Wild-type Mouse Embryonic Fibroblasts Research Articles

Loss of p53 in stromal fibroblasts enhances tumor cell proliferation through nitric-oxide-mediated cyclooxygenase 2 activation

Overexpression of cyclooxygenase 2 (COX-2) by stromal fibroblasts plays a critical role in the early stage of carcinogenesis. COX-2 expression is thought to be positively or negatively regulated by inflammatory chemical mediators or tumor suppressors. In this study, the contributions of inducible nitric oxide synthase (iNOS) and p53 to COX-2 expression were examined using mouse embryonic fibroblasts (MEFs) from wild-type, p53-deficient, iNOS-deficient, and p53/iNOS-deficient mice. These MEFs were treated with 1 μg/mL of lipopolysaccharide and 100 IU/mL of interferon gamma for up to 72 h. iNOS and COX-2 expression were analyzed by Western blotting. iNOS was induced earlier (16 h) in p53-deficient MEFs than in wild-type MEFs (48 h). Elevated expression of COX-2 was sustained for a longer duration in the p53-deficient MEFs. In contrast, COX-2 expression was reduced earlier in the iNOS-deficient MEFs. Addition of an exogenous NO donor (0.8 mM of S-nitroso-l-glutathione) to the iNOS-deficient MEFs augmented COX-2 expression. Co-culture with stimulated p53-deficient MEFs promoted cell proliferation of mouse rectal polyploid carcinoma CMT93 cells, but treatment with a COX-2-specific inhibitor counteracted this effect. These results suggest that loss of function of the p53 gene in stromal fibroblasts enhances COX-2 expression by enhancing iNOS expression and the resultant production of NO, contributing to the promotion of tumor growth.

An epigenomic role of Fe65 in the cellular response to DNA damage

Previous findings describe Fe65 as a key protein in the cellular response to genotoxic stress. However, the precise molecular mechanism by which Fe65 contributes to DNA damage signaling remains unclear. In this study, we hypothesized that the transcriptional activity of Fe65 may contribute to DNA damage pathways by regulating gene expression patterns activated in response to genotoxic stress. To address this hypothesis, we mapped the global binding profile of Fe65 by chromatin immunoprecipitation (ChIP)-sequencing in the SK-N-SH cells exposed to genotoxic stress. Unexpectedly, the genome-wide location analysis showed a substantial enrichment of Fe65 in the promoter regions of coding genes linked to DNA damage signaling pathways. To further investigate the role of Fe65 in the transcriptional regulation of putative coding target genes identified by ChIP-seq, we performed microarray assays using wild-type (WT) or Fe65 deficient mouse embryonic fibroblasts (MEFs) exposed to oxidative stress with multiple recovery times. Gene ontology analysis of the Fe65-depedent transcriptome suggested that Fe65 modulates the expression of genes critical for DNA damage response. Motif enrichment analysis of regulatory regions occupied by Fe65 revealed a strong correlation with key transcription factors involved in DNA damage signaling pathways, including E2F1, p53, and Jun. Comparison of ChIP-sequencing results with microarray results ultimately identified 248 Fe65-depedent target genes, the majority of which were known regulators of cell cycle, cell death, and DNA replication and repair pathways. We validated the target genes identified by in silico analysis by qPCR experiments. Collectively, our results provide strong evidence that Fe65 plays a role in DNA damage response and cell viability by epigenomic regulation of specific transcriptional programs activated upon genotoxic stress.

The drs tumor suppressor regulates glucose metabolism via lactate dehydrogenase-B.

Previously, we showed that drs contributes to suppression of malignant tumor formation in drs-knockout (KO) mice. In this study, we demonstrate the regulation of glucose metabolism by drs using comparisons of drs-KO and wild-type (WT) mouse embryonic fibroblasts (MEFs). Extracellular acidification, lactate concentration, and glucose consumption in drs-KO cells were significantly greater than those in WT cells. Metabolomic analyses also confirmed enhanced glycolysis in drs-KO cells. Among glycolysis-regulating proteins, expression of lactate dehydrogenase (LDH)-B was upregulated at the post-transcriptional level in drs-KO cells and increased LDH-B expression, LDH activity, and acidification of culture medium in drs-KO cells were suppressed by retroviral rescue of drs, indicating that LDH-B plays a critical role for glycolysis regulation mediated by drs. In WT cells transformed by activated K-ras, expression of endogenous drs mRNA was markedly suppressed and LDH-B expression was increased. In human cancer cell lines with low drs expression, LDH-B expression was increased. Database analyses also showed the correlation between downregulation of drs and upregulation of LDH-B in human colorectal cancer and lung adenocarcinoma tissues. Furthermore, an LDH inhibitor suppressed anchorage-independent growth of human cancer cells and MEF cells transformed by activated K-ras. These results indicate that drs regulates glucose metabolism via LDH-B. Downregulating drs may contribute to the Warburg effect, which is closely associated with malignant progression of cancer cells.

Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEF(la) (MEF(lowAPE1)), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEF(la) cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEF(la) was lower than that in the wild-type MEF (MEF(wt)), indicating the low DNA damage stress in MEF(la) under the normal growth condition. Oxidative phosphorylation activity in MEF(la) was lower than in MEF(wt), while the glycolysis rates in MEF(la) were higher than in MEF(wt). In addition, we observed decreased intracellular oxidative stress in MEF(la). These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability.

Dual stimuli polysaccharide nanovesicles for conjugated and physically loaded doxorubicin delivery in breast cancer cells.

The present work reports the development of pH and enzyme dual responsive polysaccharide vesicular nano-scaffolds for the administration of doxorubicin via physical loading and polymer-drug conjugation to breast cancer cells. Dextran was suitably modified with a renewable resource 3-pentadecyl phenol unit through imine and aliphatic ester chemical linkages that acted as pH and esterase enzyme stimuli, respectively. These dual responsive polysaccharide derivatives self-organized into 200 ± 10 nm diameter nano-vesicles in water. The water soluble anticancer drug doxorubicin (DOX·HCl) was encapsulated in the hydrophilic pocket to produce core-loaded polysaccharide vesicles whereas chemical conjugation produced DOX anchored at the hydrophobic layer of the dextran nano-vesicles. In vitro studies revealed that about 70-80% of the drug was retained under circulatory conditions at pH = 7.4 and 37 °C. At a low pH of 6.0 to 5.0 and in the presence of esterase; both imine and ester linkages were cleaved instantaneously to release 100% of the loaded drugs. Cytotoxicity assays on Wild Type Mouse Embryonic Fibroblasts (WTMEFs) confirmed the non-toxicity of the newly developed dextran derivatives at up to 500 μg mL(-1) in PBS. MTT assays on fibroblast cells revealed that DOX·HCl loaded nano-vesicles exhibited better killing abilities than DOX conjugated polymer nano-vesicles. Both DOX loaded and DOX conjugated nano-vesicles were found to show significant killing in breast cancer cells (MCF 7). Confocal microscopy images confirmed the uptake of DOX loaded (or conjugated) nano-vesicles by cells compared to free DOX. Thus, the newly developed pH and enzyme dual responsive polysaccharide vesicular assemblies are potential drug vectors for the administration of DOX in both loaded and chemically conjugated forms for the efficient killing of breast cancer cells.

Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice.

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on hypoxia-inducible factor 1 (HIF-1), which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia-responsive element reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO-mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

Sphingolipids inhibit vimentin-dependent cell migration.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

Core-shell polymer nanoparticles for prevention of GSH drug detoxification and cisplatin delivery to breast cancer cells.

Platinum drug delivery against the detoxification of cytoplasmic thiols is urgently required for achieving efficacy in breast cancer treatment that is over expressed by glutathione (GSH, thiol-oligopeptide). GSH-resistant polymer-cisplatin core-shell nanoparticles were custom designed based on biodegradable carboxylic functional polycaprolactone (PCL)-block-poly(ethylene glycol) diblock copolymers. The core of the nanoparticle was fixed as 100 carboxylic units and the shell part was varied using various molecular weight poly(ethylene glycol) monomethyl ethers (MW of PEGs = 100-5000 g mol(-1)) as initiator in the ring-opening polymerization. The complexation of cisplatin aquo species with the diblocks produced core-shell nanoparticles of 75 nm core with precise size control the particles up to 190 nm. The core-shell nanoparticles were found to be stable in saline solution and PBS and they exhibited enhanced stability with increase in the PEG shell thickness at the periphery. The hydrophobic PCL layer on the periphery of the cisplatin core behaved as a protecting layer against the cytoplasmic thiol residues (GSH and cysteine) and exhibited <5% of drug detoxification. In vitro drug-release studies revealed that the core-shell nanoparticles were ruptured upon exposure to lysosomal enzymes like esterase at the intracellular compartments. Cytotoxicity studies were performed both in normal wild-type mouse embryonic fibroblast cells (Wt-MEFs), and breast cancer (MCF-7) and cervical cancer (HeLa) cell lines. Free cisplatin and polymer drug core-shell nanoparticles showed similar cytotoxicity effects in the HeLa cells. In MCF-7 cells, the free cisplatin drug exhibited 50% cell death whereas complete cell death (100%) was accomplished by the polymer-cisplatin core-shell nanoparticles. Confocal microscopic images confirmed that the core-shell nanoparticles were taken up by the MCF-7 and HeLa cells and they were accumulated both at the cytoplasm as well at peri-nuclear environments. The present investigation lays a new foundation for the polymer-based core-shell nanoparticles approach for overcoming detoxification in platinum drugs for the treatment of GSH over-expressed breast cancer cells.

