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BBA - Molecular Cell Research (v.1823, #2)

Editorial Board (pp. i).

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2 by Kyung D. Moon; Xiaoying Zhang; Qing Zhou; Robert L. Geahlen (pp. 199-205).

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.► Syk binds tensin2 in a yeast two-hybrid assay. ► Syk binds the C-terminal SH2 and PTB domains of tensin2. ► Syk does not bind tensin1. ► Syk localizes in cells with tensin2 in cytoplasmic structures. ► Syk clustered by binding tensin2 is activated.

Keywords: Syk; Tensin2; Tyrosine phosphorylation; Signal transduction

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2 by Kyung D. Moon; Xiaoying Zhang; Qing Zhou; Robert L. Geahlen (pp. 199-205).

Keywords: Syk; Tensin2; Tyrosine phosphorylation; Signal transduction

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2 by Kyung D. Moon; Xiaoying Zhang; Qing Zhou; Robert L. Geahlen (pp. 199-205).

Keywords: Syk; Tensin2; Tyrosine phosphorylation; Signal transduction

The SH2-domain of SHIP1 interacts with the SHIP1 C-terminus: Impact on SHIP1/Ig-α interaction by Oindrilla Mukherjee; Lars Weingarten; Inken Padberg; Catrin Pracht; Rileen Sinha; Hochdorfer Thomas Hochdörfer; Stephan Kuppig; Rolf Backofen; Michael Reth; Michael Huber (pp. 206-214).

The SH2-containing inositol 5′-phosphatase, SHIP1, negatively regulates signal transduction from the B cell antigen receptor (BCR). The mode of coupling between SHIP1 and the BCR has not been elucidated so far. In comparison to wild-type cells, B cells expressing a mutant IgD- or IgM-BCR containing a C-terminally truncated Ig-α respond to pervanadate stimulation with markedly reduced tyrosine phosphorylation of SHIP1 and augmented activation of protein kinase B. This indicates that SHIP1 is capable of interacting with the C-terminus of Ig-α. Employing a system of fluorescence resonance energy transfer in S2 cells, we can clearly demonstrate interaction between the SH2-domain of SHIP1 and Ig-α. Furthermore, a fluorescently labeled SH2-domain of SHIP1 translocates to the plasma membrane in an Ig-α-dependent manner. Interestingly, whereas the SHIP1 SH2-domain can be pulled-down with phospho-peptides corresponding to the immunoreceptor tyrosine-based activation motif (ITAM) of Ig-α from detergent lysates, no interaction between full-length SHIP1 and the phosphorylated Ig-α ITAM can be observed. Further studies show that the SH2-domain of SHIP1 can bind to the C-terminus of the SHIP1 molecule, most probably by inter- as well as intra-molecular means, and that this interaction regulates the association between different forms of SHIP1 and Ig-α.► SHIP1 is a crucial negative regulator of B cell antigen receptor signal transduction. ► The SH2-domain of SHIP1 binds to the phospho-ITAM of Ig-α in vitro and in vivo. ► The SH2-domain of SHIP1 binds to the phospho-Y1020 in its C-terminus. ► Thus, SHIP1 is able to dimerize and potentially form larger oligomers. ► This intermolecular interaction of SHIP1 competes against SHIP1/Ig-α interaction.

Keywords: BCR/SHIP1/Syk/tyrosine phosphorylation; Interaction

Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry by Miho Suzuki; Satoshi Tanaka; Yoichiro Ito; Makiko Inoue; Takafumi Sakai; Koichi Nishigaki (pp. 215-226).

Sensing systems based on Förster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein–protein interactions, changes in conformation, and Ca²⁺ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.► We developed color variable FRET-based bioprobes to monitor caspase-3 activity. ► The bioprobes were demonstrated to function as sensor molecules inside cells for detecting the target enzyme activity by flow cytometric analysis. ► Sensor molecules like that will be available to study dynamics of cellar events together with other fluorescent indices.

Keywords: Abbreviations; DTT; dithiothreitol; DsRed; Discosoma; red fluorescent protein; FACS; fluorescence-activated cell sorting; FLIM; fluorescence lifetime imaging; FRET; Förster resonance energy transfer; GFP; green fluorescent protein; Qdot; quantum dot; PBS; phosphate-buffered saline; RFP; red fluorescent proteinApoptosis; Caspase-3; Flow cytometry; Förster resonance energy transfer; Green fluorescent protein; Paracrine signaling

Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: Implications for cellular Aβ metabolism by María C. Leal; Ezequiel I. Surace; María P. Holgado; Carina C. Ferrari; Rodolfo Tarelli; Fernando Pitossi; Thomas Wisniewski; Castano Eduardo M. Castaño; Laura Morelli (pp. 227-235).

Cerebral amyloid β (Aβ) accumulation is pathogenically associated with sporadic Alzheimer's disease (SAD). BACE-1 is involved in Aβ generation while insulin-degrading enzyme (IDE) partakes in Aβ proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aβ accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at −379/−372 and −310−303 from the first translation start site in the −575/−19 (556bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aβ metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD.► We determined IDE transcript levels in human brain and cell cultures over-expressing NICD, HES-1 or Hey-1 cDNAs. ► We detected extracellular Aβ accumulation in N2aSW cells after NICD or Hey-1 transfection. ► HES-1 and Hey-1 bind to the consensus domain located at position −310/−305 of human IDE promoter. ► Site-directed mutagenesis of the −310/−305 binding site reverts the inhibition of IDE transcription ► Notch signaling exerts an inhibitory effect on IDE transcription in vitro (N2aSW cells) and in vivo (Tg2576 mice).

Keywords: Amyloid β metabolism; Notch signaling; HES; Hey; Transcriptional repressors; IDE gene promoter

Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants by Ao Yin; Vladimir Korzh; Zhiyuan Gong (pp. 236-244).

Wnt signaling plays critical roles in development of both tetrapod lung and fish swimbladder, which are the two evolutionary homologous organs. Our previous data reveal that down-regulation of Wnt signaling leads to defective swimbladder development. However, the effects of up-regulation of Wnt signaling on swimbladder development remain unclear. By knockdown of the Wnt protein inhibitory gene wif1, we demonstrated that up-regulation of Wnt signaling also resulted in perturbed development of the swimbladder. Specifically, the growth of epithelium and mesenchyme was greatly inhibited, the smooth muscle differentiation was abolished, and the organization of mesothelium was disturbed. Furthermore, our data reveal that it is the reduced cell proliferation, but not enhanced apoptosis, that contributes to the disturbance of swimbladder development in wif1 morphants. Blocking Wnt signaling by the Wnt antagonist IWR-1 did not affect wif1 expression in the swimbladder, but complete suppression of Hedgehog signaling in smo− /⁻ mutants abolished wif expression, consistent with our earlier report of a negative feedback regulation of Wnt signaling in the swimbladder by the Hedgehog signaling. Our works established the importance of proper level of Wnt signaling for normal development of swimbladder in zebrafish.► The Wnt inhibitor Wif1 is expressed in developing swimbladder in zebrafish. ► Knockdown of Wif1 affects the development of all three tissue layers of swimbladder. ► Hedgehog signaling, but not Wnt signaling, negatively regulates wif1 expression. ► A proper level of Wnt signaling is important for normal swimbladder development.

Keywords: Wnt signaling; wif1; Swimbladder; Hedgehog signaling; IWR-1; Zebrafish

Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants by Ao Yin; Vladimir Korzh; Zhiyuan Gong (pp. 236-244).

Keywords: Wnt signaling; wif1; Swimbladder; Hedgehog signaling; IWR-1; Zebrafish

Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants by Ao Yin; Vladimir Korzh; Zhiyuan Gong (pp. 236-244).

Keywords: Wnt signaling; wif1; Swimbladder; Hedgehog signaling; IWR-1; Zebrafish

Sphingosine 1-phosphate-mediated α_1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions by Jean A. Castillo-Badillo; Molina-Munoz Tzindilú Molina-Muñoz; Romero-Avila M. Teresa Romero-Ávila; Vazquez-Macias Aleida Vázquez-Macías; Richard Rivera; Jerold Chun; Garcia-Sainz J. Adolfo García-Sáinz (pp. 245-254).

Sphingosine-1-phosphate-induced α_1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α_1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P₁ receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α_1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor–α_1B-adrenergic receptor crosstalk.Display Omitted► Sphingosine-1-phosphate induces α_1B-adrenergic receptor phosphorylation. ► IGF-I and EGF also induce this effect and increase sphingosine kinase activity. ► A sphingosine kinase-1 dominant-negative mutant reduced growth factor action. ► Similar effects are produced by S1P₁ receptor knockdown. ► Sphingosine-1-phosphate is an autocrine mediator in the growth factor's effects.

