BBA - Molecular Cell Research (v.1823, #4)

Knock-down of Pdcd4 stimulates angiogenesis via up-regulation of angiopoietin-2 by Sebastian Krug; Johannes Huth; Friederike Göke; Malte Buchholz; Thomas M. Gress; Rüdiger Göke; Brigitte Lankat-Buttgereit (789-799).
The tumor suppressor Pdcd4 is involved in multiple pathways. Considering its biological action conflicting data in the literature exist and, consequently, our own studies point to a cell type specific action of Pdcd4. In the present study, using several Pdcd4 knock down cell lines we succeeded to identify angiopoietin-2 (Ang-2) as a gene up-regulated on the mRNA and protein level. The subsequent enhanced peptide secretion forced wild type Bon-1 cells in a neoplastic direction demonstrated by increased proliferation and colony formation while cell adhesion was decreased. Most likely, the stimulation of Ang-2 is in part mediated by increased activation of AP-1 but different signal transduction pathways may also be involved since we found opposite activation of PI3K/Akt/mTOR and MAPK7ERK pathways (both known to regulate in Ang-2 expression). Ang-2 is a modulator of vascular remodeling. Therefore, we analyzed the effect of supernatants from Pdcd4 knock-down cell lines on endothelial cells. Again, we detected reduced cell adhesion and increased colony formation. Probably, the most impressive effect was described on tube formation in a model for angiogenesis. Tube length and junctions of endothelial cells treated with conditioned medium from Pdcd4 knock-down cells were considerably increased. Both, up-regulation of Ang-2 and down-regulation of Pdcd4 are described for many tumors. However, this is the first study showing a direct impact of Pdcd4 on Ang-2 levels and, thereby, angiogenesis. Our data suggest a completely new mechanism for Pdcd4 to act as a tumor suppressor rendering Pdcd4 an attractive target for new therapeutic strategies in cancer treatment.► Angiopoietin-2 is a gene regulated by Pdcd4 levels in a multitude of cell lines. ► Regulation of Angiopoietin-2 by Pdcd4 is attained by diverse mechanisms depending on the cell line. ► Ang-2 release by reduced Pdcd4 in tumor cells increased tube formation of endothelial cells. ► Pdcd4 may influence neoangiogenesis in tumors by regulating angiopoietin-2.
Keywords: Pdcd4; Tumor suppressor; Angiogenesis; Angiopoietin-2;

Evidence that AMP-activated protein kinase can negatively modulate Ornithine decarboxylase activity in cardiac myoblasts by Catherine L. Passariello; Davide Gottardi; Silvia Cetrullo; Maddalena Zini; Gabriele Campana; Benedetta Tantini; Carla Pignatti; Flavio Flamigni; Carlo Guarnieri; Claudio M. Caldarera; Claudio Stefanelli (800-807).
The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30 min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.► Isoproterenol rapidly increases ODC activity and AMPKα phosphorylation in H9c2 cardiomyoblasts. ► Downregulation of AMPK by siRNA leads to superinduction of ODC. ► The modulation of ODC by AMPK is a mechanism that can regulate cell growth processes.
Keywords: AMP-activated protein kinase (AMPK); Ornithine decarboxylase (ODC); Cardiac hypertrophy; Isoproterenol;

Identification of a nuclear carbonic anhydrase in Caenorhabditis elegans by Teresa A. Sherman; Sharath C. Rongali; Tori A. Matthews; Jason Pfeiffer; Keith Nehrke (808-817).
Carbonic anhydrases (CA) catalyze the inter-conversion of CO2 with HCO3 and H+, and are involved in a wide variety of physiologic processes such as anion transport, pH regulation, and water balance. In mammals there are sixteen members of the classical α-type CA family, while the simple genetic model organism Caenorhabditis elegans codes for six αCA isoforms (cah-1 through cah-6).Fluorescent reporter constructs were used to analyze gene promoter usage, splice variation, and protein localization in transgenic worms. Catalytic activity of recombinant CA proteins was assessed using Hansson's histochemistry. CA's ability to regulate pH as a function of CO2 and HCO3 was measured using dynamic fluorescent imaging of genetically-targeted biosensors.Each of the six CA genes was found to be expressed in a distinct repertoire of cell types. Surprisingly, worms also expressed a catalytically-active CA splice variant, cah-4a, in which an alternative first exon targeted the protein to the nucleus. Cah-4a expression was restricted mainly to the nervous system, where it was found in nearly all neurons, and recombinant CAH-4A protein could regulate pH in the nucleus.In addition to establishing C. elegans as a platform for studying αCA function, this is the first example of a nuclear-targeted αCA in any organism to date.A classical αCA isoform is targeted exclusively to the nucleus where its activity may impact nuclear physiologic and pathophysiologic responses.► Model organism research can uncover novel physiologic functions of well-studied proteins. ► Caenorhabditis elegans codes for six classical α-type carbonic anhydrases. ► Carbonic anhydrases are generally cytoprotective. ► One of the worm carbonic anhydrase gene products is targeted exclusively to the nucleus. ► Nuclear-targeting may be important for pH regulation or oxidative stress resistance.
Keywords: Caenorhabditis elegans; Carbonic anhydrase; Nuclear targeting; pH regulation; Neurobiology;

