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BBA - Molecular Cell Research (v.1833, #6)

Editorial Board (pp. i).

Vimentin phosphorylation and assembly are regulated by the small GTPase Rab7a by Laura Cogli; Cinzia Progida; Roberta Bramato; Cecilia Bucci (pp. 1283-1293).

Intermediate filaments are cytoskeletal elements important for cell architecture. Recently it has been discovered that intermediate filaments are highly dynamic and that they are fundamental for organelle positioning, transport and function thus being an important regulatory component of membrane traffic. We have identified, using the yeast two-hybrid system, vimentin, a class III intermediate filament protein, as a Rab7a interacting protein. Rab7a is a member of the Rab family of small GTPases and it controls vesicular membrane traffic to late endosomes and lysosomes. In addition, Rab7a is important for maturation of phagosomes and autophagic vacuoles. We confirmed the interaction in HeLa cells by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using bacterially expressed recombinant proteins. Immunofluorescence analysis on HeLa cells indicate that Rab7a-positive vesicles sometimes overlap with vimentin filaments. Overexpression of Rab7a causes an increase in vimentin phosphorylation at different sites and causes redistribution of vimentin in the soluble fraction. Consistently, Rab7a silencing causes an increase of vimentin present in the insoluble fraction (assembled). Also, expression of Charcot–Marie–Tooth 2B-causing Rab7a mutant proteins induces vimentin phosphorylation and increases the amount of vimentin in the soluble fraction. Thus, modulation of expression levels of Rab7a wt or expression of Rab7a mutant proteins changes the assembly of vimentin and its phosphorylation state indicating that Rab7a is important for the regulation of vimentin function.► We searched for new Rab7a interacting proteins and we found vimentin. ► We demonstrated that Rab7a interacts directly with vimentin. ► Rab7a influences vimentin's phosphorylation and soluble/insoluble ratio. ► Rab7a regulates vimentin organization and function.

Keywords: Abbreviations; GST; Glutathione-S-Transferase; HA; Hemagglutinin; EGFP; Enhanced Green Fluorescent ProteinRab7a; Vimentin; Rab protein; Intermediate filaments; Phosphorylation; Two-hybrid

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca2⁺ channel activity by Nathalie Nguyen; Michael Biet; Élie Simard; Beliveau Éric Béliveau; Nancy Francoeur; Gaétan Guillemette; Robert Dumaine; Michel Grandbois; Guylain Boulay (pp. 1294-1303).

STIM1 plays a crucial role in Ca2⁺ homeostasis, particularly in replenishing the intracellular Ca2⁺ store following its depletion. In cardiomyocytes, the Ca2⁺ content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca2⁺ imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca2⁺ oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca2⁺ channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca2⁺ overload that elicits arrhythmogenic events.•Knock down of STIM1 induces irregular spontaneous Ca2⁺ oscillations in HL-1 cells.•Knock down of STIM1 causes a Ca2⁺ overload in the sarcoplasmic reticulum.•STIM1 physically interacts with T-VDCC channel.•STIM1 negatively regulates T-VDCC activity.•Knock down of STIM1 destabilizes the contractile rhythmicity of HL-1 cells.

Keywords: Abbreviations; [Ca; 2; ⁺; ]; _i; concentration of intracellular free Ca; 2; ⁺; AFM; atomic force microscopy; CICR; Ca; 2; ⁺; -induced Ca; 2; ⁺; release; CRAC; Ca; 2; ⁺; -release activated Ca; 2; ⁺; channel; I; _Ca; Ca; 2; ⁺; current; I; _Ca,T; T-type Ca; 2; ⁺; current; L-VDCC; L-type voltage-dependent calcium channel; RI; rhythmicity index; RyR2; cardiac ryanodine receptor; SERCA; sarco/endoplasmic reticulum Ca; 2; ⁺; ATPase pump; SOCE; store-operated Ca; 2; ⁺; entry; SR; sarcoplasmic reticulum; STIM1; stromal interaction molecule 1; T-VDCC; T-type voltage-dependent calcium channel; VDCC; voltage-dependent Ca; 2; ⁺; channelSTIM1; T-type calcium channel; Contractility; Ca; 2; ⁺; overload

Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion by Louis Tong; Evelyn Png; Hou AiHua; Siew Sian Yong; Hui Ling Yeo; Andri Riau; Earnest Mendoz; Shyam S. Chaurasia; Chwee Teck Lim; Ting Wai Yiu; Siiri E. Iismaa (pp. 1304-1315).

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing.•Wound closure was delayed in transglutaminase (TG)-2 deleted compared to wild type mice.•TG-2 silencing led to reduced epithelial cell adhesion, spreading and velocity of migration.•TG-2 silencing reduced the activity of rho family kinases, i.e., rac and cdc42.•TG-2 involvement in phosphorylation of adhesion proteins explains its role in cell adhesion and migration.•TG-2 paxillin pathway may be how TG-2 function in epithelial adhesion and migration.

Keywords: Abbreviations; Tg; TransglutaminaseMigration; Adhesion; Transglutaminase; Cornea; Epithelium; Wound healing

p70S6 kinase is a target of the novel proteasome inhibitor 3,3′-diamino-4′-methoxyflavone during apoptosis in human myeloid tumor cells by Marion Piedfer; Sandrine Bouchet; Ruoping Tang; Christian Billard; Daniel Dauzonne; Brigitte Bauvois (pp. 1316-1328).

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3′-diamino-4′-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser¹³⁶), activation of caspases -8, -9, and -3 and cleavage of the caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr³⁸⁹-phosphorylation and (ii) protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent apoptosis and P70S6K degradation were simultaneously prevented by the caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the proteasome inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated proteasome inhibition and apoptosis of leukemia cells.► 3,3′-diamino-4′-methoxyflavone (DD1) induces human myeloid tumor-selective apoptosis. ► DD1-mediated proteasome inhibition dictates apoptosis. ► The mechanism relies on Bax activation and caspase-dependent p70S6 kinase degradation. ► Crosstalk by flavone and bortezomib contributes to additive apoptotic effects. ► Proteasome inhibition by DD1 could help in overcoming multidrug resistance in tumors.