Keywords: Abbreviations; NA; noradrenaline; AR; adrenergic receptor; PKC; protein kinase C; PI3K; phosphoinositide 3-kinase; S1P; sphingosine 1-phosphate; SPHK-1; sphingosine kinase-1; eGFP; enhanced green fluorescent proteinα; _1B; -Adrenoceptor; α; _1B; -Adrenergic receptor; Sphingosine-1-phosphate; EGF receptor; Transactivation

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6 by Masashi Muroi; Ken-ichi Tanamoto (pp. 255-263).

TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys⁶³-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKβ, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu⁵⁴⁴, Glu⁵⁸⁷, Glu⁷⁰⁶) of IRAK-1 but not its catalytic site (Asp³⁴⁰). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys⁴⁸- and Lys⁶³-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys⁶³-linked polyubiquitination on IRAK-1 is reported to compete with Lys⁴⁸-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.► Stimulation of a macrophage cell line with Toll-like receptor ligands decreased TRAF6 protein. ► The decrease in TRAF6 protein was proteasome-dependent. ► Ubiquitinated IRAK-1 was involved in the decrease in TRAF6 protein. ► We proposed a negative regulatory role of IRAK-1 on Toll-like receptor signaling.

Keywords: Abbreviations; ALLN; N-acetyl-leucyl-leucyl-norleucinal; IRAK; IL-1R-associated kinase; Pam; ₃; CSK; ₄; Tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys; TLR; Toll-like receptor; TIR; Toll/IL-1 receptor domainToll-like receptor; IL-1 receptor; Innate immunity; NF-κB

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6 by Masashi Muroi; Ken-ichi Tanamoto (pp. 255-263).

Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: A possible model for early stage carcinogenesis by Kristen P. Nickens; Ying Han; Harini Shandilya; Ashley Larrimore; Gary F. Gerard; Eric Kaldjian; Steven R. Patierno; Susan Ceryak (pp. 264-272).

Acquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis.► Normal cells made death-resistant after exposure to Cr(VI) ► Resistant cells retain Bcl-2 expression and show no caspase 3 cleavage after Cr(VI) ► Resistant cells do not undergo mitochondrial-mediated apoptosis after Cr(VI) exposure ► Resistant cells exhibit less basal mtDNA damage ► Mitochondrial dysregulation may be indicative of early stage carcinogenesis

Keywords: Abbreviations; Cr(VI); hexavalent chromium; TGA; terminal growth arrest; ROS; reactive oxygen species; SMAC/DIABLO; second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein; hTERT; human telomerase; mtDNA; mitochondrial DNA; ∆ψm; mitochondrial membrane potential; ND1; NADH dehydrogenase 1Death-resistance; Genotoxin; Chromium; Mitochondria; Early-stage carcinogenesis

Stimulation of Ca2⁺-sensing receptor inhibits the basolateral 50-pS K channels in the thick ascending limb of rat kidney by Shumin Kong; Chengbiao Zhang; Wennan Li; Lijun Wang; Haiyan Luan; Wen-Hui Wang; Ruimin Gu (pp. 273-281).

We used the patch-clamp technique to study the effect of changing the external Ca2⁺ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2⁺ concentration from 1mM to 2 or 3mM inhibited the basolateral 50-pS K channels while decreasing external Ca2⁺ to 10μM increased the 50-pS K channel activity. The effect of the external Ca2⁺ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2⁺ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2⁺-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2⁺ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analog increased the 50-pS K channel activity but did not block the effect of raising the external Ca2⁺ on the K channels. Neither inhibition of phospholipase A2 (PLA₂) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2⁺ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2⁺. We conclude that the external Ca2⁺ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2⁺ is mediated by the CaSR which stimulates PLC–PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2⁺ level modulates the reabsorption of divalent cations.► The patch-clamp experiments were performed in the thick ascending limb (TAL). ► Stimulation of Ca2⁺-receptor by raising external Ca2⁺ inhibits basolateral K channels in the TAL. ► The basolateral K channel activity determines transepithelial voltage (V_te). ► V_te provides the driving force for transepithelial Ca2⁺ absorption in the TAL. ► Regulation of Ca2⁺ absorption in the TAL may be achieved by modulating basolateral K channels.

Keywords: External Ca; 2; ⁺; Inwardly-rectifying K channel; Phospholipase C; PKC

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase by Wei Wu; Ming-Qing Dong; Xing-Gang Wu; Hai-Ying Sun; Hung-Fat Tse; Chu-Pak Lau; Gui-Rong Li (pp. 282-289).

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10μM) or the Src-family inhibitor PP2 (10μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr⁹⁰, Try³⁴⁴, and Try⁴⁸⁵. This effect is likely involved in regulating neuronal activity and/or tumor growth.► The EGFR kinase inhibitor AG556 suppresses hEAG1 current. ► Tyrosine phosphorylation of the channel protein is also inhibited by AG556. ► These inhibitory effects are countered by the PTP inhibitor orthovanadate. ► Tyr90, Try344 and Try485 are responsible for the tyrosine phosphorylation.

Keywords: Ion channel modulation; Signal transduction; Protein tyrosine phosphorylation; Protein tyrosine kinase; EGFR kinase

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase by Wei Wu; Ming-Qing Dong; Xing-Gang Wu; Hai-Ying Sun; Hung-Fat Tse; Chu-Pak Lau; Gui-Rong Li (pp. 282-289).

Keywords: Ion channel modulation; Signal transduction; Protein tyrosine phosphorylation; Protein tyrosine kinase; EGFR kinase

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase by Wei Wu; Ming-Qing Dong; Xing-Gang Wu; Hai-Ying Sun; Hung-Fat Tse; Chu-Pak Lau; Gui-Rong Li (pp. 282-289).

Keywords: Ion channel modulation; Signal transduction; Protein tyrosine phosphorylation; Protein tyrosine kinase; EGFR kinase

PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9 by Peta Bradbury; Maha Mahmassani; Jessie Zhong; Kylie Turner; Andre Paul; Nicole M. Verrills; Geraldine M. O'Neill (pp. 290-297).

The mesenchymal mode of cancer cell invasion characterized by active adhesion turnover and a polarized actin cytoskeleton, is critically regulated by the adaptor protein NEDD9/HEF1/Cas-L. While it is known that NEDD9 is subject to extensive phosphorylation modification, the molecules that determine NEDD9 phosphorylation to stimulate adhesion turnover and mesenchymal cell morphologies are currently unknown. Earlier studies have suggested that the serine/threonine phosphatase PP2A regulates interconversion between a low molecular mass NEDD9 phosphoform and higher molecular mass phosphoforms. However, previous studies have used chemical inhibitors to block PP2A activity. In the present study we therefore aimed to specifically inhibit PP2A activity via siRNA and dominant negative approaches to investigate the effect of PP2A on interconversion between 115kDa and 105kDa NEDD9 and determine the functional consequence of PP2A activity for NEDD9 function. Strikingly, we find that while the phosphatase inhibitor Calyculin A indeed abrogates detachment-induced dephosphorylation of the 115kDa NEDD9 phosphoform, PP2A depletion does not inhibit 115kDa to 105kDa interconversion. Our data suggest instead that PP2A targets discrete NEDD9 phosphorylation modifications separate to the events that mediate interconversion between the two forms. Functionally, PP2A depletion increases NEDD9 mediated cell spreading and mutation of S369 in the serine-rich region of NEDD9 to aspartate mimics this effect. Importantly, mutation of S369 to alanine abrogates the ability of dominant negative PP2A to increase NEDD9-mediated cell spreading. Collectively, our data reveal that the tumour suppressor PP2A may act via S369 to regulated NEDD9-mediated cell spreading.► PP2A phosphatase regulates 105kDa NEDD9 phosphoform. ► PP2A interacts with the Serine-Rich region (SRR) of NEDD9. ► Constitutive phosphorylation of NEDD9 S369 in the SRR promotes cell spreading. ► The tumour suppressor PP2A suppresses NEDD9-mediated spreading and switch to mesenchymal morphologies.

Keywords: NEDD9; PP2A; Serine; Phosphatase; Phosphorylation; Cell morphology

Role of PCDH10 and its hypermethylation in human gastric cancer by Zesong Li; James C.S. Chim; Mo Yang; Jieyu Ye; Benjamin C.Y. Wong; Liang Qiao (pp. 298-305).

Epigenetic changes of genomic DNA are involved in the development and progression of many cancers. Aberrant methylation of CpG islands in the promoter regions of certain tumor-suppressor genes (TSG) is frequently observed in cancer cells. Protocadherin 10 (PCDH10), a member of the cadherin superfamily, is a recently identified putative TSG. PCDH10 is frequently silenced in many solid tumors. However, the role of PCDH10 in gastric cancer is largely unknown. In this study, we examined the expression and methylation status of PCDH10 in gastric cancer cells and tissues by real time PCR and methylation-specific PCR (MSP), and then investigated the biological function of PCDH10. We found that the expression of PCDH10 was markedly reduced in gastric cancer cells and tissues. The reduced expression correlated with hypermethylation of this gene in its promoter region, as demonstrated by MSP and bisulfite genomic sequencing (BGS) analysis. In addition, pharmacological demethylation using 5-Aza restored the expression of PCDH10 in gastric cancer cells. Over-expression of PCDH10 in gastric cancer cells suppressed cell proliferation and migration, but did not cause marked apoptosis. Over-expression of PCDH10 also suppressed growth of xenograft tumors in nude mice. Thus, PCDH10 functions as a TSG in gastric cancer, and might be a useful target for cancer therapy.► Protocadherin 10 (PCDH10) is a member of the cadherin superfamily and has recently been identified as a putative TSG. ► PCDH10 is frequently silenced in many solid tumors but its role in gastric cancer is largely unknown. ► We examined the expression and methylation of PCDH10 in gastric cancer cells and tissues and investigated its biological function. ► PCDH10 functions as a TSG and may be a potential therapeutic target for gastric cancer.

Keywords: Abbreviations; 5-AZA; 5-Aza-2′-deoxycytidine; BGS; bisulfite genomic sequencing; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GC; gastric cancer; MSP; methylation-specific PCR; nt; nucleotide; PCDH10; protocadherin 10; PCR; polymerase chain reaction; RT-PCR; reverse transcription polymerase chain reaction; TSG; tumor-suppressor genesGastric cancer; PCDH10; Hypermethylation; Tumor-suppressor gene

Role of PCDH10 and its hypermethylation in human gastric cancer by Zesong Li; James C.S. Chim; Mo Yang; Jieyu Ye; Benjamin C.Y. Wong; Liang Qiao (pp. 298-305).

Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity by Daniel R. Ambruso; Michael A. Ellison; Gail W. Thurman; Thomas L. Leto (pp. 306-315).

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O₂⁻). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the K_m for NADPH, but increased the V_max for O₂⁻ production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.► Phagocyte NADPH oxidase (phox) is crucial to microbicidal activity of neutrophils. ► Modulation by peroxiredoxin 6 (Prdx6) presents a novel paradigm for phox. ► Prdx6 moves to plasma membrane and enhances activity in neutrophils and K562 line. ► Prdx6 retains phox complex in phox competent K562 cells. ► Prdx6 is critical for optimal phox activity.

Keywords: Abbreviations; fMLP; formyl-methionyl-leucyl-phenylalanine; H; ₂; O; ₂; hydrogen peroxide; KRPD; Krebs Ringers phosphate with dextrose; NADPH oxidase; NADPH oxidase 2; O; ₂; ⁻; superoxide anion; PMA; phorbol myristate acetate; Prdx; peroxiredoxin; Prdx6; peroxiredoxin 6; phox; phagocyte oxidase; PLA2; phospholipase A2; rh; recombinant human; RLUs; relative light units; ROS; reactive oxygen species; siRNA; short interfering RNA; SOD; superoxide dismutaseNeutrophil; NADPH oxidase; Peroxiredoxin 6; K562 cell

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells by Dhanendra Tomar; Rochika Singh; Arun Kumar Singh; Chirayu D. Pandya; Rajesh Singh (pp. 316-326).

Autophagy is one of the cellular adaptive processes that provide protection against many pathological conditions like infection, cancer, neurodegeneration, and aging. Recent evidences suggest that ubiquitination plays an important role in degradation of proteins or defective organelle either through proteasome or autophagy. In this study, we describe the role of TRIM13, ER resident ubiquitin E3 ligase in induction of autophagy and its role during ER stress. The ectopic expression of TRIM13 in HEK-293 cells induces autophagy. Domain mapping showed that coiled-coil (CC) domain is required for induction of autophagy. TRIM13 is stabilized during ER stress, interacts with p62/SQSTM1 and co-localizes with DFCP1. TRIM13 regulates initiation of autophagy during ER stress and decreases the clonogenic ability of the cells. This study for the first time demonstrates the role of TRIM13 in induction of autophagy which may play an important role in regulation of ER stress and may act as tumor suppressor.► TRIM13 induces autophagy. ► CC domain of TRIM13 is essential for autophagy induction. ► TRIM13 interacts with p62 and co-localizes with DFCP1. ► TRIM13 regulates ER stress induced autophagy. ► TRIM13 decreases clonogenic ability of cells via regulation of autophagy.

Keywords: Autophagy; ER stress; TRIM; Ubiquitin E3 ligase; Ubiquitin proteasome system

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells by Dhanendra Tomar; Rochika Singh; Arun Kumar Singh; Chirayu D. Pandya; Rajesh Singh (pp. 316-326).

Keywords: Autophagy; ER stress; TRIM; Ubiquitin E3 ligase; Ubiquitin proteasome system

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells by Dhanendra Tomar; Rochika Singh; Arun Kumar Singh; Chirayu D. Pandya; Rajesh Singh (pp. 316-326).

Keywords: Autophagy; ER stress; TRIM; Ubiquitin E3 ligase; Ubiquitin proteasome system

The ER–mitochondria interface: The social network of cell death by Stefan Grimm (pp. 327-334).

When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2⁺ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, “mitochondria-associated membranes”, are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP₃R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.

Keywords: Mitochondria; Endoplasmic reticulum; Interface; Cell death; Calcium; Signalling

The ER–mitochondria interface: The social network of cell death by Stefan Grimm (pp. 327-334).

Keywords: Mitochondria; Endoplasmic reticulum; Interface; Cell death; Calcium; Signalling

The ER–mitochondria interface: The social network of cell death by Stefan Grimm (pp. 327-334).

Keywords: Mitochondria; Endoplasmic reticulum; Interface; Cell death; Calcium; Signalling

Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31 by Hannah M. Heath-Engel; Bing Wang; Gordon C. Shore (pp. 335-347).

Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca²⁺ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca²⁺ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca²⁺ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca²⁺ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death.► Ectopic expression of p20 leads to paraptosis-like cell death in E1A/DNp53 BMK cells. ► This p20-induced pathway is characterized by a rise in ER Ca²⁺ and ER vacuolization. ► Cell death and ER vacuolization can be delayed by Bcl2b5 in the absence of Bax/Bak. ► ER localized Bcl2 can exert a pro-survival effect in the absence of Bax/Bak.

Keywords: Bap31; Bcl-2; Apoptosis; Paraptosis; Vacuolization; Calcium

Tom34: A cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import by Pierre Faou; Nicholas J. Hoogenraad (pp. 348-357).

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex. We identify Tom34 as a cochaperone of Hsp70/Hsp90 in mitochondrial protein import. We show that Tom34 is an integral component with Hsp70 and Hsp90 in the large complex. We also demonstrate the role of Tom34 in the mitochondrial import process, as the addition of an excess of Tom34 prevents efficient mitochondrial translocation of precursor proteins that have requirements for Hsp70/Hsp90. Tom34 exhibits an affinity for mitochondrial preproteins of the Tom70 translocation pathway as demonstrated by binding assays using in vitro translated proteins as baits. In addition, we examined the specificity and the size of different complex cytosolic machines. Separation of different radiolabeled cell-free translated proteins on Native-PAGE showed the presence of a high molecular weight complex which binds hydrophobic proteins. Importantly we show that the formation of the chaperone cytosolic complex that mediates the targeting of proteins to the mitochondria contains Tom34 and assembles in the presence of a fully translated substrate protein.► Tom34 is a cochaperone of Hsp70 and Hsp90>Tom34 play a role in the import of mitochondrial preproteins. ► Mitochondrial proteins with high degree of hydrophobicity have affinity for Tom34. ► A large size cytosolic protein complex is formed with selected newly-synthesized proteins.

Keywords: Abbreviations; TOM; translocase of the outer mitochondria membrane; TPR; tetratricopeptide repeat domain; RL; reticulocyte lysate; Hop; Hsp70/Hsp90 organizing protein; Hip; Hsc70-interacting protein; PiC; phosphate carrier; AAC; ADP/ATP carrier protein; pOTC; pre-ornithine transcarbamylase; ISP; Rieske iron–sulfur protein; BAP31; B-cell receptor associated protein 31; PDI; protein disulfide isomerase; GR; glucocorticoid receptor; RNaseA; Ribonuclease A; GST; glutathione S-transferase; DHFR; dihydrofolate reductase; CMA; chaperone-mediated autophagy; LAMP-2A; lysosome-associated membrane glycoprotein 2Tom34; Mitochondria; Protein import; Chaperone; Protein translocation

Tom34: A cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import by Pierre Faou; Nicholas J. Hoogenraad (pp. 348-357).

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor by Elizabeth Dumas; Catherine Martel; Paul-Eduard Neagoe; Arnaud Bonnefoy; Martin G. Sirois (pp. 358-367).

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25nM) and PAF (100nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001–50nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25nM) or PAF (100nM) with Ang1 (10nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.► First report of Ang1 pro-survival activity on neutrophils. ► Ang2 has no effect on neutrophil viability, apoptosis and necrosis. ► Combination of Ang1 with IL-8 or PAF has an additive effect on neutrophil viability. ► Ang1 pro-survival activity is Tie-2-dependent. ► Ang1-induced neutrophil viability is IL-8-dependent and PAF-independent.

Keywords: Angiopoietin; Interleukin-8; Neutrophil; Viability; Apoptosis; Inflammation

Nicotinic stimulation induces Tristetraprolin over-production and attenuates inflammation in muscle by Brian C. Geyer; Shani Ben Ari; Shahar Barbash; Jacqueline Kilbourne; Tsafrir S. Mor; Hermona Soreq (pp. 368-378).

Cholinergic signaling suppresses inflammation in blood and brain and attenuates apoptosis in other tissues, but whether it blocks inflammation in skeletal muscle under toxicant exposure, injuries and diseases remained unexplored. Here, we report nicotinic attenuation of inflammation and alteration of apoptotic protein expression pattern in murine muscle tissue and cultured myotubes, involving the RNA-binding protein, Tristetraprolin, and the anti-apoptotic protein, Mcl-1. In muscles and C2C12 myotubes, cholinergic excitation by exposure to nicotine or the organophosphorous pesticide, Paraoxon, induced Tristetraprolin overproduction while reducing pro-inflammatory transcripts such as IL-6, CXCL1 (KC) and CCL2 (MCP-1). Furthermore, nicotinic excitation under exposure to the bacterial endotoxin LPS attenuated over-expression of the CCL2 and suppressed the transcriptional activity of NF-ĸB and AP-1. Tristetraprolin was essential for this anti-inflammatory effect of nicotine in basal conditions. However, its knockdown also impaired the pro-inflammatory response to LPS. Finally, in vivo administration of Paraoxon or recombinant Acetylcholinesterase, leading respectively to either gain or loss of cholinergic signaling, modified muscle expression of key mRNA processing factors and several of their apoptosis-related targets. Specifically, cholinergic imbalances enhanced the kinase activators of the Serine–Arginine splicing kinases, Clk1 and Clk3. Moreover, Paraoxon raised the levels of the anti-apoptotic protein, Mcl-1, through a previously unrecognized polyadenylation site selection mechanism, producing longer, less stable Mcl-1 mRNA transcripts. Together, our findings demonstrate that in addition to activating muscle function, acetylcholine regulates muscle inflammation and cell survival, and point to Tristetraprolin and the choice of Mcl-1 mRNA polyadenylation sites as potential key players in muscle reactions to insults.► Cholinergic stimulation induces anti-inflammatory response in skeletal muscle cells. ► Tristetraprolin is an essential player in the cholinergic anti-inflammatory pathway in muscles. ► Cholinergic signaling regulates the expression of mRNA processing factors and their apoptosis-related targets. ► Cholinergic imbalances in the neuromuscular junction alter polyA site selection in the anti-apoptotic Mcl-1 gene. ► Together, cholinergic signaling in muscle cells regulates inflammatory and apoptosis-related transcripts.

Keywords: Abbreviations; ACh; acetylcholine; AChE; Acetylcholinesterase; AChE-R; Acetylcholinesterase readthrough variant; AChRα7; α7 nicotinic receptor; ARE; AU-rich elements; DMD; Duchenne muscular dystrophy; miR; microRNA; NMJ; neuromuscular junction; PO; Paraoxon; RACE; rapid amplification of cDNA 3′ ends; shRNA; short hairpin RNA; TTP; tristetraprolin; UTR; untranslated regionTristetraprolin; Muscle; Inflammation; Acetylcholinesterase; Chemokine; mRNA processing

Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators by Gonzalez-Arenas Aliesha González-Arenas; Valeria Hansberg-Pastor; Hernandez-Hernandez Olivia Tania Hernández-Hernández; Gonzalez-Garcia Tania Karina González-García; Joshua Henderson-Villalpando; Lemus-Hernandez Diana Lemus-Hernández; Aglaé Cruz-Barrios; Rivas-Suarez Mariana Rivas-Suárez; Ignacio Camacho-Arroyo (pp. 379-386).

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.► ERα activation induces proliferation of human astrocytoma cell lines. ► The coactivators SRC-1 and SRC-3 are necessary to increase astrocytoma proliferation mediated by ERα. ► ERα regulates the expression of genes involved in cell cycle, angiogenesis and metastasis of human astrocytomas.

Keywords: Astrocytoma; Coactivator; ERα; SRC-1; SRC-3; RNA interference

Protein kinase Cδ negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro by Minsoon Kim; Ji-hyun Ju; Kibeom Jang; Sunhwa Oh; Jieun Song; Chul Geun Kim; Incheol Shin (pp. 387-397).

Protein kinase Cδ (PKCδ) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKCδ on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKCδ down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKCδ (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKCδ exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKCδ as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKCδ and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKCδ, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKCδ and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKCδ resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKCδ silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKCδ can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.► Protein kinase Cδ down-regulated NICD-mediated transcription. ► Protein kinase Cδ inhibited nuclear localization of NICD. ► Protein kinase Cδ induced up-regulation of NICD. ► Protein kinase Cδ inhibited association of NICD with its target gene promoter.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; DAPI; 4′,6-diamidino-2-phenylindole; DLL; delta-like; DTT; dithiothreitol; ER; estrogen receptor; HDAC; histone deacetylase; NICD; Notch1 intracellular domain; PKCδ; protein kinase Cδ; PKCδ K376R; kinase-dead dominant negative PKCδ; PMSF; phenylmethylsulfonyl fluoride; TPA; 12-O-tetradecanoyl-13-phorbol acetate; TSA; trichostatinNotch; Transcription; Protein Kinase Cδ

Protein phosphorylation is a prerequisite for the Ca²⁺-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca²⁺ and reactive oxygen species by Sachie Kimura; Hidetaka Kaya; Tomoko Kawarazaki; Goro Hiraoka; Eriko Senzaki; Masataka Michikawa; Kazuyuki Kuchitsu (pp. 398-405).

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca²⁺. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca²⁺-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca²⁺ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca²⁺. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca²⁺-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca²⁺-dependent activation of Rboh. Positive feedback regulation of Ca²⁺ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca²⁺-ROS signalling network.►ROS production by AtRbohF is synergistically activated by Ca²⁺ and phosphorylation. ►Protein phosphorylation-induced activation of AtRbohF is independent of Ca²⁺. ►Protein phosphorylation is prerequisite for Ca²⁺-dependent activation. ►Phosphorylation of Rboh is the initial trigger for the plant Ca²⁺-ROS signalling network.

Keywords: AtRbohD; AtRbohF; Ca; ²⁺; Positive feedback regulation; Protein phosphorylation; Reactive oxygen species (ROS)

Transglutaminase 2: A molecular Swiss army knife by Soner Gundemir; Gozde Colak; Janusz Tucholski; Gail V.W. Johnson (pp. 406-419).

Transglutaminase 2 (TG2) is the most widely distributed member of the transglutaminase family with almost all cell types in the body expressing TG2 to varying extents. In addition to being widely expressed, TG2 is an extremely versatile protein exhibiting transamidating, protein disulphide isomerase and guanine and adenine nucleotide binding and hydrolyzing activities. TG2 can also act as a protein scaffold or linker. This unique protein also undergoes extreme conformational changes and exhibits localization diversity. Being mainly a cytosolic protein; it is also found in the nucleus, associated with the cell membrane (inner and outer side) and with the mitochondria, and also in the extracellular matrix. These different activities, conformations and localization need to be carefully considered while assessing the role of TG2 in physiological and pathological processes. For example, it is becoming evident that the role of TG2 in cell death processes is dependent upon the cell type, stimuli, subcellular localization and conformational state of the protein. In this review we discuss in depth the conformational and functional diversity of TG2 in the context of its role in numerous cellular processes. In particular, we have highlighted how differential localization, conformation and activities of TG2 may distinctly mediate cell death processes.

Keywords: Transglutaminase 2; Transcription regulation; Transamidase; Calcium; GTP; Cell death

Transglutaminase 2: A molecular Swiss army knife by Soner Gundemir; Gozde Colak; Janusz Tucholski; Gail V.W. Johnson (pp. 406-419).

Keywords: Transglutaminase 2; Transcription regulation; Transamidase; Calcium; GTP; Cell death

Transglutaminase 2: A molecular Swiss army knife by Soner Gundemir; Gozde Colak; Janusz Tucholski; Gail V.W. Johnson (pp. 406-419).

Keywords: Transglutaminase 2; Transcription regulation; Transamidase; Calcium; GTP; Cell death

HOCl causes necrotic cell death in human monocyte derived macrophages through calcium dependent calpain activation by Ya-ting Tina Yang; Matthew Whiteman; Steven P. Gieseg (pp. 420-429).

The abundance of dead macrophages in close proximity to HOCl-modified proteins in advanced atherosclerotic plaques implicates HOCl in the killing of macrophages and the formation of the necrotic core region. The mechanism of HOCl mediated death of macrophages was unknown, so using human monocyte derived macrophages (HMDM) we here have shown that HOCl causes a rapid necrotic cell death characterized by loss of MTT reduction, cellular ATP and cell lysis without caspase-3 activation in HMDM cells. The HOCl causes a rise in cytosolic calcium level via the plasma membrane L- and T-type calcium channels and endoplasmic reticulum RyR channel. Blocking of the calcium channels or the addition of calpain inhibitors prevents the HOCl mediated loss of mitochondrial potential, lysosome failure and HMDM cell death. Blocking MPT-pore formation with cyclosporin A also prevents the loss of mitochondrial membrane potential, lysosomal destabilization and HMDM cell death. Blocking the calcium mitochondrial uniporter with ruthenium red also blocks the loss of mitochondrial potential but only at high concentrations. HOCl appears to cause HMDM cell death through destabilization of cytosolic calcium control resulting in the failure of both the mitochondria and lysosomes.► HOCl exposure in the uM range causes to human macrophages to undergo a calcium dependent necrotic cell death. ► HOCl causes the Ca⁺ to enter the cytoplasm via plasma membrane L- and T- type Ca⁺ channels and RyR channels from the ER. ► Blocking of Ca⁺ channels or addition of calpain inhibitors prevents mitochondrial potential loss and lysosome failure. ► HOCl cause HMDM cell death through destabilization of cytosolic calcium control.

Keywords: Abbreviations; Δ; Ψ; _m; mitochondrial membrane potential; A23187; 4-bromo-calcium ionophore A23187; ANT; adenine nucleotide translocator; EBSS; Earle's balanced salt solution; fluo-3-AM ester; fluo-3-acetoxymethyl ester; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOCl; hypochlorous acid; HMDM; human monocyte derived macrophages; HPLC; high performance liquid chromatography; MBB; monobromobimane; MTT; 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDTA; ethylenediaminetetraacetic acid; EGTA; ethylene glycol tetraacetic acid; MPO; myeloperoxidase; MPT; mitochondrial permeability transition; PBS; phosphate buffered saline; PI; propidium iodide; RyR; ryanodine receptor; TMRM; tetramethylrhodamine methyl esterHuman monocyte derived macrophage; Hypochlorous acid; Calcium ion; Mitochondrial membrane potential; Calpain; Lysosomal stability

HOCl causes necrotic cell death in human monocyte derived macrophages through calcium dependent calpain activation by Ya-ting Tina Yang; Matthew Whiteman; Steven P. Gieseg (pp. 420-429).

eIF4F complex disruption causes protein synthesis inhibition during hypoxia in nerve growth factor (NGF)-differentiated PC12 cells by Hernandez-Jimenez Macarena Hernández-Jiménez; M. Irene Ayuso; Perez-Morgado M. Isabel Pérez-Morgado; Garcia-Recio Eva M. García-Recio; Alcazar Alberto Alcázar; Martin M. Elena Martín; Gonzalez Víctor M. González (pp. 430-438).

Poor oxygenation (hypoxia) influences important physiological and pathological situations, including development, ischemia, stroke and cancer. Hypoxia induces protein synthesis inhibition that is primarily regulated at the level of initiation step. This regulation generally takes place at two stages, the phosphorylation of the subunit α of the eukaryotic initiation factor (eIF) 2 and the inhibition of the eIF4F complex availability by dephosphorylation of the inhibitory protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). The contribution of each of them is mainly dependent of the extent of the oxygen deprivation. We have evaluated the regulation of hypoxia-induced translation inhibition in nerve growth factor (NGF)-differentiated PC12 cells subjected to a low oxygen concentration (0.1%) at several times. Our findings indicate that protein synthesis inhibition occurs primarily by the disruption of eIF4F complex through 4E-BP1 dephosphorylation, which is produced by the inhibition of the mammalian target of rapamycin (mTOR) activity via the activation of REDD1 (regulated in development and DNA damage 1) protein in a hypoxia-inducible factor 1 (HIF1)-dependent manner, as well as the translocation of eIF4E to the nucleus. In addition, this mechanism is reinforced by the increase in 4E-BP1 levels, mainly at prolonged times of hypoxia.► Hypoxia induces protein synthesis inhibition in differentiated PC12 cells. ► Hypoxia-induced translation inhibition occurs primarily by eIF4F complex disruption. ► Translocation of eIF4E to the nucleus mediates eIF4F complex inhibition. ► Increased levels and dephosphorylation of 4E-BP1 mediate eIF4F complex disruption. ► Inhibition of mTOR by REDD1/HIF1 pathway produces 4E-BP1 dephosphorylation.

Keywords: Abbreviations; 4E-BP1; eukaryotic initiation factor 4E-binding protein 1; Akt; serine/threonine kinase B, also termed PKB; AMPK; AMP-activated protein kinase; eIF; eukaryotic initiation factor; eIF2α; subunit α of eIF2; HIF; Hypoxia inducible factor; mTOR; mammalian target of rapamycin; NGF; nerve growth factor; REDD1; regulated in development and DNA damage 1; TSC; tuberous sclerosis complex4E-BP1; eIF2; Hypoxia; Translation inhibition; Translation initiation

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells by Serena Calise; Sabrina Blescia; Francesca Cencetti; Caterina Bernacchioni; Chiara Donati; Paola Bruni (pp. 439-450).

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P₂ and S1P₃. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P₁ and S1P₄ in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P₄ receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P₂ was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.► S1P stimulates cell proliferation of murine satellite cells. ► Cell migration of satellite cells is also enhanced by S1P treatment. ► S1P2 and S1P3 receptors mediate the mitogenic effect. ► S1P1 and S1P4 receptor transmit the chemotactic response.

Keywords: Abbreviations; S1P; sphingosine 1-phosphate; S1PR; S1P receptor; DMEM; Dulbecco's modified Eagle's medium; FCS; fetal calf serum; BSA; bovine serum albumin; PBS; phosphate buffered saline; HS; horse serumSatellite cell; Sphingosine 1-phosphate; S1P receptor; Cell migration; Cell proliferation; Skeletal muscle regeneration

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells by Seoung Woo Shin; Sung Youl Kim; Jeen-Woo Park (pp. 451-457).

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G₁ phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.► Ursolic acid induced apoptosis in PC3 cells. ► A novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells was demonstrated. ► Autophagy functions as a survival mechanism in PC3 cells against ursolic acid-induced apoptosis.

Keywords: Prostate cancer; Ursolic acid; Autophagy; Apoptosis; Akt/mTOR

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells by Seoung Woo Shin; Sung Youl Kim; Jeen-Woo Park (pp. 451-457).

Keywords: Prostate cancer; Ursolic acid; Autophagy; Apoptosis; Akt/mTOR

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells by Seoung Woo Shin; Sung Youl Kim; Jeen-Woo Park (pp. 451-457).

Keywords: Prostate cancer; Ursolic acid; Autophagy; Apoptosis; Akt/mTOR

Akt acutely activates the cholesterogenic transcription factor SREBP-2 by Winnie Luu; Laura J. Sharpe; Julian Stevenson; Andrew J. Brown (pp. 458-464).

Akt is an essential protein kinase for cell growth, proliferation, and survival. Perturbed Akt regulation is associated with a number of human diseases, such as cancer and diabetes. Recently, evidence has emerged that Akt is involved in the regulation of the sterol-regulatory element binding proteins, which are master transcriptional regulators of lipid metabolism. This offers a means by which synthesis of new membrane can be coordinated with cell growth and proliferation. However, the link between Akt and sterol-regulatory element binding protein-2, the major isoform participating in cholesterol regulation, is relatively unexplored. In the present study, we employed insulin-like growth factor-1 as an inducer of Akt signalling, and showed that it increased sterol-regulatory element binding protein-2 activation acutely (within 1h). This insulin-like growth factor-1-induced sterol-regulatory element binding protein-2 activation was blunted when Akt was inhibited pharmacologically or molecularly with small interfering RNA. Furthermore, we employed a rapalog heterodimerisation system to specifically and rapidly activate Akt, and found that sterol-regulatory element binding protein-2 activation was increased in response to Akt activation. Together, this study provides compelling evidence that Akt contributes to the acute regulation of cholesterol metabolism through activating sterol-regulatory element binding protein-2.► IGF-1 increases SREBP-2 activation acutely. ► IGF-1 activates SREBP-2 via PI3K, as determined by two specific PI3K inhibitors. ► Pharmacological and siRNA inhibition of Akt also decreases SREBP-2 activation. ► Specifically inducing activation of Akt with rapalog increases SREBP-2 activation. ► Active Akt increases both mature (active) SREBP-2, and its downstream gene targets.

Keywords: Abbreviations; 25HC; 25-hydroxycholesterol; CHO-7; Chinese hamster ovary-7; DN-Akt; dominant-negative Akt; FKBP; FK506-binding protein; FRB; FKBP-rapamycin binding; HMGCR; 3-hydroxy-3-methylglutaryl coenzyme A reductase; IGF-1; insulin-like growth factor-1; LDLR; low density lipoprotein receptor; LPDS; lipoprotein-deficient serum; Myr; myristoylation; mTOR; mammalian Target of Rapamycin; pAkt; phosphorylated Akt; PBGD; porphobilinogen deaminase; PDK1; phosphoinositide-dependent kinase 1; PI3K; phosphatidylinositol 3-kinase; Scap; SREBP cleavage-activating protein; siRNA; small interfering RNA; SREBP; sterol-regulatory element binding proteinAkt; Cholesterol; IGF-1; PI3K; SREBP-2

Akt acutely activates the cholesterogenic transcription factor SREBP-2 by Winnie Luu; Laura J. Sharpe; Julian Stevenson; Andrew J. Brown (pp. 458-464).

Nucleo-cytoplasmic shuttling of PAK4 modulates β-catenin intracellular translocation and signaling by Yan Li; Yangguang Shao; Yuxin Tong; Tao Shen; Jian Zhang; Yanshu Li; Hui Gu; Feng Li (pp. 465-475).

The canonical Wnt/β-catenin signaling pathway plays a central role in development and cancer. The p21-activated kinase 4 (PAK4) involves in a wide range of cellular processes, including cytoskeletal reorganization, cell proliferation, gene transcription and oncogenic transformation. However, the cross talk between the Wnt and PAK4 signaling pathways is poorly understood. Here, we show that PAK4 is a nucleo-cytoplasmic shuttling protein, containing three nuclear export signals (NESs) and two nuclear localization signals (NLSs). PAK4 is exported by the chromosome region maintenance-1 (CRM-1)-dependent pathway and is imported into the nucleus in an importin α5-dependent manner. PAK4 interacts with and phosphorylates β-catenin on Ser675, which promotes the TCF/LEF transcriptional activity and stabilizes β-catenin through inhibition of its degradation. Moreover, nuclear import of PAK4 accompanies with the nuclear import of β-catenin and increased TCF/LEF transcriptional activity. We further demonstrated that PAK4 associates with the TCF/LEF transcriptional complex by ChIP assays. These findings uncover a novel role for PAK4 in modulating intracellular translocation and signaling of β-catenin.► PAK4 nuclear export is mediated by CRM-1-dependent pathway. ► PAK4 interacts with and phosphorylates β-catenin on Ser675. ► PAK4 phosphorylation of β-catenin on Ser675 promotes its transcriptional activity. ► Nuclear import of PAK4 accompanies with the nuclear import of β-catenin. ► PAK4 associates with the TCF/LEF transcriptional complex.

Keywords: PAK4; Nucleo-cytoplasmic shuttling; β-catenin; Signal transduction

A conserved cysteine cluster, essential for transcriptional activity, mediates homodimerization of human metal-responsive transcription factor-1 (MTF-1) by Gunther Viola Günther; Alisa M. Davis; Oleg Georgiev; Walter Schaffner (pp. 476-483).

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I) and is also involved in the response to hypoxia and oxidative stress. MTF-1 recognizes a specific DNA sequence motif termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. The functional domains of MTF-1 include, besides the DNA-binding and activation domains and signals for subcellular localization (NLS and NES), a cysteine cluster⁶³²CQCQCAC⁶³⁸ located near the C-terminus. Here we show that this cysteine cluster mediates homodimerization of human MTF-1, and that dimer formation in vivo is important for basal and especially metal-induced transcriptional activity. Neither nuclear translocation nor DNA binding is impaired in a mutant protein in which these cysteines are replaced by alanines. Although zinc supplementation induces MTF-1 dependent transcription it does not per se enhance dimerization, implying that actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates MTF-1 only weakly in the cell lines tested, stabilizes the dimer by inducing intermolecular disulfide bond formation and synergizes with zinc to boost MTF-1 dependent transcription.►Transcription factor MTF-1 requires dimerization for metal-induced activity. ►Dimerization is mediated by a cysteine cluster that is conserved among vertebrates. ►Copper ions stabilize dimer formation via intermolecular disulfide bond formation.

Keywords: Abbreviations; EDC; 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; MRE; metal response element; MTF-1; metal-responsive transcription factor-1; TCEP; tris; (2-carboxyethyl)phosphine; TPEN; Tetrakis(2-pyridylmethyl)ethylenediamine; VSV; vesicular stomatitis virusMTF-1; Heavy metal homeostasis; Gene expression; Homodimerization; Cysteine cluster; Metal regulatory transcription factor-1

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis by Yanbo Shi; Manik Ghosh; Gennadiy Kovtunovych; Daniel R. Crooks; Tracey A. Rouault (pp. 484-492).

Ferredoxins are iron–sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron–sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2 — formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron–sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron–sulfur clusters, but not steroidogenesis. To further assess the role of the FDX–FDXR system in mammalian iron–sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron–sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron–sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron–sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron–sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.► We show that RNAi of human FDX1 compromises iron–sulfur cluster biogenesis. ► We also show that RNAi of human FDX2 compromises iron–sulfur cluster biogenesis. ► The two homologous human ferredoxins function in iron–sulfur cluster biogenesis. ► RNAi of the human FDX reductase (FDXR) compromises iron–sulfur cluster biogenesis. ► FDX-FDXR dysfunction disturbs mitochondrial and cytosolic iron homeostasis.

Keywords: Iron–sulfur cluster biogenesis; RNAi; Mitochondrial iron accumulation; Heme biosynthesis; Cellular iron homeostasis

Two free radical pathways mediate chemical hypoxia-induced glutamate release in synaptosomes from the prefrontal cortex by Yi Dong; Wen Zhang; Bin Lai; Wen-Jie Luan; Yan-Hua Zhu; Bing-Qiao Zhao; Ping Zheng (pp. 493-504).

It has been known that the inhibition of mitochondrial cytochrome c oxidase is one of the earliest events occurring under hypoxia and this inhibition can lead to neuronal damages. Thus, the cytochrome c oxidase inhibitor sodium cyanide (NaCN) is widely used to produce a model of chemical hypoxia by inhibiting this enzyme. However, the downstream signaling pathways of the inhibition of the cytochrome c oxidase remain to be studied. In the present paper, we used sodium cyanide to mimic the inhibition of the mitochondrial cytochrome c oxidase and studied its effect on glutamate release in synaptosomes from the prefrontal cortex using on-line fluorimetry. We also further investigated the mechanisms underlying the enhancing effect of sodium cyanide on glutamate release using pharmacological approaches combined with other techniques. The results showed that sodium cyanide significantly increased glutamate release from synaptosomes of prefrontal cortex; the broad-spectrum free radical scavenger MnTBAP and melatonin completely abolished the effect of sodium cyanide on glutamate release; the H₂O₂–NMDA receptor pathway mediated one part, whereas the lipid peroxyl radicals-ATP synthase pathway mediated another part of the sodium cyanide-induced glutamate release; scavenging H₂O₂ and enhancing ATP synthase activity could completely abolish the sodium cyanide-induced glutamate release.► H₂O₂–NMDA pathway mediated one part of the NaCN-induced glutamate release. ► Lipid peroxyl radicals-ATP synthase pathway mediated another part of the NaCN-induced glutamate release. ► Scavenging H₂O₂ and enhancing ATP synthase activity could completely abolish the NaCN-induced glutamate release.

