BBA - Molecular Cell Research (v.1863, #11)

Regulation of MAP kinase Hog1 by calmodulin during hyperosmotic stress by Jiyoung Kim; Junsang Oh; Gi-Ho Sung (2551-2559).
Mitogen-activated protein kinase (Hog1 in yeast and ortholog p38 in human cells) plays a critical role in the signal transduction pathway that is rapidly activated under multiple stress conditions. Environmental stress stimuli such as hyperosmotic stress cause changes in cellular ATP metabolism required for hyperosmotic stress tolerance. Furthermore, hyperosmotic stress induces rapid Ca2 + signals in eukaryotic cells. These Ca2 + signals can be decoded by Ca2 + sensor calmodulin (CaM). By using genetic and biochemical approaches, we demonstrate that Hog1 is a novel CaM-binding protein, and that CaM-binding to Hog1 is involved in the mediation of the hyperosmotic stress signaling pathway. In addition, we show that p38α, a human ortholog of Hog1, interacts with CaM, suggesting that the CaM-binding feature of Hog1/p38α is evolutionarily conserved in eukaryotic cells. Hog1 is likely involved in cellular ATP regulation through CaM signaling during hyperosmotic stress. Therefore, this work suggests that Hog1 plays an important role in connecting CaM signaling with the hyperosmotic stress pathway by directly interacting with CaM in Saccharomyces cerevisiae.Display Omitted
Keywords: Calmodulin; Hog1; Hyperosmotic stress; MAP kinase; p38; Yeast;

Heat shock protein-70kDa (Hsp70) is a member of molecular chaperone family, involved in the proper folding of various proteins. Hsp70 is important for tumor cell survival and is also reported to be involved in enhancing the drug resistance of various cancer types. Hsp70 controls apoptosis both upstream and downstream of the mitochondria by regulating the mitochondrial membrane permeabilization (MMP) and apoptosome formation respectively. In the present study, we have elucidated the role of Hsp70 in Gambogic acid (GA) induced apoptosis in bladder cancer cells. We observed that functional inhibition of Hsp70 by Pifithrin-μ switches GA induced caspase dependent (apoptotic) cell death to caspase independent cell death. However, this cell death was not essentially necrotic in nature, as shown by the observations like intact plasma membranes, cytochrome-c release and no significant effect on nuclear condensation/fragmentation. Inhibition of Hsp70 by Pifithrin-μ shows differential effect on MMP. GA induced MMP and cytochrome-c release was inhibited by Pifithrin-μ at 12 h but enhanced at 24 h. Pifithrin-μ also reverted back GA inhibited autophagy which resulted in the degradation of accumulated ubiquitinated proteins. Our results demonstrate that Hsp70 plays an important role in GA induced apoptosis by regulating caspase activation. Therefore, inhibition of Hsp70 may hamper with the caspase dependent apoptotic pathways induced by most anti-cancer drugs and reduce their efficacy. However, the combination therapy with Pifithrin-μ may be particularly useful in targeting apoptotic resistant cancer cells as Pifithrin-μ may initiate alternative cell death program in these resistant cells.
Keywords: Hsp70; Caspase; Programmed cell death; Apoptosis; Autophagy; Necrosis; Bladder cancer;

Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation by Héctor Diez; Ma José Benitez; Silvia Fernandez; Ignacio Torres-Aleman; Juan José Garrido; Francisco Wandosell (2574-2583).
PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase > Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels.Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3.
Keywords: Akt; PI3K; mTORC1; Signalling; Axon establishment; Polarity;