Inhibiting IκBβ-NFκB signaling attenuates the expression of select pro-inflammatory genes.

Multiple mediators of septic shock are regulated by the transcription factor nuclear factor κB (NFκB). However, complete NFκB inhibition can exacerbate disease, necessitating evaluation of targeted strategies to attenuate the pro-inflammatory response. Here, we demonstrate that in murine macrophages, low-dose NFκB inhibitors specifically attenuates lipopolysaccharide (LPS)-induced IκBβ degradation and the expression of a select subset of target genes (encoding IL1β, IL6, IL12β). Gain- and loss-of-function experiments demonstrate the necessary and sufficient role of inhibitor of NFκB family member IκBβ (also known as NFKBIB) in the expression of these genes. Furthermore, both fibroblasts and macrophages isolated from IκBβ overexpressing mice demonstrate attenuated LPS-induced IκBβ-NFκB signaling and IL1β, IL6 and IL12β expression. Further confirming the role of IκBβ and its NFκB subunit binding partner cRel in LPS-induced gene expression, pre-treatment of wild-type mouse embryonic fibroblasts with a cell-permeable peptide containing the cRel nuclear localization sequence attenuated IL6 expression. We prove that LPS-induced IκBβ-NFκB signaling can be selectively modulated to attenuate the expression of select pro-inflammatory target genes, thus providing therapeutic insights for patients exposed to systemic inflammatory stress.

Rapid nuclear transit and impaired degradation of amyloid β and glypican-1-derived heparan sulfate in Tg2576 mouse fibroblasts.

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from S-nitrosylated glypican-1 is generated in endosomes by an endogenously or ascorbate induced S-nitrosothiol-catalyzed reaction. Expression and processing of amyloid precursor protein (APP) is required to initiate formation and endosome-to-nucleus translocation of anMan-containing HS in wild-type mouse embryonic fibroblasts (WT MEF). HS is then transported to autophagosomes and finally degraded in lysosomes. To investigate how APP-derived amyloid β (Aβ) peptide affects intracellular trafficking of HS, we have studied nuclear transit as well as autophagosome/lysosome targeting and degradation in transgenic Alzheimer disease mouse (Tg2576) MEF which produce increased amounts of Aβ. Deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody showed anMan staining in the nuclei of Tg2576 MEF after 5 min of ascorbate treatment and after 15 min in WT MEF. There was also greater nuclear accumulation of HS in Tg2576 MEF as determined by (35)S-sulfate-labeling experiments. Tg2576 MEF was less sensitive to inhibition of NO production and copper-chelation than WT MEF. By using APP- and Aβ-recognizing antibodies, we observed nuclear translocation of Aβ peptide in Tg2576 MEF but not in WT MEF. HS remained in the nucleus of WT MEF for at least 8 h and was then transported to autophagosomes. By 8 h, HS had disappeared from the nuclei of Tg2576 MEF but colocalized poorly with the autophagosome marker LC3. Aβ also disappeared rapidly from the nuclei of Tg2576 MEF. Initially, it appeared in acidic vesicles and later it accumulated extracellularly. Thus, in Tg2576 MEF there is nuclear accumulation as well as secretion of Aβ and impaired degradation of HS.

Apoptolidins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A

Apoptolidin A was first isolated as a secondary metabolite of a Nocardiopsis sp. and is the founding member of a family of potential selective cancer cell toxins. We now report the isolation, production and pharmacological characterization of apoptolidins A and C from an alternate actinomycete producer, an Amycolatopsis sp. from soil samples collected in Indonesia. We investigated the action of apoptolidins A and C in representative human glioblastoma cells, lung cancer cells and mouse embryonic fibroblasts (MEFs) to better understand the mechanism of action of the known apoptolidins. Shifts in cellular metabolism in intact cells and the status of the AMP-activated protein kinase (AMPK) stress pathway in response to apoptolidin A were entirely consistent with the actions of an ATP synthase inhibitor. We find the metabolic phenotype of the cell to be a critical determinant of apoptolidin sensitivity and the likely basis for cancer cell selectivity. The apoptolidins induce indirect activation of AMPK and trigger autophagy in sensitive cell types without significant inhibition of mTORC1. Human U87-MG glioblastoma cells and wild type MEFs showed increased phosphorylation of AMPK (Thr172), ACC (Ser79) and ULK1 (Ser555), whereas AMPKα-null MEFs and more glycolytic SF-295 glioblastoma cells lacked this response. Although both are reported to be selective inhibitors of mitochondrial ATP synthase, differences between apoptolidin- and oligomycin A-induced responses in cells indicate that the action of these macrolides is not identical.

AZD5363 Inhibits Inflammatory Synergy between Interleukin-17 and Insulin/Insulin-Like Growth Factor 1.

In the United States, one-third of population is affected by obesity and almost 29 million people are suffering from type 2 diabetes. Obese people have elevated serum levels of insulin, insulin-like growth factor 1 (IGF1), and interleukin-17 (IL-17). Insulin and IGF1 are known to enhance IL-17-induced expression of inflammatory cytokines and chemokines, which may contribute to the chronic inflammatory status observed in obese people. We have previously demonstrated that insulin/IGF1 signaling pathway crosstalks with IL-17-activated nuclear factor-κB pathway through inhibiting glycogen synthase kinase 3β (GSK3β) activity. However, it is unclear whether GSK3α also plays a role and whether this crosstalk can be manipulated by AZD5363, a novel pan-Akt inhibitor that has been shown to increase glycogen synthase kinase 3 activity through reducing phosphorylation of GSK3α and GSK3β. In this study, we investigated IL-17-induced expression of C-X-C motif ligand 1 (Cxcl1), C-C motif ligand 20 (Ccl20), and interleukin-6 (Il-6) in wild-type, GSK3α−/−, and GSK3β−/− mouse embryonic fibroblast cells as well as in mouse prostate tissues by real-time quantitative PCR. We examined the proteins involved in the signaling pathways by Western blot analysis. We found that insulin and IGF1 enhanced IL-17-induced expression of Cxcl1, Ccl20, and Il-6, which was associated with increased phosphorylation of GSK3α and GSK3β in the presence of insulin and IGF1. AZD5363 inhibited the synergy between IL-17 and insulin/IGF1 through reducing phosphorylation of GSK3α and GSK3β by inhibiting Akt function. These findings imply that the cooperative crosstalk of IL-17 and insulin/IGF1 in initiating inflammatory responses may be alleviated by AZD5363.

Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

Oncogenic Ras inhibits IRF1 to promote viral oncolysis.

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1(-/-) MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl–CIN85–endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

Loss of the tumor suppressor Hace1 leads to ROS-dependent glutamine addiction.