Keywords: Sodium cyanide; Synaptosome; Glutamate release; Free radical; NMDA receptor; ATP synthase

Rituximab inhibits Kv1.3 channels in human B lymphoma cells via activation of FcγRIIB receptors by Li-Hua Wang; Ning Wang; Xiao-Yu Lu; Bing-Chen Liu; Murali K. Yanda; John Z. Song; Helena M. Dai; Yu-Liang Sun; Hui-Fang Bao; Douglas C. Eaton; He-Ping Ma (pp. 505-513).

Kv1.3 channels play an important role in modulating lymphocyte proliferation and apoptosis. We hypothesized that Kv1.3 channels in B lymphocytes might be regulated by rituximab, an antibody to CD20, a drug for treatments of B-cell lymphomas and autoimmune diseases. Using both whole-cell and cell-attached patch-clamp techniques, we found that rituximab inhibited Kv1.3 channels in Daudi human B lymphoma cells by promoting the channel inactivation at a concentration which was much greater than that required for activation of CD20. The effect of rituximab on Kv1.3 channels was abolished after selective blockade of FcγRIIB receptors with anti-FcγRIIB antibody. Western blot experiments showed that Daudi B cells expressed both Kv1.3 channel and the low affinity Fc receptor, FcγRIIB, which could be activated by the Fc region of rituximab. In contrast, normal lymphocytes expressed less Kv1.3 channels with faster inactivation. Confocal microscopy and flow cytometry data showed that rituximab induced apoptosis of Daudi B cells and that the effect was attenuated by blockade of FcγRIIB receptors and partially mimicked by inhibition of Kv1.3 channels. These results suggest that in addition to previously described complement-dependent cytotoxicity, rituximab also induces apoptosis of malignant B lymphocyte by stimulating FcγRIIB receptors and inhibiting Kv1.3 channels.► Both FcγRIIB receptor and Kv1.3 channel are expressed in malignant Daudi B cells. ► Compared to normal lymphocytes, Kv1.3 channel is highly expressed in Daudi cells. ► Kv1.3 channel in Daudi cells does not inactivate completely as normal lymphocytes. ► Rituximab inhibits Kv1.3 channel by promoting its inactivation via FcγRIIB receptor. ► Rituximab reduces membrane potential and induces apoptosis via FcγRIIB receptor.

Keywords: Voltage-dependent potassium channel; Fc receptor; Rituximab; Apoptosis; Patch-clamp technique; Confocal microscopy

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial–mesenchymal transition by Mi-Sook Lee; Hwang-Phill Kim; Tae-You Kim; Jung Weon Lee (pp. 514-523).

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-MET amplification, or epithelial–mesenchymal transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27^Kip1 stabilization. Gefitinib-resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27^Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27^Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell–cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27^Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells.► Gefitinib resistance showed sustained TM4SF5 expression and EMT upon gefitinib treatment. ► T790M EGFR introduction into gefitinib-sensitive cells showed EMT and increased TM4SF5 level. ► Regulation of TM4SF5 expression resulted in modulation of gefitinib resistance. ► Suppression of TM4SF5-mediated effects also resulted in reduced gefitinib resistance.

Keywords: Epithelial–mesenchymal transition; Drug resistance; EGFR; Lung cancer; Tetraspanin

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial–mesenchymal transition by Mi-Sook Lee; Hwang-Phill Kim; Tae-You Kim; Jung Weon Lee (pp. 514-523).

Keywords: Epithelial–mesenchymal transition; Drug resistance; EGFR; Lung cancer; Tetraspanin

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial–mesenchymal transition by Mi-Sook Lee; Hwang-Phill Kim; Tae-You Kim; Jung Weon Lee (pp. 514-523).

Keywords: Epithelial–mesenchymal transition; Drug resistance; EGFR; Lung cancer; Tetraspanin

Reduced protein stability of human DJ-1/PARK7 L166P, linked to autosomal recessive Parkinson disease, is due to direct endoproteolytic cleavage by the proteasome by Beatriz Alvarez-Castelao; Munoz Carolina Muñoz; Sanchez Isabel Sánchez; Marc Goethals; Joël Vandekerckhove; Castano José G. Castaño (pp. 524-533).

Parkinson's disease (PD) is characterized by dopaminergic dysfunction and degeneration. DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. In this study, we found that human DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A were stable proteins in cells, only the L166P mutant was unstable. In parallel, the former were not degraded and the L166P mutant was directly degraded in vitro by proteasome-mediated endoproteolytic cleavage. Furthermore, genetic evidence in fission yeast showed the direct involvement of proteasome in the degradation of human DJ-1 L166P and the corresponding L169P mutant of SPAC22E12.03c, the human orthologue of DJ-1 in Schizosaccharomyces Pombe, as their protein levels were increased at restrictive temperature in fission yeast (mts4 and pts1-732) harboring temperature sensitive mutations in proteasomal subunits. In total, our results provide evidence that direct proteasomal endoproteolytic cleavage of DJ-1 L166P is the mechanism of degradation contributing to the loss-of-function of the mutant protein, a property not shared by other DJ-1 missense mutants associated with PD.► DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. ► DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A are stable proteins in the cell. ► DJ-1 L166P mutant is unstable because of its direct endoproteolytic degradation by the proteasome. ► Genetic evidence obtained in yeast shows the direct role of proteasome in degradation of DJ-1 L166P.

Keywords: DJ-1; Proteasome; Parkinson; Proteolysis; Pombe; Ubiquitin

Localization and functional requirement of yeast Na⁺/H⁺ exchanger, Nhx1p, in the endocytic and protein recycling pathway by Ai Kojima; Junko Y. Toshima; Chisa Kanno; Chie Kawata; Jiro Toshima (pp. 534-543).

Acidification of the lumen of intracellular organelles is important for post-transcriptional processing, endosomal maturation, receptor recycling, and vesicle trafficking, being regulated by an intricate balance between H⁺ influx through vacuolar-type H⁺-ATPase and efflux through ion channels and transporters, such as the Na⁺/H⁺ exchanger (NHE). The eukaryotic NHE family comprises two major subgroups, one residing in the plasma membrane and the other in intracellular organelles. While mammalian intracellular NHE isoforms are localized to various organelles, including the mid-trans-Golgi compartments, early and late endosomes, and recycling endosomes, Nhx1p, the sole NHE in yeast, has been reported to be localized predominantly to the late endosomal/prevacuolar compartment. Here, using live cell imaging, we demonstrated that Nhx1p is localized to the trans-Golgi network compartments, late endosomes, and recycling endosomes, similar to mammalian intracellular NHE isoforms. Loss of Nhx1p led to accumulation of components of the retromer and endosomal sorting complex required for transport complexes, but not trans-Golgi compartments, in aberrant prevacuolar compartments. Importantly, Nhx1p was also required for recycling of the plasma membrane vesicle SNAP receptor Snc1p. These observations suggest that Nhx1p plays an important role in regulation of the luminal pH of various intracellular organelles, and that this regulation is critical for the protein recycling pathway as well as the endocytic pathway.► Nhx1p is localized to the trans-Golgi/TGN, endocytic, and recycling compartments. ► The dynamics of Nhx1p-residing compartments partly depend on actin cytoskeleton. ► Loss of Nhx1p leads to accumulation of some organelle markers in prevacuolar region. ► Nhx1p is required for protein trafficking in the endocytic and recycling pathway.

Keywords: Abbreviations; LatA; Latrunculin A; GFP; green fluorescent protein; mCherry; monomeric Cherry; NHE; The Na; ⁺; /H; ⁺; exchanger; VPS; vacuolar protein sorting; PVC; prevacuolar compartments; ER; endoplasmic reticulum; ESCRT; endosomal sorting complex required for transport; v-SNARE; vesicle SNAP receptor; TGN; trans; -Golgi networkEndocytosis; Protein sorting; NHE; Nhx1p; Recycling; Endosome

Zinc pyrithione induces ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells by Robert E. Carraway; Paul R. Dobner (pp. 544-557).