Keywords: Abbreviations; AML; acute myeloid leukemia; DD1; 3,3′-diamino-4′-methoxyflavone; DNFAA; 2′,3-dinitroflavone-8-acetic acid; 4EBP1; eukaryotic translation initiation factor 4E binding protein 1; FCS; fetal calf serum; FITC; fluorescein isothiocyanate; LPS; lipopolysaccharide; MAPK; mitogen activated protein kinase; MMP; mitochondrial membrane potential; mTOR; mammalian target of rapamycin; PARP-1; poly ADP-ribose polymerase-1; PDK2; pyruvate dehydrogenase kinase isoform 2; PI; propidium iodide; PI3K; phosphoinositide-3-kinase; P70S6K; 70; kDa ribosomal protein S6 kinase; rh; recombinant humanApoptosis; Myeloid cell; Proteasome; Caspase; Bax; P70S6K

N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization by M.G. De Napoli; N. de Miguel; M. Lebrun; S.N.J. Moreno; S.O. Angel; M.M. Corvi (pp. 1329-1337).

Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.► TgHSP20 is synthesized and is palmitoylated in three cysteine residues. ► N-terminal palmitoylation determines its IMC localization. ► Incomplete palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. ► TgHSP20 palmitoylation is not responsible for its interaction with daughter cells IMCs.

Keywords: Toxoplasma gondii; Small heat shock protein 20; Palmitoylation; Inner membrane complex; Endodyogeny

Transcriptional activation of Odf2/Cenexin by cell cycle arrest and the stress activated signaling pathway (JNK pathway) by Nadin Pletz; Anja Medack; Riess Eva Maria Rieß; Kefei Yang; Zahra Basir Kazerouni; Huber Daniela Hüber; Sigrid Hoyer-Fender (pp. 1338-1346).

The centrosome/basal body protein ODF2/Cenexin is necessary for the formation of the primary cilium. Primary cilia are essential organelles that sense and transduce environmental signals. Primary cilia are therefore critical for embryonic and postnatal development as well as for tissue homeostasis in adulthood. Impaired function of primary cilia causes severe human diseases. ODF2 deficiency prevents formation of the primary cilium and is embryonically lethal. To explore the regulation of primary cilia formation we analyzed the promoter region of Odf2 and its transcriptional activity. In cycling cells, Odf2 transcription is depressed but becomes up-regulated in quiescent cells. Low transcriptional activity is mediated by sequences located upstream from the basal promoter, and neither transcription factors with predicted binding sites in the Odf2 promoter nor Rfx3 or Foxj, which are known to control ciliary gene expression, could activate Odf2 transcription. However, co-expression of either C/EBPα, c-Jun or c-Jun and its regulator MEKK1 enhances Odf2 transcription in cycling cells. Our results provide the first analysis of transcriptional regulation of a ciliary gene. Furthermore, we suggest that transcription of even more ciliary genes is largely inhibited in cycling cells but could be activated by cell cycle arrest and by the stress signaling JNK pathway.•It is the first analysis of transcriptional regulation of an essential ciliary gene.•Transcription of Odf2 is repressed in cycling cells.•Up-regulation of Odf2 in quiescent cells is coincident with cilia formation.•Transcription of Odf2 is activated by the stress signaling (JNK) pathway.

Keywords: ODF2/Cenexin; Primary cilium; Transcriptional regulation; G0-arrest; C/EBPα; Stress signaling pathway

Brg1-dependent epigenetic control of vascular smooth muscle cell proliferation by hydrogen sulfide by Li Li; Die Liu; Dingfang Bu; Stella Chen; Jianxin Wu; Chaoshu Tang; Junbao Du; Hongfang Jin (pp. 1347-1355).

Hydrogen sulfide (H₂S) can modulate the proliferation of vascular smooth muscle cells. This study was designed to investigate the epigenetic control of vascular smooth muscle cell proliferation in response to H₂S. Microarray analysis indicated that Brahma-related gene 1 ( Brg1) and proliferation-related genes including proliferating cell nuclear antigen ( Pcna), neurotrophin 3 ( Ntf3) and platelet-derived growth factor subunit A ( Pdgfα) were significantly downregulated by H₂S in endothelin-1-stimulated proliferative vascular smooth muscle cells. Brg1 is the central catalytic subunit of the SWI/SNF apparatus (an ATP-dependent chromatin remodeling complex). Overexpression and knockdown of Brg1 confirmed that Brg1 was crucial for H₂S-induced inhibition of vascular smooth muscle cell proliferation. A luciferase reporter assay, real-time PCR and Western blotting demonstrated that H₂S inhibited Brg1 transcription and expression. A DNase I hypersensitivity assay revealed that H₂S reversed endothelin-1-stimulated Pcna, Ntf3 and Pdgfα chromatin remodeling and vascular smooth muscle cell proliferation. A chromatin immunoprecipitation assay indicated that H₂S inhibited the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoters. The results of this study indicate that H₂S inhibits vascular smooth muscle cell proliferation via an epigenetic mechanism involving the inhibition of Brg1 transcription and expression, and by reducing the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoter regions.Display Omitted•We investigated the mechanisms by which H₂S inhibits VSMC proliferation.•H₂S downregulated Brg1 and the proliferation-related genes Pcna, Ntf3 and Pdgfα.•H₂S reversed ET-1-induced Pcna, Ntf3 and Pdgfα chromatin remodeling.•H₂S reduced the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoters.•H₂S inhibited VSMC proliferation via an epigenetic mechanism by repressing Brg1.

Keywords: Abbreviations; VSMCs; vascular smooth muscle cells; H; ₂; S; hydrogen sulfide; CSE; cystathionine γ-lyase; CBS; cystathionine β-synthase; MAPK; mitogen-activated protein kinase; Brg1; Brahma-related gene 1; Brm; Brahma; RNAi; RNA interference; RASMCs; rat aortic smooth muscle cells; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; CCK-8; cell counting kit-8; NaHS; sodium hydrosulfide; ET-1; endothelin-1; Pdgfα; platelet-derived growth factor subunit A; Ntf3; neurotrophin 3; Pcna; proliferating cell nuclear antigen; HSs; hypersensitive sites; ChIP; chromatin immunoprecipitation; Erk; extracellular signal-regulated kinase; SRF; serum response factor; MRTFA; myocardin-related transcription factor A Brg1; Epigenetic; Hydrogen sulfide; Vascular smooth muscle cells; Proliferation

Hsp90 inhibition by PU-H71 induces apoptosis through endoplasmic reticulum stress and mitochondrial pathway in cancer cells and overcomes the resistance conferred by Bcl-2 by Cindy Gallerne; Alexandre Prola; Christophe Lemaire (pp. 1356-1366).

Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment because of its role in stabilizing the active form of a wide range of client oncoproteins. This study investigated the mechanism of apoptosis induced by the purine-scaffold Hsp90 inhibitor PU-H71 in different human cancer cell lines and examined the role of Bcl-2 and Bax in this process. We demonstrated that Hsp90 inhibition by PU-H71 generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of Grp94, Grp78, ATF4 and CHOP. In response to PU-H71-induced ER stress, apoptosis was triggered in melanoma, cervix, colon, liver and lung cancer cells, but not in normal human fibroblasts. Apoptosis was executed through the mitochondrial pathway as shown by down-regulation of Bcl-2, up-regulation and activation of Bax, permeabilization of mitochondrial membranes, release of cytochrome c and activation of caspases. We also found that, in contrast to the ER stressor thapsigargin, PU-H71 induced apoptosis in cells overexpressing Bcl-2 and thus overcame the resistance conferred by this anti-apoptotic protein. In addition, although Bax deficiency rendered cells resistant to PU-H71, combined treatment with the anticancer drugs cisplatin or melphalan greatly sensitized these cells to PU-H71. Taken together, these data suggest that inhibition of Hsp90 by PU-H71 is a promising strategy for cancer treatment, particularly in the case of tumors resistant to conventional chemotherapy.► Hsp90 inhibition by PU-H71 induces ER-stress mediated apoptosis in human cancer cells. ► Apoptosis occurs through the mitochondrial pathway and requires Bax. ► PU-H71 exerts no toxicity toward healthy cells. ► PU-H71 overcomes Bcl-2 mediated resistance to apoptosis. ► Combination of PU-H71 and alkylating agents sensitizes resistant cells to apoptosis.

Keywords: Abbreviations; Cisp; cisplatin; ΔΨm; mitochondrial transmembrane potential; Hsp90; heat shock protein 90; IM; inner membrane; IP3R; inositol 1,4,5-triphosphate receptor; MLP; melphalan; MMP; mitochondrial membrane permeabilization; OM; outer membrane; PI; propidium iodide; PU-H71; 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine; RT; room temperature; TG; thapsigargin; UPR; Unfolded Protein Response; vMIA; viral mitochondrial inhibitor of apoptosisPU-H71; ER stress; Bcl-2; Bax; Mitochondrial apoptosis; Cancer

Platelet-derived growth factor negatively regulates the insulin-like growth factor signaling pathway through the coordinated action of phosphatidylinositol 3-kinase and protein kinase C beta I by Claudine Lassarre; Christine Legay; Manale Karam; Jean-Marc Ricort (pp. 1367-1377).

We recently described that epidermal and fibroblast growth factors (EGF and FGF) regulate the IGF-I signaling pathway at the level of IRS-1 through the cooperative action of two independent signaling pathways; one dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and the other on protein kinase D1 (PKD1) (Karam et al. [22]). To determine whether this mechanism could be generalized to another tyrosine kinase receptor-dependent growth factor, the effect of platelet-derived growth factor (PDGF) on the IGF-I signaling pathway was studied. PDGF inhibited IGF-I-stimulated IRS-1 tyrosine phosphorylation and subsequent IGF-I-induced PI 3-kinase activity, and stimulated IRS-1 serine 307 phosphorylation. These effects were mediated through a PI 3-kinase-dependent but extracellular signal-regulated kinase (ERK)-independent signaling pathway. However, PDGF-induced IRS-1 serine 307 phosphorylation was not sufficient per se to inhibit the IGF-I signaling but required another independent pathway. Noteworthy, although acutely stimulated by PDGF, and contrary to what we previously described (Karam et al. [22]), PKD1 did not associate with IRS-1and did not inhibit the IGF-I signaling in response to PDGF. However, we identified PKCβI as a new regulatory partner of PI 3-kinase for PDGF-induced inhibition of the IGF-I signaling pathway. Therefore, our results reinforce the idea that a coordinated action of two independent pathways seems absolutely necessary to negatively regulate IRS-1. Moreover, they also demonstrated that, depending of the cross-talk considered, subtle and specific regulatory mechanisms occur at the level of IRS-1 and that a unique regulatory model is not conceivable.► PI 3-kinase and PKCβI cooperate to inhibit the IGF-I pathway at the level of IRS-1 in response to PDGF. ► PKD1 is not involved in the regulation of the IGF-I signaling in response to PDGF. ► PI 3-kinase, but not PKCβI, induces the Ser307 phosphorylation of IRS-1 in response to PDGF. ► IRS-1 Ser307 phosphorylation is not sufficient per se to inhibit the IGF signaling. ► Down-regulation of the IGF-I signaling implicates different regulatory mechanisms depending on the cross-talk considered.

Keywords: Abbreviations; EGF; epidermal growth factor; ERK; extracellular signal-regulated kinase; FGF; fibroblast growth factor; IGF; insulin-like growth factor; IGF-IR; type 1 IGF receptor; IR; insulin receptor; IRS; insulin receptor substrate; PI 3-kinase; phosphatidylinositol 3-kinase; PKC; protein kinase C; PKD1; protein kinase D1Insulin-like growth factor; Signaling pathway cross-talk; Tyrosine kinase receptor dependent signaling pathway; Platelet-derived growth factor; Protein kinase C beta I

MgcRacGAP, a cytoskeleton regulator, inhibits HIF-1 transcriptional activity by blocking its dimerization by Aggeliki Lyberopoulou; Ilias Mylonis; George Papachristos; Dimitrios Sagris; Alkmini Kalousi; Christina Befani; Panagiotis Liakos; George Simos; Eleni Georgatsou (pp. 1378-1387).