The CCAAT box-binding transcription factor NF-Y regulates basal expression of human proteasome genes by Haiming Xu; Jiejun Fu; Seung-Wook Ha; Donghong Ju; Jianpu Zheng; Li Li; Youming Xie (818-825).
Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.► A large set of human proteasome genes contains the CCAAT box in their promoters. ► The transcription factor NF-Y binds CCAAT box-containing proteasome genes. ► Knockdown of NF-Y reduces proteasome expression and compromises the cellular proteasome activity. ► Knockdown of NF-Y sensitizes breast cancer cells to the proteasome inhibitor MG132.
Keywords: Proteasome; Protein degradation; NF-Y transcription factor; CCAAT box; Rpn4; Cancer therapy;

Emerging evidence from therapeutic trials in humans and animal models suggests that in the treatment of depression, antidepressants play a role by targeting the glutamatergic system. Fluvoxamine is one of the widely used SSRIs which has been considered to target monoamine neurotransmitter reuptake mechanisms. However, whether fluvoxamine has an effect on the glutamate release is still unclear. The present experiment studied the effect of fluvoxamine on presynaptic glutamate release in prelimbic cortex, both in control C57BL/6 mice and chronic restraint stress C57BL/6 mice, and further investigated the mechanism underlying this effect by using patch clamp, on-line fluorimetry, pharmacological approaches combined with other techniques. The results showed that fluvoxamine increased the glutamate release in the depression model mice but it had no effect on the glutamate release in the control mice. The mechanism underlying these effects in depression model mice was that, fluvoxamine firstly activated presynaptic 5-HT3 receptors, which transiently increased the Ca2+ concentration. The increase of Ca2+ concentration via 5-HT3 receptors caused the activation of sigma-1 receptors, which were activated by fluvoxamine. The activation of sigma-1 receptors increased the intrasynaptosomal Ca2+ concentration significantly through the outflow of endoplasmic reticulum calcium and finally activated PKC. These results suggested that fluvoxamine may have a selective effect and different mechanism based on the condition of animal.► Fluvoxamine could increase presynaptic glutamate release in chronic restraint stress C57 BL/6 mice. ► The effect of fluvoxamine is mediated by activating presynaptic 5-HT3 receptors and σ1 receptors. ► The mechanisms involved two phase of the elevation of Ca2+ concentration and the activation of PKC pathway.
Keywords: Fluvoxamine; Glutamate release; Sigma-1 receptors; 5-HT3 receptors; Synaptosomes;

Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2 by Jelena Kocić; Juan F. Santibañez; Aleksandra Krstić; Slavko Mojsilović; Ivana Okić Đorđević; Drenka Trivanović; Vesna Ilić; Diana Bugarski (838-849).
The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment.► The involvement of IL-17 in the C2C12 cell differentiation was investigated. ► IL-17 inhibits migration without affecting the proliferation of C2C12 cells. ► IL-17 inhibits myogenic differentiation of C2C12 mesenchymal cells by activating ERK1,2. ► IL-17 induces osteogenic differentiation of C2C12 mesenchymal cells by activating ERK1,2. ► IL-17 modulates C2C12 cell commitment to myogenic and osteogenic lineages.
Keywords: C2C12; Myoblast; IL-17; Myogenic differentiation; Osteogenic differentiation; ERK1,2;

Mechanisms of FGFR-mediated carcinogenesis by Imran Ahmad; Tomoko Iwata; Hing Y. Leung (850-860).
In this review, the evidence for a role of fibroblast growth factor receptor (FGFR) mediated signalling in carcinogenesis are considered and relevant underlying mechanisms highlighted. FGF signalling mediated by FGFR follows a classic receptor tyrosine kinase signalling pathway and its deregulation at various points of its cascade could result in malignancy. Here we review the accumulating reports that revealed the association of FGF/FGFRs to various types of cancer at a genetic level, along with in vitro and in vivo evidences available so far, which indicates the functional involvement of FGF signalling in tumour formation and progression. An increasing number of drugs against the FGF pathways is currently in clinical testing. We will discuss the strategies for future FGF research in cancer and translational approaches.► Role for FGFR mediated signalling in cancer. ► FGFR as a target of therapy. ► Future perspectives.
Keywords: FGF; FGFR; Receptor tyrosine kinase; Signalling; Cancer; Cancer therapy;