Zinc dyshomeostasis can induce cell death. However, the mechanisms involved have not been fully elucidated in prostate cancer (PCa) cells, which differ dramatically from normal cells in their zinc handling ability. Here, we studied the effects of the ionophore Zn-pyrithione (ZP) and the chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Both compounds induced cell death at micromolar concentrations when incubated with androgen-dependent (LNCaP), androgen-independent (PC3, DU145) and androgen-sensitive (C4-2) PCa cell-lines. Compared to PCa cells, RWPE1 prostate epithelial cells were less sensitive to ZP and more sensitive to TPEN, but total cellular zinc levels were changed similarly. ZnSO₄ enhanced the toxicity of ZP, but inhibited the effects of TPEN as expected. The morphological/biochemical responses to ZP and TPEN differed. ZP decreased ATP levels and stimulated ERK, AKT and PKC phosphorylation. DNA laddering was observed only at low doses of ZP but all doses of TPEN. TPEN activated caspase 3/7 and induced PARP-cleavage, DNA-fragmentation, ROS-formation and apoptotic bodies. PKC and ERK-pathway inhibitors, and antioxidants protected against ZP-induced but not TPEN-induced death. Inhibitors of MPTP-opening protected both. Cell death in response to TPEN (but not ZP) was diminished by a calpain inhibitor and largely prevented by a caspase 3 inhibitor. Overall, the results indicated primarily a necrotic cell death for ZP and an apoptotic cell death for TPEN. The enhanced sensitivity of PCa cells to ZP and the apparent ability of ZP and TPEN to kill quiescent and rapidly dividing cells in a p53-independent manner suggest that ZP/TPEN might be used to develop adjunct treatments for PCa.► Zinc chelator TPEN induces apoptosis in prostate cancer cells. ► Zinc pyrithione induces ERK-, ROS- and PKC-dependent necrosis. ► Normal prostate epithelial cells are less sensitive to zinc pyrithione.

Keywords: Zinc; Prostate cancer; Apoptosis; Necrosis; Ionophore; Chelator

Zinc pyrithione induces ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells by Robert E. Carraway; Paul R. Dobner (pp. 544-557).

Keywords: Zinc; Prostate cancer; Apoptosis; Necrosis; Ionophore; Chelator

Zinc pyrithione induces ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells by Robert E. Carraway; Paul R. Dobner (pp. 544-557).

Keywords: Zinc; Prostate cancer; Apoptosis; Necrosis; Ionophore; Chelator

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway by Manale Karam; Claudine Lassarre; Christine Legay; Jean-Marc Ricort (pp. 558-569).

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.► PI 3-kinase and PKD1cooperate to inhibit the IGF-I pathway at the level of IRS-1. ► PKD1 is activated independently of PI 3-kinase. ► PKD1 interacts with IRS-1 in response to EGF and FGF. ► PI 3-kinase, but not PKD1, induces the ser-307 phosphorylation of IRS-1. ► IRS-1 Ser-307 phosphorylation is not sufficient per se to inhibit the IGF signaling.

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 D1Tyrosine kinase receptor; Signaling pathways cross-talk; Insulin receptor substrate 1; Insulin-like growth factor; Protein kinase D1; Phosphatidylinositol 3-kinase

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells by Juan I. Barrasa; Nieves Olmo; Santiago-Gomez Angélica Santiago-Gómez; Emilio Lecona; Patrick Anglard; Javier Turnay; M. Antonia Lizarbe (pp. 570-581).

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.► MMP-11 is expressed in epithelial human colon adenocarcinoma cells. ► Transcriptional activity requires binding of Sp1/Smad2 complexes. ► HDAC inhibitors increase the acetylation status of Smad2. ► Smad2 acetylation promotes interaction with Sp1 and MMP-11 transcriptional activity. ► ERK pathway activation by HDAC inhibitors is required for upregulation of MMP-11.

Keywords: Abbreviations; HDAC; histone deacetylase; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; TIMP; tissue inhibitor of metalloproteinases; TSA; trichostatin AButyrate; Colon adenocarcinoma; Histone-deacetylase inhibitor; MAP kinase; Metalloproteinase

Involvement of ERK–RSK cascade in phenylephrine-induced phosphorylation of GATA4 by Tao Li; Zhiqiang Liu; Xiaoqing Hu; Kangtao Ma; Chunyan Zhou (pp. 582-592).

GATA4 has been characterized as a crucial regulator of cardiac development and hypertrophy. Multiple signaling pathways involving MAPK contribute to GATA4 activation via direct phosphorylation. MSK and RSK are two kinase families mediating signal transduction downstream of the MAPK cascade. In this study, we investigated the effects of MSK and RSK on GATA4 activation. Overexpression of RSK2 greatly increased phosphorylation of GATA4 at Ser261. This phosphorylation enhanced its transcriptional and DNA binding activity. RSK-dependent phosphorylation of GATA4 also led to enhanced interaction with NKX2.5 and p300. Sequential phosphorylation of the ERK–RSK–GATA4 cascade and nuclear accumulation of RSK in cardiomyocytes were observed after phenylephrine treatment. Inhibition of RSK using the small molecule SL0101 abrogated GATA4 phosphorylation at Ser261, ultimately leading to a repression of fetal cardiac genes. Adenovirus-mediated overexpression of MSK1 had no direct effect on GATA4 phosphorylation but increased GATA4 expression. Together with GATA4 phosphorylation at Ser105 by ERK1/2, our findings show dual phosphorylation of GATA4 by the ERK–RSK cascade and suggest that MSK and RSK have distinct effects in PE-induced cardiac hypertrophic response.► Overexpression of RSK2 increases phosphorylation level of GATA4 at Ser261. ► Phosphorylation of GATA4 at Ser261 enhances its transcriptional activities. ► RSK-dependent phosphorylation of GATA4 enhances its interaction with NKX2.5 and p300. ► Phenylephrine triggers a sequential phosphorylation of ERK–RSK–GATA4 cascade. ► Inhibition of RSK abrogates GATA4 phosphorylation at Ser261 triggered by PE.

Keywords: Abbreviations; Anf; atrial natriuretic factor; β-Mhc; β-myosin heavy chain; PE; phenylephrine; MSK; mitogen- and stress-activated protein kinase; RSK; ribosomal S6 kinase; NRVMs; neonatal rat ventricular myocytes; Co-IP; coimmunoprecipitation; GST; glutathione-S-transferase; EMSA; electrophoretic mobility-shift assay; ChIP; chromatin immunoprecipitationGATA4; ERK; RSK; Phosphorylation; Cardiac hypertrophy

Involvement of ERK–RSK cascade in phenylephrine-induced phosphorylation of GATA4 by Tao Li; Zhiqiang Liu; Xiaoqing Hu; Kangtao Ma; Chunyan Zhou (pp. 582-592).

Evidence for activation of mutated p53 by apigenin in human pancreatic cancer by Jonathan C. King; Qing-Yi Lu; Gang Li; Aune Moro; Hiroki Takahashi; Monica Chen; Vay Liang W. Go; Howard A. Reber; Guido Eibl; O. Joe Hines (pp. 593-604).

Pancreatic cancer is an exceedingly lethal disease with a five-year survival that ranks among the lowest of gastrointestinal malignancies. Part of its lethality is attributable to a generally poor response to existing chemotherapeutic regimens. New therapeutic approaches are urgently needed. We aimed to elucidate the anti-neoplastic mechanisms of apigenin—an abundant, naturally-occurring plant flavonoid—with a particular focus on p53 function. Pancreatic cancer cells (BxPC-3, MiaPaCa-2) experienced dose and time-dependent growth inhibition and increased apoptosis with apigenin treatment. p53 post-translational modification, nuclear translocation, DNA binding, and upregulation of p21 and PUMA were all enhanced by apigenin treatment despite mutated p53 in both cell lines. Transcription-dependent p53 activity was reversed by pifithrin-α, a specific DNA binding inhibitor of p53, but not growth inhibition or apoptosis suggesting transcription-independent p53 activity. This was supported by immunoprecipitation assays which demonstrated disassociation of p53/BclXL and PUMA/BclXL and formation of complexes with Bak followed by cytochrome c release. Treated animals grew smaller tumors with increased cellular apoptosis than those fed control diet. These results suggest that despite deactivating mutation, p53 retains some of its function which is augmented following treatment with apigenin. Cell cycle arrest and apoptosis induction may be mediated by transcription-independent p53 function via interactions with BclXL and PUMA. Further study of flavonoids as chemotherapeutics is warranted.► Mutant p53 exhibits signs of enhanced function following treatment with apigenin. ► Apigenin-induced apoptosis involves activation of PUMA and mitochondrial cell death. ► p53 and PUMA act in parallel, independent of transcription-dependent p53 function. ► Apigenin is non-toxic and pharmacologically active in vivo.

Keywords: Pancreatic cancer; Apoptosis; p53; PUMA

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