Shikonin induces apoptosis of lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling antagonized by p300 by Yun-Ji Jeung; Han-Gyeul Kim; Jiwon Ahn; Ho-Joon Lee; Sae-Bhom Lee; Misun Won; Cho-Rock Jung; Joo-Young Im; Bo-Kyung Kim; Seung-Kiel Park; Myung Jin Son; Kyung-Sook Chung (2584-2593).
Shikonin derivatives exert powerful cytotoxic effects including induction of apoptosis. Here, we demonstrate the cytotoxic efficacy of shikonin in vivo in xenograft models, which did not affect body weight as well as its reduction of cell viability in vitro using several non-small cell lung cancer (NSCLC) cell lines. We found that inhibition of AKT by shikonin activated the forkhead box (FOX)O3a/early growth response protein (EGR)1 signaling cascade and enhanced the expression of the target gene Bim, leading to apoptosis in lung cancer cells. Overexpression of wild-type or a constitutively active mutant of FOXO3a enhanced shikonin-induced Bim expression. The NAD+-dependent histone deacetylase sirtuin (SIRT)1 amplified the pro-apoptotic effect by deacetylating FOXO3a, which induced EGR1 binding to the Bim promoter and activated Bim expression. Meanwhile, PI3K/AKT activity was enhanced, whereas that of FOXO3a was reduced and p300 was upregulated by treatment with a sublethal dose of shikonin. FOXO3a acetylation was enhanced by p300 overexpression, while shikonin-induced Bim expression was suppressed by p300 overexpression, which promoted cell survival. FOXO3a acetylation was increased by p300 overexpression and treatment with SIRT1 inhibitor, improving cell survival. In addition, shikonin-induced FOXO3a nuclear localization was blocked by AKT activation and SIRT1 inhibition, which blocked Bim expression and conferred resistance to the cytotoxic effects of shikonin. The EGR1 increase induced by shikonin was restored by pretreatment with SIRT1 inhibitor. These results suggest that shikonin induces apoptosis in some lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling, and that AKT and p300 negatively regulate this process via Bim upregulation.
Keywords: Shikonin-induced apoptosis; AKT; p300; FOXO3a; EGR1; SIRT1;

Diapause (developmental arrest) is characterized by dramatic depression of metabolic activity and profoundly extends insect lifespan, similar to the Caenorhabditis elegans dauer stage and Drosophila longevity; however, the molecular mechanism of low metabolism in insect diapause is unclear. Here, we show that HIF-1α expression is significantly increased in diapause-destined pupal brains compared to nondiapause-destined pupal brains and that HIF-1α negatively regulates mitochondrial biogenesis. HIF-1α mediates this effect by inhibiting c-Myc activity via proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A (TFAM), which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA replication, is activated by the binding of c-Myc to the TFAM promoter, thereby inducing transcription. Loss of TFAM expression is a major factor contributing to reducing the mitochondrial activity. Thus, the HIF-1α-c-Myc-TFAM signaling pathway participates in the regulation of mitochondrial activity for insect diapause or lifespan extension.
Keywords: HIF-1α; C-Myc; TFAM; Developmental arrest; Lifespan extension; Helicoverpa armigera;

Adenoviral CCN gene transfers induce in vitro and in vivo endoplasmic reticulum stress and unfolded protein response by Erawan Borkham-Kamphorst; Bettina Therese Steffen; Eddy Van de Leur; Lidia Tihaa; Ute Haas; Marius M. Woitok; Steffen K. Meurer; Ralf Weiskirchen (2604-2612).
The endoplasmic reticulum (ER) is primarily recognized as the site of synthesis and folding of secreted membrane-bound and certain organelle-targeted proteins. Optimum protein folding requires several factors, including ATP, Ca2 + and an oxidizing environment to allow disulphide-bond formation. ER is highly sensitive to stress that perturb cellular energy levels, the redox state or the Ca2 + concentration. Such stresses reduce the protein folding capacity of the ER, resulting in the accumulation and aggregation of unfolded proteins, a condition referred to as unfolded protein response (UPR). Matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover. They exhibit a similar type of organization and share a closely related primary structure, including 38 conserved cysteine residues. Since CCN1/CYR61 overexpression in hepatic stellate cells (HSC) induces ER stress-related apoptosis, we endeavored to investigate whether the adenovirus mediated gene transfer of other members of CCN proteins incurs ER stress in primary HSC and hepatocytes. We found Ad5-CMV-CCN2, Ad5-CMV-CCN3 and Ad5-CMV-CCN4 to induce ER stress and UPR comparable to Ad5-CMV-CCN1. UPR is a pro-survival response to reduce accumulation of unfolded proteins and restore normal ER functioning. If, however protein aggregation is persistent and the stress cannot be resolved, signaling switches from pro-survival to pro-apoptosis. The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. Adenoviral gene transfer induced massive amounts of matricellular proteins proving to effectively mitigate liver fibrosis if targeted cell specific in HSC and myofibroblasts.
Keywords: CCN proteins; ER stress; unfolded protein response; hepatic stellate cells; hepatocytes; matricellular proteins;

