BBA - Molecular Cell Research (v.1863, #9)

The glucagon-like peptide-1 analogue exendin-4 reverses impaired intracellular Ca2 + signalling in steatotic hepatocytes by Eunüs S. Ali; Jin Hua; Claire H. Wilson; George A. Tallis; Fiona H. Zhou; Grigori Y. Rychkov; Greg J. Barritt (2135-2146).
The release of Ca2 + from the endoplasmic reticulum (ER) and subsequent replenishment of ER Ca2 + by Ca2 + entry through store-operated Ca2 + channels (SOCE) play critical roles in the regulation of liver metabolism by adrenaline, glucagon and other hormones. Both ER Ca2 + release and Ca2 + entry are severely inhibited in steatotic hepatocytes. Exendin-4, a slowly-metabolised glucagon-like peptide-1 (GLP-1) analogue, is known to reduce liver glucose output and liver lipid, but the mechanisms involved are not well understood. The aim of this study was to determine whether exendin-4 alters intracellular Ca2 + homeostasis in steatotic hepatocytes, and to evaluate the mechanisms involved. Exendin-4 completely reversed lipid-induced inhibition of SOCE in steatotic liver cells, but did not reverse lipid-induced inhibition of ER Ca2 + release. The action of exendin-4 on Ca2 + entry was rapid in onset and was mimicked by GLP-1 or dibutyryl cyclic AMP. In steatotic liver cells, exendin-4 caused a rapid decrease in lipid (half time 6.5 min), inhibited the accumulation of lipid in liver cells incubated in the presence of palmitate plus the SOCE inhibitor BTP-2, and enhanced the formation of cyclic AMP. Hormone-stimulated accumulation of extracellular glucose in glycogen replete steatotic liver cells was inhibited compared to that in non-steatotic cells, and this effect of lipid was reversed by exendin-4. It is concluded that, in steatotic hepatocytes, exendin-4 reverses the lipid-induced inhibition of SOCE leading to restoration of hormone-regulated cytoplasmic Ca2 + signalling. The mechanism may involve GLP-1 receptors, cyclic AMP, lipolysis, decreased diacylglycerol and decreased activity of protein kinase C.Display Omitted
Keywords: Liver; Steatosis; Exendin-4; GLP-1; Intracellular Ca2 +;  Cyclic AMP; Store operated Ca2 + entry;

Ste20-like kinase, SLK, activates the heat shock factor 1 – Hsp70 pathway by Andrey V. Cybulsky; Julie Guillemette; Joan Papillon (2147-2155).
Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-μ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury.
Keywords: Cell death; Ischemia-reperfusion; Phosphorylation; Protein kinases; Signal transduction;

Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). Biochemical studies indicate that the transduction mechanism at ORNs is mediated by cyclic adenosine monophosphate (cAMP) and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. However, the interaction between these two second messenger systems during olfactory perception or adaptation processes is much less understood. Here, we used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca2+-response. In-vivo functional bioluminescence Ca2+-imaging indicates that a single 5 s application of an odor increased Ca2 +-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca2 +-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response.
Keywords: Olfactory perception; Odor adaptation; cAMP; InsP3 receptor; Ryanodine receptor; Second messengers;

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration by Alvaro Alvarez; Raúl Lagos-Cabré; Milene Kong; Areli Cárdenas; Francesca Burgos-Bravo; Pascal Schneider; Andrew F.G. Quest; Lisette Leyton (2175-2188).
Our previous reports indicate that ligand-induced αVβ3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca2 + to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVβ3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVβ3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca2 +, hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVβ3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca2 + entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.Display Omitted
Keywords: Cell migration; Thy-1; ATP; Calcium; Connexins; Pannexins;

