we found attenuated cell attachment with ECIS measurements, while it was supported by overexpression of wild type NHERF2.ConclusionsPivotal role of NHERF2 in the phosphorylation process of ERM in pulmonary artery endothelial cells is shown. We propose that NHERF2 provides a common anchoring surface for ERM and Rho kinase 2. Our results demonstrate the essential role of NHERF2 in endothelial cell adhesion/migration and angiogenesis.

The Transcriptional Targets of Mutant FOXL2 in Granulosa Cell Tumours

BackgroundDespite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets.MethodsThe expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Total RNA was hybridised to Affymetrix U133 Plus 2 microarrays. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2.ResultsThe overexpression of wildtype and mutant FOXL2 in COV434, and the silencing of mutant FOXL2 expression in KGN, has shown that mutant FOXL2 is able to differentially regulate the expression of many genes, including two well known FOXL2 targets, StAR and CYP19A. We have shown that many of the genes regulated by mutant FOXL2 are clustered into functional annotations of cell death, proliferation, and tumourigenesis. Furthermore, TGF-β signalling was found to be enriched when using the gene annotation tools GATHER and GeneSetDB. This enrichment was still significant after performing a robust permutation analysis.ConclusionGiven that many of the transcriptional targets of mutant FOXL2 are known TGF-β signalling genes, we suggest that deregulation of this key antiproliferative pathway is one way mutant FOXL2 contributes to the pathogenesis of adult-type GCTs. We believe this pathway should be a target for future therapeutic interventions, if outcomes for women with GCTs are to improve.

Abstract LB-280: RNP biogenesis factor Thoc1 is targeted for ubiquitination by NEDD4-1 E3 ligase

Abstract Thoc1 encodes an essential protein component (pThoc1) of the THO complex that regulates the packaging of nascent transcripts into ribonucleoprotein complexes (RNPs). Loss of pThoc1 compromises transcriptional elongation, RNA processing, and RNA export. Up-regulation of pThoc1 expression is common in a number of different cancers relative to normal tissue (Guo et al., 2005, Yang et al., 2008) and Thoc1 is preferentially required by neoplastic cells for growth and viability (Li et al., 2007). It is unclear how Thoc1 protein or the THO complex is normally regulated. In this study we examined whether pThoc1 is modified and regulated by ubiquitination because it is a low abundant protein in normal tissue. We found that both endogenous and ectopically expressed pThoc1 are polyubiquitinylated in Hela cells. Importantly, we identified that NEDD4-1 (neural precursor cell expressed developmentally down-regulated protein 4) is an E3 ligase that can mediate pThoc1 ubiquitination. This conclusion was reached based on several lines of evidence. First, overexpression of wild type but not the enzyme-dead NEDD4-1 increased pThoc1 ubiquitination. Second, pThoc1 ubiquitination was significantly reduced in Nedd4 knock out murine embryonic fibroblasts (MEFs) compared to the wild type MEFs. Third, inducible knockdown of NEDD4-1 expression in HeLa cells caused significant reduction in pThoc1 ubiquitination. These results indicate that pThoc1 is a novel substrate of NEDD4-1 E3 ligase in cells. Regulation of pThoc1 functions by NEDD4-1 mediated ubiquitination merits further investigation. This study was supported by grant # NIH R01 CA125665 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-280. doi:1538-7445.AM2012-LB-280

Abstract 2193: WT1 mediated regulation of growth control genes in leukemia

Abstract Prognosis and therapy of acute leukemia is influenced by leukemia-specific genetic alterations, thus highlighting the importance of identifying novel prognostic markers. One such potential marker, the Wilms’ tumor gene (WT1), a zinc finger transcription factor, is expressed in leukemic blasts and has been found to be mutated in approximately 10 percent of leukemia cases. Work in other tumor systems has shown that WT1 up-regulates expression of genes promoting cancer progression, including the angiogenic and mitogenic factor VEGF. Microarray studies have also correlated expression of JAG1 (Jagged 1) and CCNA1 (Cyclin A) with WT1 in Acute Myeloid Leukemia (AML) samples. We have identified potential WT1 binding sites within the promoters of both JAG1 and CCNA1 and have compared the expression of these genes to that of WT1, using real time quantitative PCR (QRTPCR), in pediatric leukemia and normal bone marrow. Compared to normal bone marrow, we observed lower than normal levels of WT1 in a majority of pediatric ALL samples, associated with lower than normal levels of VEGF, JAG-1 and CCNA-1. Conversely in pediatric AML(M3) we observed elevated levels of WT1 associated with elevated JAG-1 and CCNA-1. Mutations of the WT1 zinc finger (ZF) DNA binding domain have also been described in poor prognosis leukemias. To identify novel ZF mutations we sequenced WT1 in twelve pediatric acute leukemia samples. No ZF domain mutations were identified among these samples with high WT1 expression. However, a well-described SNP (rs 16754, also in exon 7), identified as a good prognostic marker in Cytogenetically Normal AML, was observed either as a homozygous or heterozygous variant of the WT1 gene. Because a majority of WT1 mutations identified in leukemias are frameshift mutations leading to a truncated protein lacking the ZF DNA binding domain, we created a truncation mutant of isoform A-WT1 lacking the ZF domain and assessed its function in K562 cells. Using QRTPCR we quantified the effect of over-expression of wild type and mutant WT1 on VEGF, JAG1 and CCNA1 expression. Compared to the vector control, wild type WT1 upregulated levels of JAG1 and CCNA1 but not VEGF mRNA. Mutant WT1 did not upregulate JAG1 or CCNA1. Our data and other studies have shown that JAG1 is elevated in AML patient samples, implicating the NOTCH 1 pathway that promotes cell proliferation and blocks differentiation. Thus, these results suggest that WT1 may act as an oncogene in K562 cells, in part by upregulating the growth promoting NOTCH-1 ligand, JAG1. Similarly, cell cycle regulation by WT1, demonstrated in other systems, may be mediated by transcriptional regulation of CCNA1. WT1 may contribute to leukemogenesis through transcriptional regulation of genes controlling cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2193. doi:1538-7445.AM2012-2193

Claudin 18 is a novel negative regulator of bone resorption and osteoclast differentiation

Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18(-/-) mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18(-/-) mice without changes in bone formation. Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation but not macrophage colony-stimulating factor (MCSF)-induced bone marrow macrophage (BMM) proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation.