BBA - Molecular Cell Research (v.1823, #8)

PKC signaling inhibits osteogenic differentiation through the regulation of Msx2 function by Hyung Min Jeong; Yun-Hye Jin; You Hee Choi; Jinah Yum; Joong-Kook Choi; Chang-Yeol Yeo; Kwang-Youl Lee (1225-1232).
Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, β, δ, and ζ) interacted with Msx2, and PKCβ phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.► PKC activity suppresses osteoblast differentiation in vitro. ► PKCs affect the expression of Msx2 protein. ► Msx2 interacts with PKCs and is phosphorylated by PKCβ in vitro. ► PKC activity prevents Msx2 ubiquitination. ► PKC inhibition relieves the suppression of Runx2 transcriptional activity by Msx2.
Keywords: Osteoblast differentiation; PKC; Msx2; Runx2; Protein stability;

The APP intracellular domain (AICD) inhibits Wnt signalling and promotes neurite outgrowth by Fangfang Zhou; Kai Gong; Bo Song; Tuo Ma; Theo van Laar; Yandao Gong; Long Zhang (1233-1241).
β- and γ-secretase cleave the amyloid precursor protein (APP) to release the amyloidogenic β-amyloid peptides (Aβ) and the APP intracellular domain (AICD). Aβ has been widely believed to initiate pathogenic cascades culminating in Alzheimer's disease (AD). However, the physiological functions of the AICD remain elusive. In this study, we found the AICD to strongly inhibit Wnt-induced transcriptional reporter activity, and to counteract Wnt-induced c-Myc expression. Loss of the AICD resulted in an increased responsiveness to Wnt/β-catenin-mediated transcription. Mechanically, the AICD was found to interact with glycogen synthase kinase 3 beta (GSK3β) and promote its kinase activity. The subsequent AICD-strengthened Axin–GSK3β complex potentiates β-catenin poly-ubiquitination. Functional studies in N2a mouse neuroblastoma cells, rat pheochromocytoma PC12 cells and primary neurons showed that the AICD facilitated neurite outgrowth. And AICD antagonised Wnt3a-suppressed growth arrest and neurite outgrowth in N2a and PC12 cells. Taken together, our results identify the AICD as a novel inhibitory factor of the canonical Wnt signalling pathway and suggest its regulatory role in neuronal cell proliferation and differentiation.► AICD inhibits canonical Wnt signalling and reduces Wnt induced c-Myc expression. ► AICD antagonises Wnt signalling in GSK3β kinase activity dependent manner. ► AICD associates with and activates GSK3β. ► AICD cooperates with NGF to suppress neuronal cell proliferation. ► AICD antagonises Wnt suppressed and promotes NGF induced neurite outgrowth.
Keywords: APP intracellular domain (AICD); Wnt signalling; Neurite outgrowth;

Capacitative and non-capacitative signaling complexes in human platelets by Alejandro Berna-Erro; Carmen Galan; Natalia Dionisio; Luis J. Gomez; Gines M. Salido; Juan A. Rosado (1242-1251).
Discharge of the intracellular Ca2+ stores activates Ca2+ entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca2+ signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca2+ stores with thapsigargin, independently of changes in cytosolic Ca2+ concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca2+ entry. These results indicate that Ca2+ store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca2+ influx in human platelets.► Ca2+ store depletion results in a complex, probably regulated by STIMs, including Orai1 and Orai2. ► Orai3 does not associate with STIM1 or STIM2 upon Ca2+ store depletion. ► The association of Orai3 with Orai1 or Orai2 is reduced after Ca2+ store discharge. ► The diacylglycerol analog OAG stimulates specific association of Orai3 with TRPC3. ► Activation of Orai3 leads to non-capacitative Ca2+ entry in human platelets.
Keywords: Orai1; Orai2; Orai3; STIM1; STIM2; TRPC;

REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation by Monica Baiula; Gioia Carbonari; Samantha D. Dattoli; Maria Calienni; Andrea Bedini; Santi Spampinato (1252-1263).
REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-XL whereas in HeLa cells overexpressing REST, the reduction of Bcl-XL mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.► Transcription factor REST is elevated in HeLa cells exposed to EGF or serum. ► REST down-regulation by antisense increases apoptosis induced by serum withdrawal. ► REST overexpression reduces apoptosis caused by exposure to an anti-Fas antibody. ► Histone H3 acetylation is increased in antisense-treated HeLa cells. ► Histone H3 acetylation is reduced in REST overexpressing cells.
Keywords: Epidermal growth factor; HeLa cell; REST; Apoptosis; Histone H3;

Inhibitory role of adiponectin peptide I on rat choroidal neovascularization by Valeriy V. Lyzogubov; Ruslana G. Tytarenko; Nalini S. Bora; Puran S. Bora (1264-1272).
Age-related macular degeneration (AMD) is a leading cause of central blindness in the elderly population. The wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied the effects of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density were investigated by microscopy. Immunohistochemical staining (IHC) for von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV.► Adiponectin peptide I (APNpI) inhibited area of CNV by 4 folds. ► The expression of VEGF and VEGF-R2 decreased after APNpI treatment. ► Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats. ► APNpI inhibited formation of new vessels in rat model of CNV. ► APNpI may have therapeutic use for AMD treatment.
Keywords: Adiponectin; Adiponectin receptors; VEGF; Angiogenesis; Macular degeneration; Neovascularization;

Mechanisms underlying the protein-kinase mediated regulation of the HERG potassium channel synthesis by Yamini Krishnan; Yan Li; Renjian Zheng; Vikram Kanda; Thomas V. McDonald (1273-1284).
The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.► HERG channel synthesis is increased by protein kinase A (PKA) activity. ► Channel phosphorylation is necessary but not sufficient for increased protein abundance. ► PKA enhancement of HERG requires an additional protein co-factor. ► Kinase activity does not elevate HERG translation from low to high rates. ► Increased HERG abundance does not result from proteasome or lysosome inhibition.
Keywords: HERG; Protein kinase A; Cyclic-AMP; Protein translation; Protein kinase C; Potassium channel;

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK1-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130Cas, PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.► Yes is activated by pancreatic stimulants activating phospholipase C or PKC. ► 60% CCK-maximal stimulation is by high- and 40% by low-affinity receptor state. ► CCK/TPA stimulate Yes's association with Pyk2, p125FAK and their phosphorylated forms. ► CCK/TPA stimulate Yes's association with PKCδ, Shc, PKD, p130CAS, PI3K, and PTEN. ► Thapsigargin and a PKC inhibitor, GF109203X inhibit TPA-/CCK-stimulated Yes.
Keywords: Src; Yes activation; Pancreatic acinus; CCK; Signaling; Pancreatic growth factor;

Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells by Aleksandra Wiczk; Dagmara Hofman; Grażyna Konopa; Anna Herman-Antosiewicz (1295-1305).
Sulforaphane (SFN) is a compound derived from cruciferous plants. Its anticancer properties have been demonstrated both, in cancer cell lines as well as tumors in animal models. It has been shown that SFN inhibits cell proliferation, induces apoptosis, autophagy, and sensitizes cancer cells to therapies. As induction of catabolic processes is often related to perturbation in protein synthesis we aimed to investigate the impact of SFN on this process in PC-3 human prostate cancer cells. In the present study we show that SFN inhibits protein synthesis in PC-3 cells in a dose- and time-dependent manner which is accompanied by a decreased phosphorylation of mTOR substrates. Translation inhibition is independent of mitochondria‐derived ROS as it is observed in PC-3 derivatives devoid of functional mitochondrial respiratory chain (Rho0 cells). Although SFN affects mitochondria and slightly decreases glycolysis, the ATP level is maintained on the level characteristic for control cells. Inhibition of protein synthesis might be a protective response of prostate cancer cells to save energy. However, translation inhibition contributes to the death of PC-3 cells due to decreased level of a short-lived protein, survivin. Overexpression of this anti-apoptotic factor protects PC-3 cells against SFN cytotoxicity. Protein synthesis inhibition by SFN is not restricted to prostate cancer cells as we observed similar effect in SKBR-3 breast cancer cell line.► Sulforaphane disturbs signal transduction between mTOR and its substrates. ► It results in a drop in protein synthesis in prostate and breast cancer cells. ► Translation inhibition might be a protective response to save ATP level. ► Prolonged translation inhibition leads to depletion of anti-apoptotic survivin.
Keywords: Sulforaphane; Prostate cancer; mTOR; S6K1; Protein synthesis; Survivin;

Earlier studies showed that 2-methoxyestradiol (2ME2), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G2/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME2-induced cell cycle arrest. We found that 2ME2 can selectively induce mitotic prometaphase arrest, but not G2 phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12–24 h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME2-induced prometaphase arrest. The 2ME2-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME2-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME2 causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.► 2ME2 induces prometaphase arrest, but not G2 arrest, in human breast cancer cells. ► Up-regulation of cyclin B1 and Cdc2 by 2ME2 mediates mitotic prometaphase arrest. ► MAD2 and JNK1 are key upstream mediators of 2ME2-induced cell cycle changes.
Keywords: 2-Methoxyestradiol; Anti-microtubule agents; Mitotic prometaphase arrest; Cyclin-dependent kinases; MAD2;

Thymidine phosphorylase inhibits vascular smooth muscle cell proliferation via upregulation of STAT3 by Hong Yue; Kuniyoshi Tanaka; Tatsuhiko Furukawa; Sadashiva S. Karnik; Wei Li (1316-1323).
Dysregulated growth and motility of vascular smooth muscle cells (VSMC) play important role in obstructive vascular diseases. We previously reported that gene transfer of thymidine phosphorylase (TP) into rat VSMC inhibits cell proliferation and attenuates balloon injury induced neointimal hyperplasia; however, the mechanism remains unclear. The current study identified a signaling pathway that mediates effect of TP inhibited VSMC proliferation with a TP activity-dependent manner. Rat VSMC overexpressing human TP gene (C2) or control empty vector (PC) were used. Serum stimulation induced constitutive STAT3 phosphorylation at tyrosine705 in C2 cell but not in PC, which was independent of JAK2 signaling pathway. Inhibition of Src family kinases activity inhibited STAT3 phosphorylation in C2 cells. Lyn activity was higher in C2 cell than in PC. SiRNA based gene knockdown of Lyn significantly decreased serum induced STAT3 phosphorylation in C2 and dramatically increased proliferation of this cell, suggesting that Lyn plays a pivotal role in TP inhibited VSMC proliferation. Unphosphorylated STAT3 (U-STAT3) expression was significantly increased in C2 cells, which may be due to the increased STAT3 transcription. Gene transfection of mouse wild‐type or Y705F mutant STAT3 into PC cell or mouse primary cultured VSMC significantly reduced proliferation of these cells, suggesting that overexpression of U-STAT3 inhibits VSMC proliferation. We conclude that Lyn mediates TP induced STAT3 activation, which subsequently contributes to upregulate expression of U-STAT3. The U-STAT3 plays a critical role in inhibiting VSMC proliferation.► Inhibition of vascular smooth muscle proliferation benefits atherosclerotic diseases. ► Thymidine phosphorylase inhibits vascular smooth muscle cell proliferation. ► We found thymidine phosphorylase impacts STAT3 activity via Lyn. ► We found that an unphosphorylated STAT3 inhibits VSMC proliferation.
Keywords: Thymidine phosphorylase; Vascular smooth muscle cell proliferation; STAT3;

Cohesin: A guardian of genome integrity by Gunjan D. Mehta; Syed Meraj Azhar Rizvi; Santanu Kumar Ghosh (1324-1342).
Ability to reproduce is one of the hallmark features of all life forms by which new organisms are produced from their progenitors. During this process each cell duplicates its genome and passes a copy of its genome to the daughter cells along with the cellular matrix. Unlike bacteria, in eukaryotes there is a definite time gap between when the genome is duplicated and when it is physically separated. Therefore, for precise halving of the duplicated genome into two, it is required that each pair of duplicated chromosomes, termed sister chromatids, should be paired together in a binary fashion from the moment they are generated. This pairing function between the duplicated genome is primarily provided by a multimeric protein complex, called cohesin. Thus, genome integrity largely depends on cohesin as it ensures faithful chromosome segregation by holding the sister chromatids glued together from S phase to anaphase. In this review, we have discussed the life cycle of cohesin during both mitotic and meiotic cell divisions including the structure and architecture of cohesin complex, relevance of cohesin associated proteins, mechanism of cohesin loading onto the chromatin, cohesion establishment and the mechanism of cohesin disassembly during anaphase to separate the sister chromatids. We have also focused on the role of posttranslational modifications in cohesin biology. For better understanding of the complexity of the cohesin regulatory network to the readers, we have presented an interactome profiling of cohesin core subunits in budding yeast during mitosis and meiosis.► Discussed the latest research on life cycle of cohesin with special emphasis on S. cerevisiae. ► Comparison between budding yeast cohesin/cohesion mechanism with that of the other model eukaryotes. ► Interactome table of cohesin has been provided to understand the complexity of its regulatory network. ► Tabular compilation to highlight the role of posttranslational modifications in cohesin biology.
Keywords: Cohesin; Chromosome; Yeast; Centromere;

zVAD-fmk upregulates caspase-9 cleavage and activity in etoposide-induced cell death of mouse embryonic fibroblasts by Aida Rodríguez-Enfedaque; Elisabeth Delmas; Arnaud Guillaume; Sébastien Gaumer; Bernard Mignotte; Jean-Luc Vayssière; Flore Renaud (1343-1352).
Caspases are key effectors of programmed cell death. Down- and up-regulation of their activity are involved in different pathologies. In most cells, zVAD-fmk prevents apoptosis. However, unexpected effects of zVAD-fmk have been characterized in different laboratories, cell models and cell death processes. We have previously shown that zVAD-fmk accelerates p53-dependent apoptosis in rat embryonic fibroblasts. In this study, we pursued our investigations on zVAD-fmk effects and focused our study at the mitochondrial level in mouse embryonic fibroblasts (MEFs). In both primary and immortalized (by AgT or 3T9 protocol) MEFs, zVAD-fmk increased etoposide-induced loss of ΔΨm. This increase correlated with an increase of the number of apoptotic cells in primary and 3T9 MEFs, but did not in AgT MEFs. In both types of immortalized MEFs, zVAD-fmk regulated neither p53 levels nor transcriptional activities, suggesting that zVAD-fmk acts downstream of p53. In MEFs, zVAD-fmk increased p53-dependent loss of ΔΨm, cytochrome c release and caspase-9 activity. Indeed, zVAD-fmk inhibited effector caspases (caspases-3, -6, -7) as expected but increased caspase-9 cleavage and activity in etoposide-treated MEFs. Q-VD-OPh, another caspase inhibitor, also increased both loss of ΔΨm and caspase-9 cleavage in etoposide-treated MEFs. Invalidation of bax and bak suppressed p53-dependent cell death and zVAD-fmk regulation of this process. Invalidation of caspase-9 did not inhibit mitochondrial membrane depolarization but suppressed zVAD-fmk amplification of this process. Altogether, our data suggest that caspase-9 activity is up-regulated by zVAD-fmk and is involved in an amplification loop of etoposide-induced cell death at the mitochondrial level in MEFs.► zVAD-fmk increases etoposide-induced mitochondrial membrane depolarization in MEFs. ► zVAD-fmk increases cytochrome c release after etoposide-treatment. ► zVAD-fmk downregulates effector caspases but upregulates caspase-9. ► Q-VD-OPh increased etoposide-induced loss of ΔΨm and caspase-9 cleavage in 3T9 MEFs. ► Caspase-9 activates an amplification loop at the mitochondrial level
Keywords: Caspase-9; zVAD-fmk; p53-dependent cell death; Mouse embryonic fibroblast;

De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1 by Anne-Laure Mahul-Mellier; Christoph Datler; Evangelos Pazarentzos; Bevan Lin; Wanwisa Chaisaklert; Ghada Abuali; Stefan Grimm (1353-1365).
Dynamic ubiquitination impacts on the degradation of proteins by the proteasome as well as on their effects as signalling factors. Of the many cellular responses that are regulated by changes in ubiquitination, apoptosis has garnered special attention. We have found that USP2a and USP2c, two isoforms of the ubiquitin-specific protease USP2, cause cell death upon ectopic expression. We show that both USP2 isoforms can control the ubiquitination status of many proteins but from a panel of potential targets only the protein level of RIP1 was increased by these enzymes. This effect is responsible for the activity of USP2a and USP2c to cause cell death. Both enzymes likewise de-ubiquitinate TRAF2, a ubiquitin-ligase in the TNFR1 complex. Whilst this and the similar sub‐cellular localisations of both enzyme isoforms indicate a substantial overlap of activities, inactivation by RNAi revealed that only the knock-down of USP2c resulted in apoptosis, whilst targeting USP2a did not have any consequence on the cells' survival. Consequently, we focussed our studies on USP2a and found that TRAF2 inhibits USP2a's effect on K48- but not on K63-linked ubiquitin chains. Hence, the ratio between USP2a and TRAF2 protein levels determines the cells' sensitivity to cell death.
Keywords: Apoptosis; Ubiquitin;