Symmetry breaking in spreading RAT2 fibroblasts requires the MAPK/ERK pathway scaffold RACK1 that integrates FAK, p190A-RhoGAP and ERK2 signaling by Zuzana Klímová; Vojtěch Bráborec; Miloslava Maninová; Josef Čáslavský; Michael J. Weber; Tomáš Vomastek (2189-2200).
The spreading of adhering cells is a morphogenetic process during which cells break spherical or radial symmetry and adopt migratory polarity with spatially segregated protruding cell front and non-protruding cell rear. The organization and regulation of these symmetry-breaking events, which are both complex and stochastic, are not fully understood. Here we show that in radially spreading cells, symmetry breaking commences with the development of discrete non-protruding regions characterized by large but sparse focal adhesions and long peripheral actin bundles. Establishment of this non-protruding static region specifies the distally oriented protruding cell front and thus determines the polarity axis and the direction of cell migration. The development of non-protruding regions requires ERK2 and the ERK pathway scaffold protein RACK1. RACK1 promotes adhesion-mediated activation of ERK2 that in turn inhibits p190A-RhoGAP signaling by reducing the peripheral localization of p190A-RhoGAP. We propose that sustained ERK signaling at the prospective cell rear induces p190A-RhoGAP depletion from the cell periphery resulting in peripheral actin bundles and cell rear formation. Since cell adhesion activates both ERK and p190A-RhoGAP signaling this constitutes a spatially confined incoherent feed-forward signaling circuit.
Keywords: RACK1; ERK; FAK; p190A-RhoGAP; Polarity; Feed-forward loop;

A hemagglutinin isolated from Northeast China black beans induced mitochondrial dysfunction and apoptosis in colorectal cancer cells by Xiuli Dan; Tzi Bun Ng; Jack Ho Wong; Yau Sang Chan; Randy Chi Fai Cheung; Wai Yee Chan (2201-2211).
Incidence of colorectal cancer is closely related with the lifestyle, especially the dietary habits of patients. Epidemiological researches have demonstrated a negative correlation between legume consumption and colorectal cancer incidence. Lectins/hemagglutinins are a type of carbohydrate binding proteins which are abundantly stored in legumes. Their eminent pH-stability allows them to survive digestion and remain active in the intestine where they may have direct contact with colorectal tumors. It is therefore interesting to explore the direct interaction between lectins/hemagglutinins and colorectal cancer. In the present research, we reported a detailed research on the interaction between a hemagglutinin isolated from an edible legume with two colorectal cancer cell lines. This hemagglutinin (NCBBH) was found to first bind to tumor cell membrane as early as 30 min post treatment and was gradually transported inside the cytoplasm within 3 h, with some of it localized in the Golgi apparatus and some in the lysosomes. After its entrance, the hemagglutinin induced aggregation of the Golgi apparatus, which in turn adversely affected the transportation of protein from endoplasmic reticulum (ER) to the Golgi apparatus, resulting in protein accumulation in ER and ER stress. The hemagglutinin-treated cells also manifested severe mitochondrial malformation and membrane depolarization, accompanied by obvious apoptosis characteristics, like chromatin condensation, phosphatidylserine exposure and caspase activation. Collectively, our results indicate that the hemaggltuinin could successfully enter the cytoplasm of colorectal cancer cells and adversely affect their growth, providing a mechanism in support of the application of edible legumes to the prevention and treatment of colorectal cancer.
Keywords: Hemagglutinin; Mitochondrial dysfunction; Apoptosis; Colorectal cancer; Legume;