PKCδ and ε regulate the morphological integrity of the ER–Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus by Taichi Sugawara; Daiki Nakatsu; Hiroaki Kii; Nobuhiko Maiya; Atsuhiro Adachi; Akitsugu Yamamoto; Fumi Kano; Masayuki Murata (861-875).
The ER–Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.► Kinase inhibitor screening reveals that rottlerin disperses ERGIC morphology. ► Rottlerin inhibits the anterograde and retrograde transports via the Golgi. ► PKCδ and ε regulate ERGIC morphology. ► PKCδ and ε don't regulate the anterograde and retrograde transports via the Golgi.
Keywords: ER–Golgi intermediate compartment; Visualization; Vesicular transport; Kinase;

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice by Jung-Hyun Kim; Eung-Ryoung Lee; Kilsoo Jeon; Hye Yeon Choi; Hyejin Lim; Su-Jeong Kim; Han-Jung Chae; Seung Hwa Park; SangUk Kim; Young Rok Seo; Jin-Hoi Kim; Ssang-Goo Cho (876-888).
Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.► BI-1 shows most effective suppression of stress-induced apoptosis, compared with other BI-1 family members. ► BI-1 overexpression leads to the induction of ERK 1/2 phosphorylation in a sustained pattern. ► ERK activation is important for the anti-apoptotic function of BI-1. ► Transgenic mice overexpressing BI-1 shows splenomegaly and extramedullary megakaryopoiesis.
Keywords: Bax inhibitor-1; BI-1 family protein; ERK1/2; Splenomegaly; Autoimmune response;

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer by Jie Li; Junichi Koike; Hiroyuki Kugoh; Michitsune Arita; Takahito Ohhira; Yoshinori Kikuchi; Kimihiko Funahashi; Ken Takamatsu; C. Richard Boland; Minoru Koi; Hiromichi Hemmi (889-899).
Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.► hMSH3 is down-regulated by hypoxia in a p53-dependent and -independent manner in human cells. ► HIF-1α may negatively regulate hMSH3 transcription.
Keywords: hMSH3; Down-regulation; Hypoxia; HIF-1; p53; Colorectal cancer;

Regulation of leptin expression by 17beta-estradiol in human placental cells involves membrane associated estrogen receptor alpha by Yésica P. Gambino; Antonio Pérez Pérez; José L. Dueñas; Juan Carlos Calvo; Víctor Sánchez-Margalet; Cecilia L. Varone (900-910).
The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol (E2) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E2 conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E2 also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E2 induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E2 genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E2 regulates leptin expression in placenta and support the importance of leptin in placental physiology.► We study estrogen receptor mechanisms that regulate placental leptin expression. ► We use a membrane-constrained E2 conjugate, E-BSA. ► E-BSA induced leptin expression, involving the activation of MAPK and AKT pathways. ► ERα was detected in the plasma membrane of BeWo cells. ► ERα mediates genomic and nongenomic actions of E2 involved on leptin expression.
Keywords: Leptin; 17β-estradiol; Placenta; Gene expression; Estrogen receptor;

SUMO-2/3 conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis by Markéta Častorálová; Dagmar Březinová; Martin Švéda; Jan Lipov; Tomáš Ruml; Zdeněk Knejzlík (911-919).
Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.► SUMO-2/3 conjugates accumulated under MG132 treatment result from new protein synthesis. ► Part of SUMO-2/3 conjugates accumulated under heat shock result from new protein synthesis. ► Subset of artificially truncated proteins are modified by SUMO-2/3 in HEK 293T cells.
Keywords: SUMO; SUMOylation; Protein synthesis; Heat shock; MG132; 17-AAG;

α-Lipoic acid increases tolerance of cardiomyoblasts to glucose/glucose oxidase-induced injury via ROS-dependent ERK1/2 activation by Yuzhen Yao; Rongrong Li; Yujie Ma; Xiaohui Wang; Chuanfu Li; Xiaojin Zhang; Rong Ma; Zhengnian Ding; Li Liu (920-929).
α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on d-glucose/glucose oxidase (DG/GO, 30 mM/5 mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.► α-Lipoic acid (LA) prevented d-glucose/glucose oxidase (DG/GO) induced H9c2 cell injury. ► LA activated ERK1/2 and increased ROS generation. ► Inhibition of ERK1/2 activation abolished LA-induced protection against DG/GO challenge. ► Inhibition of ROS increase abrogated LA-induced cytoprotection and ERK1/2 activation. ► Our data suggest that LA-induced cytoprotection was mediated by ROS-dependent ERK1/2 activation.
Keywords: α-lipoic acid; Apoptosis; ERK1/2; Reactive oxygen specie; High glucose;

Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p by Hiroki Inoue; Takashi Baba; Seiichi Sato; Ryuya Ohtsuki; Aya Takemori; Takuya Watanabe; Mitsuo Tagaya; Katsuko Tani (930-939).
Members of the intracellular phospholipase A1 family of proteins have been implicated in organelle biogenesis and membrane trafficking. The mammalian family comprises three members: phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLA1 is mostly cytosolic, while KIAA0725p and p125 are more stably associated with the Golgi/endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and ER exit sites, respectively. Here we show that KIAA0725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAA0725p suggested that a sterile alpha-motif (SAM), which is also present in p125, but not in cytosolic PA-PLA1, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (PI(4)P)-binding. A construct with mutations in the positively charged cluster of the SAM domain is defective in both phosphoinositide-binding and Golgi/ERGIC targeting. Consistent with the view that the PI(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sac1 reduces the association of expressed KIAA0725p with membranes. In addition, we show that deletion of the DDHD domain or introduction of point mutations at the conserved aspartate or histidine residues in the domain abolishes the phospholipase activity of KIAA0725p and PA-PLA1. Together, our results suggest that KIAA0725p is targeted to specific organelle membranes in a phosphoinositide-dependent manner, and that its SAM and DDHD domains are essential for its phosphoinositide-binding and phospholipase activity.► KIAA0725p and p125 are novel phosphoinositides-binding proteins. ► The SAM-DDHD domain in KIAA0725p is a minimal region for PI(4)P-binding. ► A positive charge cluster in the SAM domain is responsible for PI(4)P-binding. ► The lipid binding is necessary for targeting of KIAA0725p to Golgi/ERGIC membranes. ► The DDHD domain in KIAA0725p and PA-PLA1 is essential for their PLA1 activities.
Keywords: KIAA0725p; Phospholipase A1; Phosphoinositide; Phosphatidylinositol 4-phosphate; SAM domain; DDHD domain;

Receptor-independent cellular uptake of pituitary adenylate cyclase-activating polypeptide by Ngoc-Duc Doan; David Chatenet; Myriam Létourneau; Hubert Vaudry; David Vaudry; Alain Fournier (940-949).
Pituitary adenylate cyclase-activating polypeptide (PACAP), a hypophysiotropic neurohormone, participates in the regulation of pleiotropic functions. The recent discovery of intracellular PACAP receptors in the brain and the testis as well as the physico-chemical characteristics of PACAP, i.e. extended α-helix containing basic residues, prompted us to evaluate the propensity of PACAP to cross the plasma membrane in a receptor-independent manner. Using confocal microscopy and flow cytometry, we demonstrated the ability of FITC-conjugated PACAP to efficiently penetrate into the internal cell compartment by direct translocation and endocytosis through clathrin-coated pits and macropinocytosis. Our study also revealed that, once inside the cells, PACAP38 is not entirely degraded by intracellular enzymes and that a significant amount of intact PACAP38 is also able to exit cells. Moreover, using binding assay on rat nuclear fractions from various tissues, PACAP nuclear receptors were identified. We also found that PACAP stimulates calcium release in rat testis nuclei. Interestingly, PACAP27 and PACAP38 but not VIP were able to upregulate de novo DNA synthesis in testis nuclei and that this effect was abolished by PACAP(6–38). These results support the presence of PAC1 receptors at the nuclear membrane and raise questions about their role in the biological activity of the peptide. These findings contribute to the characterization of PACAP as an intracrine factor and suggest that these intracellular PAC1 binding sites, probably associated with specific biological activities, should be taken into account during the development of PACAP-based drugs.► Both PACAP isoforms cross the plasma membrane by a receptor-independent mechanism. ► Direct translocation and endocytosis are involved in cellular uptake of PACAP. ► A significant amount of intact PACAP38 is also able to exit cells. ► Specific PACAP binding sites are presence in rat tissue nuclei. ► PACAP stimulates calcium release and transcription initiation in rat testis nuclei.
Keywords: Pituitary adenylate cyclase-activating polypeptide; Translocation; Endocytosis; Intracellular receptor; Intranuclear calcium release; Intracrine factor;