Hypoxia inducible factor-1 (HIF-1), a dimeric transcription factor of the bHLH-PAS family, is comprised of HIF-1α, which is inducible by hypoxia and ARNT or HIF-1β, which is constitutively expressed. HIF-1 is involved in cellular homeostasis under hypoxia, in development and in several diseases affected by oxygen availability, particularly cancer. Since its expression is positively correlated with poor outcome prognosis for cancer patients, HIF-1 is a target for pharmaceutical therapy. We have previously shown that male germ cell Rac GTPase activating protein (MgcRacGAP), a regulator of Rho proteins which are principally involved in cytoskeletal organization, binds to HIF-1α and inhibits its transcriptional activity. In this work, we have explored the mechanism of the MgcRacGAP-mediated HIF-1 inactivation. We show that the Myo domain of MgcRacGAP, which is both necessary and sufficient for HIF-1 repression, binds to the PAS-B domain of HIF-1α. Furthermore MgcRacGAP competes with ARNT for binding to the HIF-1α PAS-B domain, as shown by in vitro binding pull down assays. In mammalian cells, ARNT overexpression can overcome the MgcRacGAP-mediated inhibition and MgcRacGAP binding to HIF-1α in vivo inhibits its dimerization with ARNT. We additionally present results indicating that MgcRacGAP binding to HIF-1α is specific, since it does not affect the transcriptional activity of HIF-2, a close evolutionary relative of HIF-1 also involved in hypoxia regulation and cancer. Our results reveal a new mechanism for HIF-1 transcriptional activity regulation, suggest a novel hypoxia-cytoskeleton link and provide new tools for selective HIF-1 inhibition.•The Myo domain of MgcRacGAP is necessary and sufficient for HIF-1 activity inhibition.•ARNT overexpression relieves MgcRacGAP-mediated HIF-1 repression.•MgcRacGAP competes with ARNT for HIF-1α binding in vitro.•MgcRacGAP competes with ARNT for HIF-1α binding in vivo.•MgcRacGAP inhibits HIF-1 (but not HIF-2) activity by interfering with its dimerization.

Keywords: Abbreviations; HIF-1; hypoxia inducible factor 1; ARNT; aryl hydrocarbon receptor nuclear translocator; MgcRacGAP; male germ cell Rac GTPase activating protein; bHLH; basic helix–loop–helix; PAS; Per-ARNT-Sim; ODD; oxygen dependent degradation; pVHL; von Hippel–Lindau protein; PHDs; prolyl hydroxylases; HREs; hypoxia responsive elements; AhR; aryl hydrocarbon receptor; ROS; reactive oxygen species; STAT3; signal transducer and activator of transcription 3; MKLP1; mitotic kinesin-like protein 1; TACC3; transforming acidic coiled-coil protein 3; Ect2; epithelial cell-transforming sequence 2; CK1; casein kinase 1; Ainp1; ARNT interacting protein 1; RACK1; receptor of activated protein kinase C 1; Hsp90; heat shock protein 90HIF-1α; HIF-2; MgcRacGAP; PAS-B; Hypoxia; Cytoskeleton

Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action by Antonella Marucci; Flora Cozzolino; Claudia Dimatteo; Maria Monti; Piero Pucci; Vincenzo Trischitta; Rosa Di Paola (pp. 1388-1395).

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3′untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3′UTR binding proteins, and investigate their role on insulin signaling.By RNA pull-down, 49 proteins bound to ENPP1-3′UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA. GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase ( PEPCK) expression, a key neoglucogenetic enzyme.Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cells.•ENPP1 inhibits insulin signaling and action.• ENPP1-3′UTR, has a role in controlling ENPP1 expression.•GALNT2 binds ENPP1-3′UTR in HepG2 cells.• GALNT2 down-regulation increases ENPP1 expression and reduces insulin signaling.•GALNT2 is a novel factor which modulates insulin action in human liver cells.

Keywords: Abbreviations; ENPP1; ectonucleotide pyrophosphatase phosphodiesterase 1; UTR; untranslated region; GALNT2; N-acetylgalactosaminyltransferase 2 gene; IR; insulin receptor; PEPCK; phosphoenolpyruvate carboxykinase; GWAS; genome wide association studies; HEK293; human embryo kidney; HepG2; human hepatoma cell line; GAPDH; glyceraldehyde 3-phosphate dehydrogenase3′UTR binding proteins; GALNT2; ENPP1; Insulin signaling

Low electric fields induce ligand-independent activation of EGF receptor and ERK via electrochemical elevation of H⁺ and ROS concentrations by Tami Wolf-Goldberg; Alexander Barbul; Nadav Ben-Dov; Rafi Korenstein (pp. 1396-1408).

Physiological electric fields are involved in many biological processes and known to elicit their effects during long exposures ranging from a few hours to days. Following exposure to electric fields of physiological amplitude, epidermal growth factor receptor (EGFR) was demonstrated to be redistributed and upregulated with further intracellular signaling such as the MAPK signaling cascade. In our study we demonstrated EGFR activation and signaling induced by short train of pulsed low electric field (LEF) (10V/cm, pulse-width 180μs, 500Hz, 2min) in serum-free medium, following 24-hour starvation, and in the absence of exogenous EGF ligand, suggesting a ligand-independent pathway for EGFR activation. This ligandless activation was further confirmed by using neutralizing antibodies (LA1) that block the EGFR ligand-binding site. EGFR activation was found to be EGFR kinase dependent, yet with no dimerization following exposure to LEF. ERK activation was found to be mainly a result of EGFR downstream signaling though it partially occurred via EGFR-independent way. We demonstrate that reactive oxygen species and especially decrease in pH generated during exposure to LEF are involved in EGFR ligandless activation. We propose a possible mechanism for the LEF-induced EGFR ligand-independent activation and show activation of other receptor tyrosine kinases following exposure to LEF.► Low electric field (LEF) is involved in biological processes affecting EGFR signaling. ► Ligand-independent EGFR activation with subsequent downstream signaling is induced following cell exposure to LEF. ► LEF exert its effects via electrochemical reactions, mostly pH decrease. ► Short exposure to LEF or low pH may significantly affect cell processes and lay down a basis for therapeutic treatments.

Keywords: Abbreviations; EF(s); Electric field(s); LEF; Low electric fields; ROS; Reactive oxygen species; H; ₂; DCF-DA; Dichlorodihydrofluorescein diacetate; DHA; Dehydroascorbic acid; RTK; Receptor tyrosine kinase; PTPs; Protein tyrosine phosphatases; DC; Direct current; FITC; Fluorescein isothiocyanate; HBSS; Hank's buffered salt solution; BSA; Bovine serum albumin; TBHP; tert; -Butyl hydroperoxide; BS; ³; Bis(sulfosuccinimidyl) suberate; DMEM; Dulbecco's modified Eagles medium; FCS; Fetal calf serum; PI; Propidium iodide; HRP; Horseradish peroxidase; PS; Phospholipid phosphatidylserine; PIP; ₂; Phosphatidylinositol 4,5-bisphosphate; MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideElectric field; EGFR; ERK; Electrochemical product; pH; ROS

Thy-1-mediated cell–cell contact induces astrocyte migration through the engagement of α_Vβ₃ integrin and syndecan-4 by Milene Kong; Munoz Nicolás Muñoz; Alejandra Valdivia; Alvaro Alvarez; Rodrigo Herrera-Molina; Cardenas Areli Cárdenas; Pascal Schneider; Keith Burridge; Andrew F.G. Quest; Lisette Leyton (pp. 1409-1420).