The mitochondrial intermembrane space (IMS) is involved in protein transport, lipid homeostasis and metal ion exchange, while further acting in signalling pathways such as apoptosis. Regulation of these processes involves protein modifications, as well as stress-induced import or release of proteins and other signalling molecules. Even though the IMS is the smallest sub-compartment of mitochondria, its redox state seems to be tightly regulated. However, the way in which this compartment participates in the cross-talk between the multiple organelles and the cytosol is far from understood. Here we focus on newly identified IMS proteins that may represent future challenges in mitochondrial research. We present an overview of the import pathways, the recently discovered new components of the IMS proteome and how these relate to key aspects of cell signalling and progress made in stem cell and cancer research.Display Omitted
Keywords: Mitochondria; Protein import; Intermembrane space; Redox signalling; Metabolism; Mitochondrial proteome;

Low dose ouabain stimulates Na―K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells by Corey J. Ketchem; Clayton D. Conner; Rebecca D. Murray; Madalyn DuPlessis; Eleanor D. Lederer; Daniel Wilkey; Michael Merchant; Syed J. Khundmiri (2624-2636).
Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of Na―K ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of Na―K ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1 μg/kg body wt./day) for 9 days and increased angiotensin II levels in cell culture media after 24 h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10 pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.
Keywords: Na―K ATPase; Ouabain; AT1R; Proteomics; FRET-TIRF microscopy; Human kidney cells;

Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca2 + entry (SOCE). In addition, Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca2+ overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca2+ transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca2+ store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca2+ stores become overloaded, Ca2+ release is enhanced, expression of the mitochondrial Ca2+ uniporter (MCU) increases and most Ca2 + released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca2+ remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly.
Keywords: Aging; Hippocampal neurons; Store-operated calcium entry; ER-mitochondria cross talking; Stim1; Orai1;

Glutathione S-transferase ϴ-subunit as a phenotypic suppressor of pmr1Δ strain, the Kluyveromyces lactis model for Hailey-Hailey disease by G. Ficociello; E. Zanni; S. Cialfi; C. Aurizi; G. Biolcati; C. Palleschi; C. Talora; D. Uccelletti (2650-2657).
Hailey–Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1, the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump.In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1, the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells.We have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δmutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca2 +) homeostasis by decreasing the high cytosolic Ca2 + levels in pmr1Δstrain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart (GSTT1/M1) was observed in lesion-derived keratinocytes from HHD patients.These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology.
Keywords: PMR1; ATP2C1; Keratinocytes; Oxidative stress; Calcium; Glutathione S-transferase;

TSC2 N-terminal lysine acetylation status affects to its stability modulating mTORC1 signaling and autophagy by Ana García-Aguilar; Carlos Guillén; Mark Nellist; Alberto Bartolomé; Manuel Benito (2658-2667).
There is a growing evidence of the role of protein acetylation in different processes controlling metabolism. Sirtuins (histone deacetylases nicotinamide adenine dinucleotide-dependent) activate autophagy playing a protective role in cell homeostasis. This study analyzes tuberous sclerosis complex (TSC2) lysine acetylation, in the regulation of mTORC1 signaling activation, autophagy and cell proliferation. Nicotinamide 5 mM (a concentration commonly used to inhibit SIRT1), increased TSC2 acetylation in its N-terminal domain, and concomitantly with an augment in its ubiquitination protein status, leading to mTORC1 activation and cell proliferation. In contrast, resveratrol (RESV), an activator of sirtuins deacetylation activity, avoided TSC2 acetylation, inhibiting mTORC1 signaling and promoting autophagy. Moreover, TSC2 in its deacetylated state was prevented from ubiquitination. Using MEF Sirt1 +/+ and Sirt1 −/− cells or a SIRT1 inhibitor (EX527) in MIN6 cells, TSC2 was hyperacetylated and neither NAM nor RESV were capable to modulate mTORC1 signaling. Then, silencing Tsc2 in MIN6 or in MEF Tsc2 −/− cells, the effects of SIRT1 modulation by NAM or RESV on mTORC1 signaling were abolished. We also observed that two TSC2 lysine mutants in its N-terminal domain, derived from TSC patients, differentially modulate mTORC1 signaling. TSC2 K599M variant presented a lower mTORC1 activity. However, with K106Q mutant, there was an activation of mTORC1 signaling at the basal state as well as in response to NAM. This study provides, for the first time, a relationship between TSC2 lysine acetylation status and its stability, representing a novel mechanism for regulating mTORC1 pathway.Display Omitted
Keywords: Acetylation; TSC2; mTORC1; Stability; Ubiquitination; Proliferation;