Negative pressure induces p120-catenin–dependent adherens junction disassembly in keratinocytes during wound healing by Ching-Hui Huang; Chih-Chin Hsu; Carl Pai-Chu Chen; Shu-Er Chow; Jong-Shyan Wang; Yu-Chiau Shyu; Mu-Jie Lu (2212-2220).
A negative-pressure of 125 mm Hg (NP) has been widely used to treat chronic wounds in modern medicine. Keratinocytes under NP treatment have shown accelerated cell movement and decreased E-cadherin expression. However, the molecular mechanism of E-cadherin regulation under NP remains incompletely understood. Therefore, we investigated the E-cadherin regulation in keratinocytes (HaCaT cells) under NP. HaCaT cells were treated at ambient pressure (AP) and NP for 12 h. Cell movement was measured by traditional and electric wound healing assays at the 2 different pressures. Mutants with overexpression of p120-catenin (p120ctn) were used to observe the effect of NP on p120ctn and E-cadherin expression during wound healing. Cell fractionation and immunoblotting data showed that NP increased Y228-phosphorylated p120ctn level and resulted in the translocation of p120ctn from the plasma membrane to cytoplasm. Immunofluorescence images revealed that NP decreased the co-localization of p120ctn and E-cadherin on the plasma membrane. Knockdown of p120ctn reduced E-cadherin expression and accelerated cell movement under AP. Overexpression of the Y228-phosphorylation-mimic p120ctn decreased E-cadherin membrane expression under both AP and NP. Phosphorylation-deficient mutants conferred restored adherens junctions (AJs) under NP. The Src inhibitor blocked the phosphorylation of p120ctn and impeded cell migration under NP. In conclusion, Src-dependent phosphorylation of p120ctn can respond rapidly to NP and contribute to E-cadherin downregulation. The NP-induced disassembly of the AJ further accelerates wound healing.
Keywords: Negative-pressure wound therapy; Wound healing; Cell movement; E-cadherin;

β-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells by Katarzyna Sobierajska; Katarzyna Wieczorek; Wojciech M. Ciszewski; Izabela Sacewicz-Hofman; Marta E. Wawro; Magdalena Wiktorska; Joanna Boncela; Izabela Papiewska-Pajak; Pawel Kwasniak; Elzbieta Wyroba; Czeslaw S. Cierniewski; Jolanta Niewiarowska (2221-2233).
Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.Display Omitted
Keywords: EMT; Snail; β-III tubulin; Colon cancer; Migration;

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis by Samuel Nyabam; Zhuo Wang; Thomas Thibault; Ayinde Oluseyi; Rameeza Basar; Lindsay Marshall; Martin Griffin (2234-2244).
Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.Display Omitted
Keywords: Tissue transglutaminase; Cystic fibrosis; Transforming growth factor β; Epithelial-mesenchymal transition; Cystic fibrosis transmembrane conductance regulator;

Effects of G6PD activity inhibition on the viability, ROS generation and mechanical properties of cervical cancer cells by Zishui Fang; Chengrui Jiang; Yi Feng; Rixin Chen; Xiaoying Lin; Zhiqiang Zhang; Luhao Han; Xiaodan Chen; Hongyi Li; Yibin Guo; Weiying Jiang (2245-2254).
Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer.
Keywords: Cervical cancer cell; G6PD deficiency; Mechanical properties; Cytoskeleton; Anti-tumor;

The Candida albicans fimbrin Sac6 regulates oxidative stress response (OSR) and morphogenesis at the transcriptional level by Bing Zhang; Qilin Yu; Yuzhou Wang; Chenpeng Xiao; Jianrong Li; Da Huo; Dan Zhang; Chang Jia; Mingchun Li (2255-2266).
The actin cytoskeleton coordinates numerous fundamental cellular processes. Fimbrins are a class of evolutionally conserved ABPs that mediate actin bundling and regulate actin dynamics and functions. In this study, we identified the fimbrin Sac6 from the important fungal pathogen, Candida albicans. Interestingly, deletion of SAC6 led to increased tolerance to oxidative stress, while its overexpression caused hyper-susceptibility to this stress. Further investigations revealed that Sac6, by interaction with actin, negatively regulated the cytosol-to-nucleus transport of the key OSR (oxidative stress response) transcription factor Cap1 and consequent expression of OSR genes. Moreover, loss of Sac6 enhanced hyphal maintenance, and its overexpression caused a defect in hyphal development, which was attributed to abnormal expression of morphogenesis-related genes. In addition, Sac6 was involved in regulation of secretion of lytic enzymes and virulence of C. albicans. This study reveals a novel mechanism by which fimbrin transcriptionally regulates OSR and morphogenesis, and sheds a novel light on the functions of actin cytoskeleton.Display Omitted
Keywords: Actin cytoskeleton; Fimbrin; Oxidative stress response; Morphogenesis; Candida albicans;