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron–astrocyte model, we have reported that the neuronal protein Thy-1 engages α_Vβ₃ integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell–cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell–cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.Display Omitted► Strong cell adhesion is opposite to cell migration. ► Neuronal Thy-1 engages integrin and syndecan-4 in astrocytes to greatly increase their adhesion. ► Thy-1 stimulation for 1h activates Rac1 and inactivates RhoA. ► Extended Thy-1 stimulation period triggers the activation of FAK-PI3K-Rac1 signaling pathway. ► Neuron–astrocyte binding dorsally engages receptors and signaling molecules to promote cell motility over a substratum.

Keywords: Thy-1; Integrin; Syndecan-4; Neuron–astrocyte interaction; Cell migration

Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons by Levent Kaya; Barbara Meissner; Maria Christine Riedl; Martin Muik; Christoph Schwarzer; Francesco Ferraguti; Bettina Sarg; Herbert Lindner; Rüdiger Schweigreiter; Hans-Günther Knaus; Christoph Romanin; Christine E. Bandtlow (pp. 1421-1433).

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [³H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2⁺ oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2⁺ dynamics the in neurons.•The reticulon protein RTN1A is an ER-resident protein of unknown function.•We identified the intracellular calcium release channel ryanodine receptor RyR2 as a specific binding partner of RTN1A.•RNT1A co-immunoprecipitates and colocalizes with the ryanodine receptor RyR2 in neurons via its N-terminal domain.•Binding of RTN1A modulates the activity of RyR2.•RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2⁺ dynamics

Keywords: Abbreviations; RTN; reticulon protein; ER; endoplasmic reticulum; RyR; ryanodine receptor; RHD; reticulon homology domain; CICR; calcium induced calcium release; IPTG; isopropyl-β-d-thiogalactopyranoside; PB; phosphate buffer; DAB; 3,3′-diaminobenzidine tetrahydrochloride dihydrate; CA; cornu ammonisReticulon; Brain; Protein–protein interaction; Calcium homeostasis

High glucose induces podocyte apoptosis by stimulating TRPC6 via elevation of reactive oxygen species by Bing-Chen Liu; Xiang Song; Xiao-Yu Lu; Daniel T. Li; Douglas C. Eaton; Bao-Zhong Shen; Xue-Qi Li; He-Ping Ma (pp. 1434-1442).

Podocyte number is significantly reduced in diabetic patients and animal models, but the mechanism remains unclear. In the present study, we found that high glucose induced apoptosis in control podocytes which express transient receptor potential canonical 6 (TRPC6) channels, but not in TRPC6 knockdown podocytes in which TRPC6 was knocked down by TRPC6 silencing short hairpin RNA (shRNA). This effect was reproduced by treatment of podocytes with the reactive oxygen species (ROS), hydrogen peroxide (H₂O₂). Single-channel data from cell-attached, patch-clamp experiments showed that both high glucose and H₂O₂ activated the TRPC6 channel in control podocytes, but not in TRPC6 knockdown podocytes. Confocal microscopy showed that high glucose elevated ROS in podocytes and that H₂O₂ reduced the membrane potential of podocytes and elevated intracellular Ca2⁺ via activation of TRPC6. Since intracellular Ca2⁺ overload induces apoptosis, H₂O₂-induced apoptosis may result from TRPC6-mediated elevation of intracellular Ca2⁺. These data together suggest that high glucose induces apoptosis in podocytes by stimulating TRPC6 via elevation of ROS.•High glucose-induced apoptosis is blocked by a generic TRPC channel blocker.•High glucose-induced apoptosis is attenuated in TRPC6 knockdown podocytes.•High glucose activates TRPC6 channels and causes oxidative stress in podocytes.•H2O2 mimics the effects of high glucose on apoptosis and TRPC6 channel activity.•H2O2 depolarizes the membrane potential and elevates intracellular Ca2+ via TRPC6.

Keywords: ROS; Patch-clamp technique; Confocal microscopy; Hydrogen peroxide; Oxidative stress; Intracellular Ca; 2; ⁺

HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation by Francesca Siepi; Veronica Gatti; Serena Camerini; Marco Crescenzi; Silvia Soddu (pp. 1443-1453).

HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354.► HIPK2 is catalytically activated by activation-loop Y354 autophosphorylation. ► Inhibition of HIPK2-Y354 phosphorylation results in out-of-target Y kinase activity. ► Out-of-target Y kinase activity delocalizes HIPK2 into cytoplasmic aggresomes. ► HIPK2 cytoplasmic aggresomes are reminiscent of similar observations in human cancers.

Keywords: Abbreviations; e.v.; empty vector; IF; immunofluorescence; IP; immunoprecipitation; IVT; in vitro; translation; moAb; monoclonal antibody; purvA; purvalanol A; TCE; total cell extracts; WB; Western blottingHIPK2; Posttranslational modification; Phosphorylation; Activation-loop Y; Subcellular localization; DYRK

Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelial–mesenchymal transition in renal tubular cells by Wei-Qing Han; Qing Zhu; Junping Hu; Pin-Lan Li; Fan Zhang; Ningjun Li (pp. 1454-1462).

Transforming growth factor beta 1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGF-β1-induced signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGF-β1-induced EMT using cultured renal tubular cells. Our results showed that TGF-β1 stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker P-cadherin, and the increase in mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Meanwhile, we found that TGF-β1 time-dependently increased HIF-1α and that HIF-1α siRNA significantly inhibited TGF-β1-induced EMT, suggesting that HIF-1α mediated TGF-β1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1:PHD2=0.21:1.0). Importantly, PHD2 mRNA and protein, but not PHD1, were decreased by TGF-β1. Furthermore, over-expression of PHD2 transgene almost fully prevented TGF-β1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGF-β1-induced EMT. Finally, Smad2/3 inhibitor SB431542 prevented TGF-β1-induced PHD2 decrease, suggesting that Smad2/3 may mediate TGF-β1-induced EMT through PHD2/HIF-1α pathway. It is concluded that TGF-β1 decreased PHD2 expression via an Smad-dependent signaling pathway, thereby leading to HIF-1α accumulation and then EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGF-β1, and may be a new therapeutic target in chronic kidney diseases.•HIF-1α siRNA blocked TGF-1β-induced EMT in renal proximal tubular cells.•The mRNA and protein levels of PHD2, an enzyme that promotes the HIF-1α degradation, were decreased by TGF-β1.•Over-expression of PHD2 transgene reduced HIF-1α levels and also blocked TGF-β1-induced EMT.•Smad2/3 inhibitor prevented TGF-β1-induced PHD2 reduction.•TGF-β1-induced EMT is mediated by PHD2/HIF-1α through Smad2/3 pathway in renal proximal tubular cells.