Arsenic, a metalloid with cytotoxic and carcinogenic effects related to the disruption of glutathione homeostasis, induces the expression of ATF4, a central transcription factor in the cellular stress response. However, the interplay between factors downstream of ATF4 is incompletely understood. In this article, we investigate the role of Tribbles homolog 3 (TRIB3), a regulatory member of the ATF4 pathway, in determining cell sensitivity to arsenite. Our results show that arsenite potently upregulates Trib3 mRNA and protein in an ATF4-dependent manner in mouse embryonic fibroblasts. Trib3-deficient cells display increased susceptibility to arsenite-induced cell death, which is rescued by re-expressing TRIB3. In cells lacking TRIB3, arsenite stress leads to markedly elevated mRNA and protein levels of Chac1, a gene that encodes a glutathione-degrading enzyme and is not previously known to be repressed by TRIB3. Analysis of the Chac1 promoter identified two regulatory elements that additively mediate the induction of Chac1 by arsenite and ATF4, as well as the robust suppression of Chac1 by TRIB3. Crucially, Chac1 silencing enhances glutathione levels and eliminates the increased susceptibility of Trib3-deficient cells to arsenite stress. Moreover, Trib3-deficient cells demonstrate an increased rate of glutathione consumption, which is abolished by Chac1 knockdown. Taken together, these data indicate that excessive Chac1 expression is detrimental to arsenite-treated cell survival and that TRIB3 is critical for restraining the pro-death potential of Chac1 during arsenite stress, representing a novel mechanism of cell viability regulation that occurs within the ATF4 pathway.Display Omitted
Keywords: arsenite; cell death; stress response; transcriptional regulation; TRB3;

A-type lamin-dependent homo-oligomerization for pY19-Caveolin-2 to function as an insulin-response epigenetic regulator by Hayeong Kwon; Jaewoong Lee; Kyuho Jeong; Donghwan Jang; Moonjeong Choi; Yunbae Pak (2681-2689).
Association of Caveolin-2 in the inner nuclear membrane specifically with A-type lamin is crucial for the maintenance of its Tyr-19 phosphorylation to promote insulin-response epigenetic activation at the nuclear periphery. Here, we identify that pY19-Caveolin-2 in the inner nuclear membrane exists as homo-oligomeric forms and the A-type lamin is required for sustenance of its oligomeric status. Oligomerization-defective and hence pY19-dephosphorylated monomeric Caveolin-2 in the inner nuclear membrane is unable to carry out Caveolin-2-mediated epigenetic activation of Egr-1 and JunB genes and transactivation of Elk-1 and STAT3 in response to insulin. The homo-oligomeric pY19-Caveolin-2 localizes in and recruits epigenetic modifiers to the A-type lamin-enriched inner nuclear membrane microdomain for the epigenetic activation. Our data show that A-type lamin-dependent Caveolin-2 homo-oligomerization in the inner nuclear membrane microdomain is a precondition for pY19-Caveolin-2-mediated insulin-response epigenetic activation at the nuclear periphery.
Keywords: Caveolin-2; Homo-oligomerization; A-type lamin; Inner nuclear membrane microdomain; Epigenetic regulators;