Membrane repair of human skeletal muscle cells requires Annexin-A5 by Romain Carmeille; Flora Bouvet; Sisareuth Tan; Coralie Croissant; Céline Gounou; Kamel Mamchaoui; Vincent Mouly; Alain R. Brisson; Anthony Bouter (2267-2279).
Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.Display Omitted
Keywords: Annexin A5; Membrane repair; Skeletal muscle; Limb girdle muscular dystrophy; Laser injury; Electron microscopy;

The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator by Vincent A. van der Mark; Hugo R. de Jonge; Jung-Chin Chang; Kam S. Ho-Mok; Suzanne Duijst; Dragana Vidović; Marianne S. Carlon; Ronald P.J. Oude Elferink; Coen C. Paulusma (2280-2288).
Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function.We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription.We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells.We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.
Keywords: ATP8B1; CFTR; Trafficking; PFIC1; Cystic fibrosis; Chloride transport;

Rapid steroid hormone actions via membrane receptors by Nofrat Schwartz; Anjali Verma; Caroline B. Bivens; Zvi Schwartz; Barbara D. Boyan (2289-2298).
Steroid hormones regulate a wide variety of physiological and developmental functions. Traditional steroid hormone signaling acts through nuclear and cytosolic receptors, altering gene transcription and subsequently regulating cellular activity. This is particularly important in hormonally-responsive cancers, where therapies that target classical steroid hormone receptors have become clinical staples in the treatment and management of disease. Much progress has been made in the last decade in detecting novel receptors and elucidating their mechanisms, particularly their rapid signaling effects and subsequent impact on tumorigenesis. Many of these receptors are membrane-bound and lack DNA-binding sites, functionally separating them from their classical cytosolic receptor counterparts. Membrane-bound receptors have been implicated in a number of pathways that disrupt the cell cycle and impact tumorigenesis. Among these are pathways that involve phospholipase D, phospholipase C, and phosphoinositide-3 kinase. The crosstalk between these pathways has been shown to affect apoptosis and proliferation in cardiac cells, osteoblasts, and chondrocytes as well as cancer cells. This review focuses on rapid signaling by 17β-estradiol and 1α,25-dihydroxy vitamin D3 to examine the integrated actions of classical and rapid steroid signaling pathways both in contrast to each other and in concert with other rapid signaling pathways. This new approach lends insight into rapid signaling by steroid hormones and its potential for use in targeted drug therapies that maximize the benefits of traditional steroid hormone-directed therapies while mitigating their less desirable effects.
Keywords: Membrane receptors; Estrogen; Vitamin D3; ERα36; Hormones; Cancer;

Molecular determinants that mediate the sorting of human ATG9A from the endoplasmic reticulum by Catherine Staudt; Florentine Gilis; Marielle Boonen; Michel Jadot (2299-2310).
ATG9A is a multispanning membrane protein required for autophagosome formation. Under basal conditions, neosynthesized ATG9A proteins travel to the Golgi apparatus and cycle between the trans-Golgi network and endosomes. In the present work, we searched for molecular determinants involved in the subcellular trafficking of human ATG9A in HeLa cells using sequential deletions and point mutations. Deletion of amino acids L340 to L354 resulted in the retention of ATG9A in the endoplasmic reticulum. In addition, we found that substitution of the L711YM713 sequence (located in the C-terminal region of ATG9A) by alanine residues severely impaired its transport through the Golgi apparatus. This defect could be corrected by oligomerization of the mutant protein with co-transfected wild-type ATG9A, suggesting that ATG9A oligomerization may help its sorting through biosynthetic compartments. Lastly, the study of the consequences of the LYM/AAA mutation on the intracellular trafficking of ATG9A highlighted that some newly synthesized ATG9A can bypass the Golgi apparatus to reach the plasma membrane. Taken together, these findings provide new insights into the intracellular pathways followed by ATG9A to reach different subcellular compartments, and into the intramolecular determinants that drive the sorting of this protein.
Keywords: ATG9A; Intracellular transport; Sorting signal; Oligomerization; Endoplasmic reticulum;