Keywords: Abbreviations; α-SMA; α-smooth muscle actin; EMT; epithelial-to-mesenchymal transition; FSP-1; fibroblast-specific protein 1; HIF-1α; hypoxia-inducible factor 1α; PHD; prolyl hydroxylase domain; TFIID; transcription factor II D; TGF-β1; transforming growth factor beta 1Prolyl hydroxylase domain-containing protein; Smad signaling pathway; Renal fibrosis

β-Estradiol-dependent activation of the JAK/STAT pathway requires p/CIP and CARM1 by N. Coughlan; G. Thillainadesan; J. Andrews; M. Isovic; J. Torchia (pp. 1463-1475).

The steroid receptor coactivator p/CIP, also known as SRC-3, is an oncogene commonly amplified in breast and ovarian cancers. p/CIP is known to associate with coactivator arginine methyltransferase 1 (CARM1) on select estrogen responsive genes. We have shown, using a ChIP-on-chip approach, that in response to stimulation with 17β-estradiol (E2), the p/CIP/CARM1 complex is recruited to 204 proximal promoters in MCF-7 cells. Many of the complex target genes have been previously implicated in signaling pathways related to oncogenesis. Jak2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the Jak2 promoter are reflective of a transcriptionally permissive gene, and modest changes in RNA and protein expression lead us to suggest that an additional factor(s) may be required for a more notable transcriptional and functional response. Bioinformatic examination of the 204 proximal promoter sequences of p/CIP/CARM1 targets supports the idea that transcription factor crosstalk is likely the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. This data may have implications towards understanding the oncogenic role of p/CIP in breast cancer and ultimately allow for the identification of new prognostic indicators and/or viable therapeutic targets.► p/CIP/CARM1 complex binds 204 proximal promoters in response to E2 in MCF-7 cells. ► Many p/CIP/CARM1 complex target genes have been implicated in cancer. ► 20% of target genes are transcriptionally altered following 12h E2-stimulation ► Jak2 is a p/CIP/CARM1 complex target gene that is transcriptionally activated by E2 ► Chromatin modifications at Jak2 promoter support transcriptional permissiveness

Keywords: Cancer; Estrogen; pCIP/CARM1; Coactivator; Chromatin

Inhibition of c-Jun NH₂-terminal kinase stimulates mu opioid receptor expression via p38 MAPK-mediated nuclear NF-κB activation in neuronal and non-neuronal cells by Yadav Wagley; Cheol Kyu Hwang; Hong-Yiou Lin; Angel F.Y. Kam; Ping-Yee Law; Horace H. Loh; Li-Na Wei (pp. 1476-1488).

Despite its potential side effects of addiction, tolerance and withdrawal symptoms, morphine is widely used for reducing moderate and severe pain. Previous studies have shown that the analgesic effect of morphine depends on mu opioid receptor (MOR) expression levels, but the regulatory mechanism of MOR is not yet fully understood. Several in vivo and in vitro studies have shown that the c-Jun NH₂-terminal kinase (JNK) pathway is closely associated with neuropathic hyperalgesia, which closely resembles the neuroplastic changes observed with morphine antinociceptive tolerance. In this study, we show that inhibition of JNK by SP600125, its inhibitory peptide, or JNK-1 siRNA induced MOR at both mRNA and protein levels in neuronal cells. This increase in MOR expression was reversed by inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway, but not by inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway. Further experiments using cell signaling inhibitors showed that MOR upregulation by JNK inhibition involved nuclear factor-kappa B (NF-κB). The p38 MAPK dependent phosphorylation of p65 NF-κB subunit in the nucleus was increased by SP600125 treatment. We also observed by chromatin immunoprecipitation (ChIP) analysis that JNK inhibition led to increased bindings of CBP and histone-3 dimethyl K4, and decreased bindings of HDAC-2, MeCP2, and histone-3 trimethyl K9 to the MOR promoter indicating a transcriptional regulation of MOR by JNK inhibition. All these results suggest a regulatory role of the p38 MAPK and NF-κB pathways in MOR gene expression and aid to our better understanding of the MOR gene regulation.•JNK inhibition by SP600125 increases the expression of mu opioid receptor.•SP600125 increases the phosphorylation of p38 MAPK.•p38 MAPK mediates the increase in nuclear phosphorylation of p65 induced by SP600125.•siRNA-directed knock-down of p65 decreases MOR expression induced by SP600125.•NF-κB regulates MOR gene expression in neuronally differentiating P19 cells.

Keywords: Abbreviations; MOR; mu opioid receptor; JNK; c-Jun NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein/extracellular signal-regulated kinase; NF-κB; nuclear factor-kappaB; DP; distal promoter; PP; proximal promoter; Oct-1; octamer-1; SOX; sry-related high-mobility-group box; PCBP; poly(C) binding protein; SP1; specificity protein 1; AP2; activator protein 2; CREB; cAMP response element binding protein; SAPK; stress-activated protein kinase; TNF-α; tumor necrosis factor-alpha; IFN-γ; interferon-gamma; DRG; dorsal root ganglion; SP600125; anthra(1,9-cd)pyrazol-6(2H)-one; QNZ; 6-amino-4-(4-henoxyphenylethylamino)quinazoline; SB203580; 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; LY294002; 2,(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; U0126; 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene; PDTC; pyrrolidine dithiocarbonate; PI3-K; phosphoinositide 3-kinase; RT-PCR; reverse transcription-polymerase chain reaction; qRT-PCR; quantitative reverse transcription-polymerase chain reaction; MeCP2; methyl-CpG-binding protein 2; H3dmK4; histone 3-dimethyl lysine 4; H3me3K9; histone 3-trimethyl lysine 9; HDAC; histone deacetylase; aceH; acetyl-histone; Brg1; Brm-related gene 1; NRSF; neuron-restrictive silencing factor; Dnmt1; DNA methyltransferasemu opioid receptor; JNK; MAPK; NF-κB

DDX3 regulates DNA damage-induced apoptosis and p53 stabilization by Mianen Sun; Tong Zhou; Eric Jonasch; Richard S. Jope (pp. 1489-1497).