HGF alleviates high glucose-induced injury in podocytes by GSK3β inhibition and autophagy restoration by Congying Zhang; Bo Hou; Siying Yu; Qi Chen; Nong Zhang; Hui Li (2690-2699).
Podocyte injury or loss plays a major role in the pathogenesis of proteinuric kidney disease including diabetic nephropathy (DN). High basal level of autophagy is critical for podocyte health. Recent studies have revealed that hepatocyte growth factor (HGF) can ameliorate podocyte injury and proteinuria. However, little is known about the impact of HGF on podocyte autophagy. In this study, we investigated whether and how HGF affects autophagy in podocytes treated with high glucose (HG) conditions. HGF significantly diminishes apoptosis, oxidative stress and autophagy impairment inflicted by HG in podocytes. These beneficial effects of HGF disappear once HGF receptor is blocked by SU11274, a specific inhibitor of c-Met. Moreover, HGF markedly suppresses HG-stimulated glycogen synthase kinase 3beta (GSK3β) activity. Accordingly, exogenous constitutively-active GSK3β overexpression using an adenoviral vector system (Ad-GSK3β-S9A) abrogates the ability of HGF to ameliorate HG-mediated podocyte injury while neither adenoviral-mediated overexpression of wild-type GSK3β (Ad-GSK3β-WT) nor adenoviral transduction of inactive GSK3β mutant (Ad-GSK3β-K85A) can counteract the protective effects of HGF on HG-treated podocytes. Collectively, these results suggest that HGF prevents HG-induced podocyte injury via an autophagy-promoting mechanism, which involves GSK3β inhibition.
Keywords: Hepatocyte growth factor; Autophagy; Podocyte; Glycogen synthase kinase 3 beta; High glucose;

Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality by Florian Christoph Sigloch; Julia Daniela Knopf; Juliane Weißer; Alejandro Gomez-Auli; Martin Lothar Biniossek; Agnese Petrera; Oliver Schilling (2700-2709).
Cathepsin B (CTSB) is a lysosomal endo- and exopeptidase that is also secreted in high amounts by malignant and non-malignant cells. We determined the effect of CTSB on the tumor cell secretome by shRNA-mediated silencing of CTSB mRNA expression and subsequent proteomic LC-MS/MS analysis of the cell supernatants. We identified significant protein changes of 17 secreted or shed proteins. Notably, we found a general reduction in protein abundance of ADAM10 substrates and lysosomal proteins. We corroborated reduced amounts of soluble ADAM10 (sADAM10) and soluble APP (sAPP) in the two cancer cell lines MDA-MB-231 and U2OS by immunoblotting. Interestingly, reductions in sADAM10 and sAPP could be reversed by re-introducing a catalytically inactive variant of CTSB, suggesting a formerly unknown non-catalytic function of the protease.
Keywords: CTSB; ADAM10; Non-catalytic function; LC-MS/MS; Secretome;

New insight into the role of metabolic reprogramming in melanoma cells harboring BRAF mutations by Anna Ferretta; Immacolata Maida; Stefania Guida; Amalia Azzariti; Letizia Porcelli; Stefania Tommasi; Paola Zanna; Tiziana Cocco; Michele Guida; Gabriella Guida (2710-2718).
This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma.Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.
Keywords: Melanoma; BRAF; PGC-1α; Lactate; MCT4; peIF2α;

Heterodimerization of the kappa opioid receptor and neurotensin receptor 1 contributes to a novel β-arrestin-2–biased pathway by Haiqing Liu; Yanjun Tian; Bingyuan Ji; Hai Lu; Qing Xin; Yunlu Jiang; Liangcai Ding; Jingmei Zhang; Jing Chen; Bo Bai (2719-2738).
Together with its endogenous ligands (dynorphin), the kappa opioid receptor (KOR) plays an important role in modulating various physiological and pharmacological responses, with a classical G protein–coupled pathway mediating analgesia and non-G protein–dependent pathway, especially the β-arrestin–dependent pathway, eliciting side effects of dysphoria, aversion, drug-seeking in addicts, or even relapse to addiction. Although mounting evidence has verified a functional overlap between dynorphin/KOR and neurotensin/neurotensin receptor 1 (NTSR1) systems, little is known about direct interaction between the two receptors. Here, we showed that KOR and NTSR1 form a heterodimer that functions as a novel pharmacological entity, and this heterodimer, in turn, brings about a switch in KOR-mediated signal transduction, from G protein–dependent to β-arrestin-2–dependent. This was simultaneously verified by analyzing a KOR mutant (196th residue) that lost the ability to dimerize with NTSR1. We also found that dual occupancy of the heterodimer forced the β-arrestin-2–dependent pathway back into Gi protein–dependent signaling, according to KOR activation. These data provide new insights into the interaction between KOR and NTSR1, and the newly discovered role of NTSR1 acting as a switch between G protein– and β-arrestin–dependent pathways of KOR also suggests a new approach for utilizing pathologically elevated dynorphin/KOR system into full play for its analgesic effect with limited side effects.
Keywords: GPCR; Kappa opioid receptor; Neurotensin receptor 1; Heterodimerization;

Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation by Masahiro Aizawa; Nozomi Sugimoto; Shinya Watanabe; Kazumasa Yoshida; Masatoshi Fujita (2739-2748).
GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.Display Omitted
Keywords: DNA replication; Chromatin; Histone chaperone; GRWD1; Cdt1; MCM;

Chemoresistance is a major barrier for tumor chemotherapy. It is well-known that chemoresistance increases with tumor progression. Chemoresistance is altered by both genetic mutations and the alteration of extracellular microenvironment. Particularly, the extracellular matrix (ECM) is remodeled during tumor progression. Therefore, ECM remodeling is expected to cause the acquisition of chemoresistance in highly malignant tumor tissue. Here, we prepared cultured cell-derived decellularized matrices that mimic native ECM in tumor tissues at different stages of malignancy, and 5-fluorouracil (5-FU) resistance was compared among these matrices. 5-FU resistance of colorectal tumor cells increased on the matrices derived from highly malignant tumor HT-29 cells, although the resistance did not increase on the matrices derived from low malignant tumor SW480 cells and normal CCD-841-CoN cells. The resistance on HT-29 cell-derived matrices increased through the activation of Akt and the upregulation of ABCB1 and ABCC1 without cell growth promotion, suggesting that ECM remodeling plays important roles in the acquisition of chemoresistance during tumor progression. It is expected that our decellularized matrices, or “staged tumorigenesis-mimicking matrices”, will become preferred cell culture substrates for in vitro analysis of comprehensive ECM roles in chemoresistance and the screening and pharmacokinetic analysis of anti-cancer drugs.
Keywords: Extracellular matrix; Decellularization; Tumor; Chemoresistance;

Ph myeloproliferative neoplasm red blood cells display deregulation of IQGAP1-Rho GTPase signaling depending on CALR/JAK2 status by Nuria Socoro-Yuste; Marie-Claire Dagher; Anne Gonzalez De Peredo; Julie Mondet; Affif Zaccaria; Florence Roux Dalvai; Isabelle Plo; Jean Yves Cahn; Pascal Mossuz (2758-2765).
Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph MPN pathogenesis.
Keywords: Myeloproliferative neoplasms; Erythrocyte proteome; IQGAP1; Rho GTPases;

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage by Kazuhiro Sakamaki; Takahiro M. Ishii; Toshiya Sakata; Kiwamu Takemoto; Chiyo Takagi; Ayako Takeuchi; Ryo Morishita; Hirotaka Takahashi; Akira Nozawa; Hajime Shinoda; Kumiko Chiba; Haruyo Sugimoto; Akiko Saito; Shuhei Tamate; Yutaka Satou; Sang-Kee Jung; Satoshi Matsuoka; Koji Koyamada; Tatsuya Sawasaki; Takeharu Nagai; Naoto Ueno (2766-2783).
Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.Display Omitted
Keywords: Apoptosis; Cell shrinkage; Field effect transistor; FRET; Patch clamp; Two-pore potassium channel;

