BBA - Molecular Cell Research (v.1833, #9)
Editorial Board (i).
Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation by Ana B. Fernández-Martínez; Francisco J. Lucio Cazaña (2029-2038).
The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A.Display Omitted
Keywords: Intracellular PGE2; RARß; HIF-1α; EGFR transactivation; VEGF;
Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes by Christy A. Foster; Gail J. Mick; Xudong Wang; Kenneth McCormick (2039-2044).
The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11β-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11β-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11β-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms.
Keywords: Adrenal; Microsome; Hexose-6-phosphate dehydrogenase; Steroidogenesis;
4F2hc-silencing impairs tumorigenicity of HeLa cells via modulation of galectin-3 and β-catenin signaling, and MMP-2 expression by Angélica Santiago-Gómez; Juan I. Barrasa; Nieves Olmo; Emilio Lecona; Hans Burghardt; Manuel Palacín; Ma Antonia Lizarbe; Javier Turnay (2045-2056).
4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with β-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with β1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/β1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of β-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial–mesenchymal transition by inhibition of β-catenin proteasomal degradation through Akt/GSK-3β signaling and enabling cell motility.
Keywords: β-catenin; CD98hc; Galectin-3; HeLa cells; Integrin signaling; Matrix metalloproteinases;
Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: Differences between primary and cancer cells by Jaganmohan Reddy Jangamreddy; Saeid Ghavami; Jerzy Grabarek; Gunnar Kratz; Emilia Wiechec; Bengt-Arne Fredriksson; Rama Krishna Rao Pariti; Artur Cieślar-Pobuda; Soumya Panigrahi; Marek J. Łos (2057-2069).
The molecular mechanism of Salinomycin's toxicity is not fully understood. Various studies reported that Ca2 +, cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.
Keywords: Cancer stem cells; Mitofusin; Mitophagy; mTOR; PGC1α; Salinomycin;
The extra-cellular signal regulated kinases ERK1 and ERK2 segregate displaying distinct spatiotemporal characteristics in activated mast cells by Anat Benado Bar-Gill; Adi Efergan; Rony Seger; Mitsunori Fukuda; Ronit Sagi-Eisenberg (2070-2082).
ERK1 and ERK2 are highly homologous isoforms that often play redundant roles in regulating cellular functions. We analyzed the spatiotemporal patterns of ERK1 and ERK2 in resting and activated mast cells. Strikingly, we identified distinct pathways for these kinases. ERK1 localized to the cytosol and translocated to the nucleus upon cell activation and kinase phosphorylation. In contrast, ERK2 distributed between the cytosol and near the microtubule organizing center (MTOC) in resting cells and accumulated further at a pericentrosomal region upon cell trigger. Pericentrosomal accumulation of ERK2 was phosphorylation independent, required an intact microtubule network and was significantly enhanced by the overexpression of Neuronal Calcium Sensor-1 (NCS-1). We also identified γ-tubulin and phosphatidylinositol 4 kinaseβ (PI4Kβ), a downstream effector of NCS-1, as novel partner proteins of ERK2. Taken together, our results imply non-redundant functions of ERK1 and ERK2 in mast cells and implicate NCS-1 and PΙ4Κβ as regulators of ERK2 trafficking.Display Omitted
Keywords: ERK1; ERK2; Mast cell; NCS-1; PI4Kβ; Tubulin;
p27 suppresses cyclooxygenase-2 expression by inhibiting p38β and p38δ-mediated CREB phosphorylation upon arsenite exposure by Xun Che; Jinyi Liu; Haishan Huang; Xiaoyi Mi; Qing Xia; Jingxia Li; Dongyun Zhang; Qingdong Ke; Jimin Gao; Chuanshu Huang (2083-2091).
p27 is a cyclin-dependent kinase (CDK) inhibitor that suppresses a cell's transition from G0 to S phase, therefore acting as a tumor suppressor. Our most recent studies demonstrate that upon arsenite exposure, p27 suppresses Hsp27 and Hsp70 expressions through the JNK2/c-Jun- and HSF-1-dependent pathways, suggesting a novel molecular mechanism underlying the tumor suppressive function of p27 in a CDK-independent manner. We found that p27-deficiency (p27 −/−) resulted in the elevation of cyclooxygenase-2 (COX-2) expression at transcriptional level, whereas the introduction of p27 brought back COX-2 expression to a level similar to that of p27 +/+ cells, suggesting that p27 exhibits an inhibitory effect on COX-2 expression. Further studies identified that p27 inhibition of COX-2 expression was specifically due to phosphorylation of transcription factor cAMP response element binding (CREB) phosphorylation mediated by p38β and p38δ. These results demonstrate a novel mechanism underlying tumor suppression effect of p27 and will contribute to the understanding of the overall mechanism of p27 tumor suppression in a CDK-independent manner.
Keywords: p27; COX-2; p38; CREB; Arsenite;
Identification of a small molecule targeting annexin A7 by Haiying Li; Ning Liu; Shengqing Wang; Li Wang; Jing Zhao; Le Su; Yun Zhang; Shangli Zhang; Zhigang Xu; Baoxiang Zhao; Junying Miao (2092-2099).
Autophagy involves multiple membrane trafficking and fusion events. Annexin A7 (ANXA7) is postulated to play a role in membrane fusion during exocytosis, while the contribution of ANXA7 to autophagy is poorly understood. Our recent studies demonstrated that ABO could promote autophagy via elevation of ANXA7 and triggering ANXA7 subcellular redistribution. However, little is known about the molecular mechanisms how ANXA7 regulates autophagy. As molecular disruption of ANXA7 in mice results in several unwished phenotypes, small molecule modulators may be efficacious in defining the mechanisms of ANXA7 action. However, so far no compounds that selectively target ANXA7 have been identified. So, we hypothesize that ABO might be a potent modulator of ANXA7. We also have detected the colocalization of ANXA7 and microtubule-associated protein 1 light chain 3 (LC3), and ANXA7 was essential for LC3 accumulation in VEC autophagy. As a GTPase, whether ANXA7 affects the phosphorylation of LC3 or other proteins needs further investigation. In this study, we performed site-directed mutagenesis and found that ABO directly bound to Thr286 of ANXA7 and inhibited its phosphorylation. By yeast two-hybrid screening, we found that ANXA7 could interact with grancalcin (GCA). ABO promoted the interaction and inhibited GCA phosphorylation, leading to the decrease of intracellular Ca2 + concentration. At the same time, ABO inhibited the phosphorylation of LC3. Hence, by identifying ABO as an unprecedented modulator of ANXA7 as well as GCA and LC3 as interacting proteins of ANXA7, we demonstrated the possible mechanisms how ANXA7 regulates autophagy for the first time.Display Omitted
Keywords: ANXA7; GCA; LC3; Endothelial cell; Autophagy;