The DEAD box protein family member DDX3 was previously identified as an inhibitor of death receptor-mediated extrinsic apoptotic signaling. However, there had been no studies of the role of DDX3 in regulating the other major type of apoptosis, intrinsic apoptotic signaling, which was examined here. Intrinsic apoptosis was induced in MCF-7 cells by treatment with staurosporine, a general kinase inhibitor, thapsigargin, which induces endoplasmic reticulum (ER) stress, and camptothecin, which causes DNA damage. Each of these treatments caused time-dependent activation of caspase-7, the predominant executioner caspase in these cells. Depletion of DDX3 using shRNA did not alter apoptotic responses to staurosporine or thapsigargin. However, caspase-7 activation induced by camptothecin was regulated by DDX3 in a manner dependent on the functional status of p53. Depletion of DDX3 abrogated camptothecin-induced caspase-7 activation in MCF-7 cells expressing functional wild-type p53, but oppositely potentiated camptothecin-mediated caspase activation in cells expressing mutant or non-functional p53, which was accompanied by increased activation of the extrinsic apoptotic signaling initiator caspase-8. In MCF-7 cells, depletion of DDX3 reduced by more than 50% camptothecin-induced p53 accumulation, and this effect was blocked by inhibition of the proteasome with MG132, indicating that DDX3 regulates p53 not at expression level but rather its stabilization after DNA damage. Co-immunoprecipitation experiments demonstrated that DDX3 associates with p53, and overexpression of DDX3 was sufficient to double the accumulation of p53 in the nucleus after DNA damage. Thus, DDX3 associates with p53, increases p53 accumulation, and positively regulates camptothecin-induced apoptotic signaling in cells expressing functional wild-type p53, whereas in cells expressing mutant or non-functional p53 DDX3 inhibits activation of the extrinsic apoptotic pathway to reduce caspase activation. These results demonstrate that DDX3 not only regulates extrinsic apoptotic signaling, as previously reported, but also selectively regulates intrinsic apoptotic signaling following DNA damage.•DDX3 promotes camptothecin-induced apoptosis in cells expressing functional wild-type p53.•DDX3 impedes apoptosis in cells with nonfunctional p53.•DDX3 associates with p53.•DDX3 promotes nuclear functional wild-type p53 accumulation and activates p21 expression.

Keywords: DDX3; p53; Apoptosis; DNA damage; Camptothecin

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock by Jérôme Lugrin; Eleonora Ciarlo; Alba Santos; Gaël Grandmaison; Isis Dos Santos; Didier Le Roy; Thierry Roger (pp. 1498-1510).

Sirtuins (SIRT1-7) are NAD⁺-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.•The sirtuin inhibitor cambinol inhibits inflammatory and immune responses.•Cambinol impairs MAPK signaling.•Cambinol protects from sepsis shock.•Cambinol-like drugs may be of clinical interest to treat inflammatory diseases.

Keywords: Abbreviations; BMDCs; bone marrow-derived dendritic cells; BMDMs; bone marrow-derived macrophages; CpG; CpG motif containing oligonucleotide 1826; DC; dendritic cell; E. coli; Escherichia coli; O18; HDAC; histone deacetylase; LPS; lipopolysaccharide; MKP; MAPK phosphatase; K. pneumonia; Klebsiella pneumoniae; Caroli; Pam; ₃; CSK; ₄; N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine; S. aureus; Staphylococcus aureus; AW7; SIRT; sirtuinSirtuin; Innate immunity; Macrophage; Cambinol; LPS; Sepsis

A novel snRNA-like transcript affects amyloidogenesis and cell cycle progression through perturbation of Fe65L1 (APBB2) alternative splicing by Ilaria Penna; Irene Vassallo; Mario Nizzari; Debora Russo; Delfina Costa; Paola Menichini; Alessandro Poggi; Claudio Russo; Giorgio Dieci; Tullio Florio; Ranieri Cancedda; Aldo Pagano (pp. 1511-1526).

FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein and the consequent amyloid β production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease etiology. However, FE65 and FE65-like proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aβ production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons.•The expression of 45A, a novel non-coding RNA, leads to APBB2 alternative splicing.•Differential APBB2 isoform expression modulates APP processing and cell cycle control.•Overexpression of 45A also leads to neuroblastoma cell tumorigenesis.•We report a novel pathway linking APP processing and cell cycle regulation.

Keywords: APBB2 alternative splicing; Cell cycle progression non-coding RNA; RNA polymerase III; Neurodegeneration; β amyloid

Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism by Darius J.R. Lane; Sherin Chikhani; Vera Richardson; Des R. Richardson (pp. 1527-1541).

Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased⁵⁹Fe uptake from transferrin by 1.5–2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased⁵⁹Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated⁵⁹Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of⁵⁹Fe uptake from⁵⁹Fe-citrate, only intracellular ascorbate is needed for transferrin⁵⁹Fe uptake. Additionally, experiments withl-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent⁵⁹Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or¹²⁵I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.► The role of ascorbate in transferrin iron (Fe-Tf) uptake is unknown. ► Ascorbate stimulates Fe-Tf uptake by a novel intracellular reductive mechanism. ► This mechanism is different to ascorbate's role in stimulating non-Tf Fe uptake. ► The reductive mechanism involved requires the classical transferrin cycle. ► These results are important since cultured cells are typically devoid of ascorbate.

Keywords: Abbreviations; BCA; bicinchoninic acid; BPS; bathophenanthroline disulfonate; BSA; bovine serum albumin; CHO; Chinese hamster ovary; CHX; cycloheximide; Dcytb; duodenal cytochrome; b; DHA; dehydroascorbate; DFO; desferrioxamine; DMT1; divalent metal transporter, isoform 1; Fe; ₂; -Tf; diferric transferrin; FTH1; ferritin heavy chain; FTL; ferritin light chain; GLUT; facilitative glucose transporter; GSH; reduced glutathione; HUVECs; human umbilical vein endothelial cells; IRE; iron-responsive element; IRP; iron-regulatory protein; LIP; labile iron pool; FeTMPyP; Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; MnTBAP; Mn(III)tetrakis(4-benzoic acid) porphyrin chloride; MnTMPyP; Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; PHE; o; -phenanthroline; Steap3; six transmembrane epithelial antigen of the prostate, isoform 3; SVCT; sodium-dependent ascorbate transporter; Tf; transferrin; TfR1; transferrin receptor 1; TfR2; transferrin receptor 2; V-ATPase; vacuolar-type H; ⁺; -ATPaseTransferrin; Transferrin receptor; Iron; Iron metabolism; Ascorbate; Ascorbic acid

CD69 overexpression by human T-cell leukemia virus type 1 Tax transactivation by Chie Ishikawa; Hirochika Kawakami; Jun-Nosuke Uchihara; Masachika Senba; Naoki Mori (pp. 1542-1552).