Interleukin-17A mediates cardiomyocyte apoptosis through Stat3-iNOS pathway by Sheng-an Su; Du Yang; Wei Zhu; Zhejun Cai; Na Zhang; Lina Zhao; Jian-an Wang; Meixiang Xiang (2784-2794).
Interleukin-17A, a pro-inflammatory cytokine, has a direct proapoptotic effect on cardiomyocytes. However, the specific mechanism has not been clarified. In the present study, an in-vitro model of cardiomyocyte apoptosis induced by IL-17A stimulation was employed and the roles of iNOS and Stat3 involved were investigated. Our data showed that the neonatal mouse cardiomyocytes express IL-17 receptors: IL-17RA and IL-17RC, but did not express IL-17A. Exogenous IL-17A significantly induces iNOS expression and hence the cardiomyocyte apoptosis. Moreover, IL-17A-induced cardiomyocyte apoptosis can be achieved directly via iNOS activation. We further showed that exogenous IL-17A simultaneously triggers Stat3 activation, which in turn inhibits IL-17A-induced iNOS expression in cardiomyocytes. And both ChIP and dual-luciferase results confirmed that Stat3 directly inhibits transcriptional activities of iNOS via binding to its specific promoter region. Consistent with these data, silencing of Stat3 in fact can aggravate IL-17A-triggered cardiomyocyte apoptosis. Finally, using an in vivo myocardial ischemia/reperfusion injury model, we verified that Stat3 inhibition increased iNOS expression and exacerbated cardiomyocyte apoptosis. Thus, our data strongly support the notion that Stat3 plays a compensatory anti-apoptotic role in IL-17A/iNOS-mediated cardiomyocyte apoptosis via inhibiting iNOS transcription, providing a novel molecular mechanism of apoptosis regulation and complicated interactions between IL-17A/iNOS and IL-17A/Stat3 signalings.
Keywords: Interleukin-17A; Cardiomyocyte; Apoptosis; Stat3; iNOS; Ischemia/reperfusion injury;

Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes by Esther Groth; Jessica Pruessmeyer; Aaron Babendreyer; Julian Schumacher; Tobias Pasqualon; Daniela Dreymueller; Shigeki Higashiyama; Inken Lorenzen; Joachim Grötzinger; Didier Cataldo; Andreas Ludwig (2795-2808).
By mediating proteolytic shedding on the cell surface the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of growth factors, cytokines and adhesion molecules. We here report that stimulation of lung epithelial A549 tumor cells with phorbol-12-myristate-13-acetate (PMA) leads to the downregulation of the surface expressed mature form of ADAM17 without affecting ADAM10 expression. This reduction could not be sufficiently explained by metalloproteinase-mediated degradation, dynamin-mediated internalization or microdomain redistribution of ADAM17. Instead, surface downregulation of ADAM17 was correlated with the presence of its mature form in exosomes. Exosomal ADAM17 release was also observed in monocytic and primary endothelial cells where it could be induced by stimulation with lipopolysaccharide. Antibody-mediated surface labelling of ADAM17 revealed that at least part of exosomal ADAM17 was oriented with the metalloproteinase domain outside and had been expressed on the cell surface. Suppression of iRHOM2-mediated ADAM17 maturation prevented surface expression and exosomal release of ADAM17. Further, deletion of the protease's C-terminus or cell treatment with a calcium chelator diminished exosomal release as well as surface downregulation of ADAM17, underlining that both processes are closely associated. Co-incubation of ADAM17 containing exosomes with cells expressing the ADAM17 substrates TGFα or amphiregulin lead to increased shedding of both substrates. This was prevented when exosomes were prepared from cells with shRNA-mediated ADAM17 knockdown. These data indicate that cell stimulation can downregulate expression of mature ADAM17 from the cell surface and induce release of exosomal ADAM17, which can then distribute and contribute to substrate shedding on more distant cells.
Keywords: Metalloproteinase; ADAM17; ADAM10; exosomes; shedding; inflammation;

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones.Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, β3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and β3 is also seen in cultures of seminiferous tubules exposed to 10 nM ouabain.At the protein level we observed a significant stimulation of β3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of β3 integrin.The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology.
Keywords: Ouabain; alpha4 isoform; sodium pump; signaling; integrin; spermatogenic cells;