Cellular transformation is associated with altered glutamine (Gln) metabolism. Tumor cells utilize Gln in the tricarboxylic acid (TCA) cycle to maintain sufficient pools of biosynthetic precursors to support rapid growth and proliferation. However, Gln metabolism also generates NADPH, and Gln-derived glutamate is used for synthesis of glutathione (GSH). Since both NADPH and GSH are antioxidants, Gln may also contribute to redox balance in transformed cells. The Hace1 E3 ligase is a tumor suppressor inactivated in diverse human cancers. Hace1 targets the Rac1 GTPase for degradation at Rac1-dependent NADPH oxidase complexes, blocking superoxide generation by the latter. Consequently, loss of Hace1 increases reactive oxygen species (ROS) levels in vitro and in vivo. Given the link between Hace1 loss and increased ROS, we investigated whether genetic inactivation of Hace1 alters Gln metabolism. We demonstrate that mouse embryonic fibroblasts (MEFs) derived from Hace1-/- mice are highly sensitive to Gln withdrawal, leading to enhanced cell death compared to wild type (wt) MEFs, and Gln depletion or chemical inhibition of Gln uptake block soft agar colony formation by Hace1-/- MEFs. Hace1-/- MEFs exhibit increased Gln uptake and ammonia secretion, and metabolic labeling using 13C-Gln revealed that Hace1 loss increases incorporation of Gln carbons into TCA cycle intermediates. Gln starvation markedly increases ROS levels in Hace1-/- but not in wt MEFs, and treatment with the antioxidant N-acetyl cysteine (NAC) or the TCA cycle intermediate oxaloacetate efficiently rescues Gln starvation-induced ROS elevation and cell death in Hace1-/- MEFs. Finally, Gln starvation increases superoxide levels in Hace1-/- MEFs, and NADPH oxidase inhibitors block the induction of superoxide and cell death by Gln starvation. Together, these results suggest that increased ROS production due to Hace1 loss leads to Gln addiction as a mechanism to cope with increased ROS-induced oxidative stress.

Abstract 2439: HSulf-1 loss in ovarian cancer cells induces lipid droplet biogenesis

Abstract We have previously shown that a frequent loss of endosulfatase HSulf-1 in ovarian cancer enhances the heparin binding growth factor signaling which subsequently induces and potentiates tumorigenesis. Our recent data indicate that knockdown of HSulf-1 in ovarian cancer cells significantly alter levels of several metabolites which is comparable to the deranged metabolite profiles in primary ovarian tumors. Here we report that the metabolites associated with lipid metabolism are altered in such a way that the cells start manifesting a “lipogenic phenotype” characterized by an increased fatty acid synthesis, storage and mitochondrial beta oxidation. The lipogenic phenotype is associated with an accumulation of cytoplasmic lipid droplets (LDs) in a stably HSulf-1 knockdown cells [OV202 (Sh1/2) and OV2223 cells]. Presence of LDs is evidenced by BODIPY staining and transmission electron microscopy (TEM) analysis in Sh1/2 and a non-transduced control (NTC) cells. Conversely, enhanced expression of HSulf-1 in TOV21G, SKOV3, and C13 cells also resulted in reducing the number of LDs. More importantly, MEFs (mouse embryonic fibroblasts) from HSulf-1 knockout mice also showed an increased LD biogenesis compared to the wild type MEFs. Microarray analysis identified cytoplasmic phospholipase A2 (cPLA2; an enzyme involved in LD biogenesis) as one of the genes that was significantly upregulated in OV202 Sh1 and Sh2 cells. We found that Sh1 and Sh2 cells show an increased phosphorylation (ser505) of cytoplasmic phospholipase A2 (p-cPLA2) compared to NTC cells. Phosphorylation at ser505 is vital for the enzyme activity of cPLA2 and also necessary for LD biogenesis. We inhibited the LD biogenesis in Sh1 cells by a pharmacological inhibitor of cPLA2 (AACOCF3 and MAFP) as well by shRNA mediated knockdown of endogenous cPLA2. Further, we stimulated NTC and Sh1 cells with heparin binding growth factor (50ng/ml) for different time points and found an elevated phosphorylation of cPLA2 and extracellular signal related kinase (ERK), which is also inhibited by a pre-treatment of cells with cPLA2 inhibitor. Interestingly, inhibition of phospho-ERK by U0126 (MEK inhibitor) also inhibits the phosphorylation of cPLA2. These findings strongly suggest that loss of HSulf-1 promotes lipid metabolism in the ovarian cancer cells via activation of cPLA2 via an ERK dependent pathway. The present work highlights a novel role HSulf-1 in regulating lipid metabolism which could lead to a better understanding of tumorigenesis and identification of new targets for tumor therapy. Citation Format: Debarshi Roy, Xiaoping He, Ashwani Khurana, Susmita Mondal, Deok-Beom Jung, Sung Kim, Thomas Dierks, Clifford Folmes, Andre Terzic, Viji Shridhar. HSulf-1 loss in ovarian cancer cells induces lipid droplet biogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2014-2439

Inhibition of BMP2-induced bone formation by the p65 subunit of NF-κB via an interaction with Smad4.

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.

Pseudomonas aeruginosa quorum-sensing molecule homoserine lactone modulates inflammatory signaling through PERK and eI-F2α.

Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.