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with the development of adult T-cell leukemia (ATL) and various inflammatory diseases. CD69 is a marker of early activation of lymphocytes. We investigated the effects of HTLV-1 infection on the expression of CD69. The CD69 gene was upregulated in all viral protein Tax-expressing HTLV-1-transformed T-cell lines, except MT-2 and peripheral blood mononuclear cells from patients with ATL compared with uninfected T-cell line, Tax-negative ATL-derived T-cell lines and normal peripheral blood mononuclear cells. Flow cytometric analysis and immunohistochemical analysis confirmed the enhanced expression of CD69 in HTLV-1-transformed T-cell lines and in ATL cells in lymph nodes and skin lesions, and its absence in MT-2 and peripheral blood mononuclear cells. CD69 expression was induced following infection of human T-cell line with HTLV-1, and specifically by Tax. Tax transcriptionally activated CD69 gene through both nuclear factor-κB and cyclic adenosine 3′,5′-monophosphate response element-binding protein signaling pathways. Detailed analysis of the CD69 promoter indicated that the Tax-induced expression of CD69 was regulated by multiple cis-acting elements and by the interplay of transcription factors of the nuclear factor-κB, early growth response and cyclic adenosine 3′,5′-monophosphate response element-binding protein families. The lack of CD69 expression in MT-2 is due to epigenetic mechanism involving deacetylation, but not methylation. We conclude that CD69 is a Tax-regulated gene, and its regulation by Tax may play a role in cellular activation and HTLV-1-induced disease pathogenesis.•Upregulation of CD69 gene in HTLV-1-transformed T-cell lines and ATL cells•Presence of CD69-positive ATL cells in lymph nodes and skin lesions•Viral Tax protein transcriptionally activated CD69 promoter through NF-κB and CREB.•Tax-induced expression of CD69 is regulated by multiple cis-acting elements.

Keywords: Abbreviations; ATL; adult T-cell leukemia; 5-Aza; 5-azacytidine; CREB; cyclic adenosine 3′,5′-monophosphate response element-binding protein; Egr; early growth response; EMSA; electrophoretic mobility shift assay; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HTLV-1; human T-cell leukemia virus type 1; IKK; IκB kinase; IL; interleukin; IL-2Rα; IL-2 receptor α chain; NF-κB; nuclear factor-κB; PBMC; peripheral blood mononuclear cell; PE; phycoerythrin; PHA; phytohemagglutinin; RT-PCR; reverse transcription-PCR; SAHA; suberoylanilide hydroxamic acidCD69; HTLV-1; Tax; ATL; NF-κB; Deacetylation

The C-terminal domain of A1/Bfl-1 regulates its anti-inflammatory function in human endothelial cells by Renata P. Guedes; Eduardo Rocha; Jerome Mahiou; Herwig P. Moll; Maria B. Arvelo; Janis M. Taube; Clayton R. Peterson; Elzbieta Kaczmarek; Christopher R. Longo; Cleide G. da Silva; Christiane Ferran (pp. 1553-1561).

A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.•Deletion of the amphipathic C-terminal domain of A1/Bfl1 (A1ΔC) is performed.•A1ΔC frees A1 from mitochondrial anchorage.•Both full-length A1 and A1ΔC are cytoprotective in human endothelial cells.•rAd. mediated overexpression of A1ΔC but not A1 inhibits NF-κB in human EC.

Keywords: Abbreviations; A1∆BH4; Deletion of the BH4 domain; A1∆C; mutant lacking the C-terminal domain of A1; A1∆∆; Deletion of the C-terminal and BH4 domains; ANOVA; analysis of variance; BAEC; bovine aortic EC; Bfl-1S; short form of A1/Bfl-1; BH; Bcl-2 homology domains; CHX; cycloheximide; EBM-2; endothelial basal medium-2; EC; endothelial cells; ECL; enhanced chemiluminescence kit; FACS; fluorescence-activated cell sorting; HA; hemagglutinin A; HDMEC; dermal microvascular EC; HEK 293; Human embryonic 293 kidney cell line; HUVEC; human umbilical vein EC; ICAM-1; intercellular adhesion molecule-1; IHC; immunohistochemistry; MOI; multiplicity of infection; NS; non-stimulated; NT; non-transduced; P; probability error; pAC.A1∆BH4; plasmid encoding A1 mutant with deletion of the BH4 domain; pAC.A1∆C; plasmid encoding A1 mutant with deletion of the C-terminal domain; pAC; pAC.CMV-pLpASR; +; plasmid; pCA.A1∆∆; plasmid encoding A1 mutant with deletion of the C-terminal and BH4 domains; rAd.A1; Recombinant adenovirus human A1; rAd.A1∆C; Recombinant adenovirus A1∆C; rAd.βgal; recombinant adenovirus β-galactosidase; RLU; relative light units; RPTEC; renal proximal tubular epithelial cells; RSV; Rous Sarcoma Virus; RSV-βgal; Rous Sarcoma Virus-β-galactosidase; RT-PCR; reverse transcription polymerase chain reaction; SEM; standard error of mean; STS; staurosporine; TNF; tumor necrosis factor; VEGF; Vascular Endothelial Growth Factor; WB; Western blotA1/Bfl-1; Mitochondria; Nuclear Factor Kappa-B; Apoptosis; C-terminal domain

Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP) by Kathleen Coughlan; Xiangping Huang; Xiangyuan He; Charlotte H.Y. Chung; Guangpu Li; Jordan Tang (pp. 1562-1571).

Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t_1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.•We developed a double labeled (GFP and DsRed) truncated APP construct.•The modified APP retains similar attributes as full length APP.•A novel technique for assessing APP processing with live cell imaging was established.•Kinetics for APP processing by Memapsin 2 was established.•A unique pool of secreted APP located intracellularly was observed.

Keywords: Abbreviations; APP106; truncated last 106 amino acids of APP; GappR; GFP-APP106-DsRed; RappG; DsRed-APP106-GFP; sGapp; soluble GFP-APP; sRapp; soluble DsRed-APP; sGappα; soluble GFP-APP alpha; sGappβ; soluble GFP-APP betaAmyloid precursor protein; Amyloid-beta; Green fluorescence protein; Beta-secretase; Gamma-secretase

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BBA - Molecular Cell Research (v.1833, #6)