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity by Sara Gutiérrez-Herrero; Vera Maia; Javier Gutiérrez-Berzal; Nuria Calzada; María Sanz; Consuelo González-Manchón; Miguel Pericacho; Sara Ortiz-Rivero; José R. González-Porras; María Arechederra; Almudena Porras; Carmen Guerrero (1366-1377).
We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.► We have generated platelet-specific transgenic mice for C3G and a C3GDeltaCat mutant. ► We have analyzed the role of C3G in platelet function by in vivo and in vitro approaches. ► Transgenic C3G increases platelet activation and aggregation via Rap1. ► Platelets expressing C3GDeltaCat are impaired in activation and aggregation. ► The number of neutrophils is increased in C3G transgenic mice.
Keywords: C3G; Platelet; Transgenic mice; Granulocyte; Neutrophil; Rap1;

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid‐mediated apoptosis through Bak activation in ovarian cancer cells by Kotaro Miyake; Joseph Bekisz; Tongmao Zhao; Christopher R. Clark; Kathryn C. Zoon (1378-1388).
Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.► tBid induced by IFN-α2a activates mitochondrial Bak. ► Bak triggers the loss of mitochondrial membrane potential leading to release of apoptosis-inducing factor (AIF) in ovarian cancer (OVCAR3 cells. ► AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. ► IFN-α2a-induced apoptosis is mediated through the mitochondria.
Keywords: IFN-α; Apoptosis; Mitochondria; AIF; Bid; Bak;

In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17β-estradiol and suppressed by insulin deprivation by Marco G. Alves; Sílvia Socorro; Joaquina Silva; Alberto Barros; Mário Sousa; José E. Cavaco; Pedro F. Oliveira (1389-1394).
Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner.hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR.hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels.Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.Display Omitted► In vitro cultured human Sertoli cells (hSCs) produce high amounts of acetate. ► Insulin deprivation completely supresses acetate production. ► DHT and E2-treated cells have distinct effect on acetate production. ► ACoA Hyd and ACoA Synt gene transcript levels are hormonally controlled. ► Acetate produced by hSCs should deserve special attention as it role is unknown.
Keywords: Human Sertoli cells; Energy metabolism; Insulin; Acetate; Acetyl-CoA;

Implication of Nrf2 and ATF4 in differential induction of CHOP by proteasome inhibition in thyroid cancer cells by Zhi-Hong Zong; Zhen-Xian Du; Ning Li; Chao Li; Qiang Zhang; Bao-Qin Liu; Yifu Guan; Hua-Qin Wang (1395-1404).
Proteasome inhibition may cause endoplasmic reticulum (ER) stress, which has been reported to be implicated in the antitumoral effects of proteasome inhibitors. CCAAT/enhancer‐binding protein homologous protein (CHOP) is induced by a variety of adverse physiological conditions including ER stress and is involved in apoptosis. We have reported that distinct induction of CHOP contributes to the responsiveness of thyroid cancer cells to proteasome inhibitors. However, the mechanism underlying differential induction of CHOP by proteasome inhibitors in thyroid cancer cells has not been well characterized. In the current study, we characterized that proteasome inhibition primarily activated the amino acid response element 1 (AARE1) on the CHOP promoter. We also demonstrated that although proteasome inhibition caused similar accumulation of activating transcription factor 4 (ATF4) in a panel of thyroid cancer cells, distinct amounts of ATF4 were recruited to the AARE1 element of CHOP promoter. In addition, we demonstrated that NF‐E2‐related factor 2 (Nrf2) was also implicated in the induction of CHOP by precluding the binding of ATF4 to the CHOP promoter. This study highlights the molecular mechanisms by which ATF4 and Nrf2 can control CHOP induction in thyroid cancer cells by proteasome inhibition.► MG132 activated AARE1, ERSE and AP-1 elements on the CHOP promoter. ► ATF4 recruitment to AARE1 was involved in CHOP induction by MG132. ► Nrf2 was involved in CHOP induction via regulation of ATF4 recruitment. ► ATF4 and Nrf2 regulated the degree of CHOP induction by MG132.
Keywords: Proteasome inhibition; CHOP; ATF4; Nrf2;