Activation of protein kinase C (PKC) via adenosine receptors is known to be involved in the cardioprotection of ischemic preconditioning (IPC). Specifically, activation of PKCε is critical for cardioprotection. There is ample evidence that PKCε resides in cardiac mitochondria. However, the signals that promote translocation of PKCε are largely unknown. The present study was designed to determine whether and how adenosine receptor activation induces translocation of PKCε to mitochondria. Freshly isolated adult rat cardiac myocytes and rat heart-derived H9c2 were used in the study. Immunofluorescence imaging of isolated mitochondria showed that PKCε but not PKCδ was localized in mitochondria and this mitochondrial localization of PKCε was significantly increased by adenosine treatment. The adenosine-induced increase in PKCε-positive mitochondria was largely prevented not only by PKC inhibitor chelerythrine, but also by the HSP90 inhibitor geldanamycin and by siRNA targeting HSP90. Immunoblot analysis from percoll-purified mitochondria further demonstrated that adenosine mediated a significant increase in mitochondrial PKCε but not PKCδ. This effect was blocked by inhibiting PKC activity with chelerythrine and bisindolylmaleimide. Furthermore, co-immunoprecipitation data showed that PKCε but not PKCδ was associated with TOM70 and HSP90, and this association was enhanced by adenosine treatment. Moreover, adenosine-induced association of PKCε with TOM70 was reduced by suppressing HSP90 expression with siRNA. In conclusion, we demonstrate that adenosine induces HSP90-dependent translocation of PKCε to mitochondria, possibly through mitochondrial import machinery TOM70. These results point out a novel mechanism in regulating PKC in mitochondria and suggest an important implication in ischemic preconditioning or postconditioning.► Adenosine receptor activation induces selective translocation of PKCε from cytosol to mitochondria. ► Adenosine-induced mitochondrial translocation requires PKC activation. ► HSP90 is critical for adenosine-mediated mitochondrial translocation of PKCε. ► Adenosine induces rapid association of PKCε, HSP90 and TOM70. ► Adenosine-induced increase in mitochondrial PKCε is through a TOM70-dependent mitochondrial import process.
Keywords: Adenosine; Protein kinase C; Mitochondria; Cardiac myocyte; Heat shock protein; Mitochondrial import;

Regulation of cell cycle transcription factor Swi5 by karyopherin Msn5 by Francisco José Taberner; Inma Quilis; Josep Sendra; María Carmen Bañó; Juan Carlos Igual (959-970).
Inactivation of S. cerevisiae β-karyopherin Msn5 causes hypersensitivity to the overexpression of mitotic cyclin Clb2 and aggravates growth defects of many mutant strains in mitotic exit, suggesting a connection between Msn5 and mitotic exit. We determined that Msn5 controlled subcellular localization of the mitotic exit transcription factor Swi5, since it was required for Swi5 nuclear export. Msn5 physically interacted with the N-terminal end of Swi5. Inactivation of Msn5 caused a severe reduction in cellular levels of Swi5 protein. This effect occurred by a post-transcriptional mechanism, since SWI5 mRNA levels were not affected. The reduced amount of Swi5 in msn5 mutant cells was not due to an increased protein degradation rate, but to a defect in Swi5 synthesis. Despite the change in localization and protein level, Swi5-regulated transcription was not defective in the msn5 mutant strain. However, a high level of Swi5 was toxic in the absence of Msn5. This deleterious effect was eliminated when Swi5 nuclear import was abrogated, suggesting that nuclear export by Msn5 is important for cell physiology, because it prevents toxic Swi5 nuclear accumulation.► Msn5 controls nuclear export of mitotic exit transcription factor Swi5 ► Msn5 physically interacts with the N-terminal region of Swi5 ► Swi5 protein level is reduced in the absence of Msn5 ► Msn5 affects Swi5 protein synthesis at a posttranscriptional level ► Swi5 nuclear export by Msn5 is important for cell physiology
Keywords: Swi5; Msn5; Cell cycle; Saccharomyces cerevisiae; β-karyopherin;

This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3′,5′-cyclic monophosphate (cAMP) stimulated [14C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.► Cyclic AMP stimulates the colocalization of SGLTs with caveolin-1 in lipid rafts of renal proximal tubule cells. ► Both Epac and PKA increase SGLT expression via ERK/p38 MAPKs and NF-κB. ► Activation of Epac and PKA increase F-actin expression for localization of SGLTs. ► Inhibitions of caveolin-1 and F-actin block the increase in cAMP-induced glucose uptake.
Keywords: Lipid rafts; Caveolin-1; Adenosine 3′,5′-cyclic monophosphate (cAMP); Na+-glucose cotransporters (SGLTs); Exchange protein directly activated by cAMP (Epac); Filamentous actin (F-actin);