Investigation of the molecular mechanism of δ-catenin ubiquitination: Implication of β-TrCP-1 as a potential E3 ligase by Hridaya Shrestha; Tingting Yuan; Yongfeng He; Pyong-Gon Moon; Nensi Shrestha; Taeyong Ryu; So-Yeon Park; Young-Chang Cho; Chan-Hyeong Lee; Moon-Chang Baek; Sayeon Cho; Shishli Simkhada; Hangun Kim; Kwonseop Kim (2311-2321).
Ubiquitination, a post-translational modification, involves the covalent attachment of ubiquitin to the target protein. The ubiquitin–proteasome pathway and the endosome–lysosome pathway control the degradation of the majority of eukaryotic proteins. Our previous study illustrated that δ-catenin ubiquitination occurs in a glycogen synthase kinase-3 (GSK-3) phosphorylation-dependent manner. However, the molecular mechanism of δ-catenin ubiquitination is still unknown. Here, we show that the lysine residues required for ubiquitination are located mainly in the C-terminal portion of δ-catenin. In addition, we provide evidence that β-TrCP-1 interacts with δ-catenin and functions as an E3 ligase, mediating δ-catenin ubiquitin–proteasome degradation. Furthermore, we prove that both the ubiquitin–proteasome pathway and the lysosome degradation pathway are involved in δ-catenin degradation. Our novel findings on the mechanism of δ-catenin ubiquitination will add a new perspective to δ-catenin degradation and the effects of δ-catenin on E-cadherin involved in epithelial cell–cell adhesion, which is implicated in prostate cancer progression.
Keywords: Ubiquitination; δ-Catenin; β-TrCP-1;

Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53 by Georges Kalouche; Céline Boucher; Annick Coste; Laurent Debussche; Cécile Orsini; Christophe Baudouin; Thomas Debeir; Xavier Vigé; William Rostène (2322-2332).
E-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients.
Keywords: EP2 receptor; ER stress; cAMP; p53; PUMA; Trabecular meshwork cells;

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9 by S. Antony Ceasar; Vinothkumar Rajan; Sergey V. Prykhozhij; Jason N. Berman; S. Ignacimuthu (2333-2344).
The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.
Keywords: Genome editing; CRISPR; Cas9; Mutation; Indel; sgRNA; Double stranded breaks;

Terminal regions of β-catenin are critical for regulating its adhesion and transcription functions by Mohd Saleem Dar; Paramjeet Singh; Gurjinder Singh; Gayatri Jamwal; Syed Sajad Hussain; Aarti Rana; Yusuf Akhter; Satdarshan P. Monga; Mohd Jamal Dar (2345-2357).
β-Catenin, the central molecule of canonical Wnt signaling pathway, has multiple binding partners and performs many roles in the cell. Apart from being a transcriptional activator, β-catenin acts as a crucial effector component of cadherin/catenin complex to physically interact with actin cytoskeleton along with α-catenin and E-cadherin for regulating cell-cell adhesion. Here, we have generated a library of β-catenin point and deletion mutants to delineate regions within β-catenin that are important for α-catenin-β-catenin interaction, nuclear localization, and transcriptional activity of β-catenin. We observed a unique mechanism for nuclear localization of β-catenin and its mutants and show that N-terminal exon-3 region and C-terminal domain of β-catenin are critical for this activity of β-catenin. Furthermore, we show HepG2 cells have high β-catenin mediated transcriptional activity due to the presence of an interstitial deletion at the N-terminal region of β-catenin. Due to this deletion mutant (hereupon called TM), GSK3β and HDAC inhibitors failed to show any impact whereas curcumin significantly inhibited β-catenin mediated transcriptional activity reiterating that TM is primarily responsible for the high transcriptional activity of HepG2 cells. Moreover, we show the recombinant TM does not physically interact with α-catenin, localizes predominantly in the nucleus, and has nearly two-fold higher transcriptional activity than the wildtype β-catenin.Fate of β-catenin wildtype and TM in HepG2 cells.Display Omitted
Keywords: Cellular signaling; Cancer; Cell proliferation and differentiation; Beta-catenin signaling; HepG2 cells; Transcription factors;