Aβ-Induced Drp1 phosphorylation through Akt activation promotes excessive mitochondrial fission leading to neuronal apoptosis by Dah Ihm Kim; Ki Hoon Lee; Amr Ahmed Gabr; Gee Euhn Choi; Jun Sung Kim; So Hee Ko; Ho Jae Han (2820-2834).
Mitochondrial dysfunction is known as one of causative factors in Alzheimer's disease (AD), inducing neuronal cell death. Mitochondria regulate their functions through changing their morphology. The present work was undertaken to investigate whether Amyloid β (Aβ) affects mitochondrial morphology in neuronal cells to induce apoptosis. Aβ treatment induced not only the fragmentation of mitochondria but also neuronal apoptosis in association with an increase in caspase-9 and -3 activity. Calcium influx induced by Aβ up-regulated the activation of Akt through CaMKII resulting in changes to the phosphorylation level of Drp1 in a time-dependent manner. Translocation of Drp1 from the cytosol to mitochondria was blocked by CB-124005 (an Akt inhibitor). Recruitment of Drp1 to mitochondria led to ROS generation and mitochondrial fission, accompanied by dysfunction of mitochondria such as loss of membrane potential and ATP production. ROS generation and mitochondrial dysfunction by Aβ were attenuated when treated with Mdivi-1, a selective Drp1 inhibitor. Furthermore, the sustained Akt activation induced not only the fragmentation of mitochondria but also the activation of mTOR, eventually suppressing autophagy. Inhibition of autophagic clearance of Aβ led to increased ROS levels and aggravating mitochondrial defects, which were blocked by Rapamycin (an mTOR inhibitor). In conclusion, sustained phosphorylation of Akt by Aβ directly activates Drp1 and inhibits autophagy through the mTOR pathway. Together, these changes elicit abundant mitochondrial fragmentation resulting in ROS-mediated neuronal apoptosis.
Keywords: Amyloid β (Aβ); Mitochondrial dynamics; Akt; Drp1; ROS; Autophagy;

The developmental transcriptome sequencing of bovine skeletal muscle reveals a long noncoding RNA, lncMD, promotes muscle differentiation by sponging miR-125b by Xiaomei Sun; Mingxun Li; Yujia Sun; Hanfang Cai; Xianyong Lan; Yongzhen Huang; Yueyu Bai; Xinglei Qi; Hong Chen (2835-2845).
Pervasive transcription of the mammalian genome generates numerous long noncoding RNAs (lncRNAs), which are of crucial importance in diverse biological processes. Recent advances in high throughput sequencing technology have helped to accelerate the pace of lncRNA discovery. However, no study on the overall expression patterns of lncRNAs during muscle development has been conducted. We reported here the first analysis of lncRNA landscape in bovine embryonic, neonatal and adult skeletal muscle using Ribo-Zero RNA-Seq, a technology which can capture both poly(A)+ and poly(A) transcripts. We finally defined 7692 high-confidence lncRNAs and uncovered 401 lncRNAs differentially expressed among three developmental stages, including lncMD, a novel muscle-specific lncRNA which is gradually up-regulated during myoblast differentiation. lncMD overexpression upregulated, whereas lncMD silencing decreased the expression of two well-established myogenic markers, myosin heavy chain (MHC) and myogenin (MyoG). In-depth analyses showed that lncMD acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2) is a direct target of miR-125b in cattle. Moreover, lncMD level was positively correlated with IGF2 mRNA level in bovine muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that lncMD acts as a ceRNA to sequester miR-125b, leading to heightened IGF2 expression and thus promotes muscle differentiation. Our findings also complement the reference genome annotation of cattle, which will likely be useful for further functional lncRNA cloning and more comprehensive studies on lncRNA regulation in muscle development.Display Omitted
Keywords: Long noncoding RNA; Competing endogenous RNA; Ribo-Zero RNA-Seq; Muscle differentiation; Cattle;

Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release by Camille Ettelaie; Mary E.W. Collier; Sophie Featherby; John Greenman; Anthony Maraveyas (2846-2857).
Restriction of tissue factor (TF) activity at the cell surface and TF release are critical for prevention of excessive coagulation. This study examined the regulation of TF dephosphorylation and its release through ubiquitination. A plasmid containing the sequence to express the tandem protein TF-tGFP was mutated to include an arginine-substitution at Lys255 within TF. MDA-MB-231 cell line, and HCAEC endothelial cells were transfected and subsequently activated with PAR2-agonist peptide. The wild-type and mutant TF-tGFP were immunoprecipitated from the cell lysates and the ubiquitination and phosphorylation state of TF examined. Analysis of the proteins showed that arginine-substitution of Lys255 within TF prevented its ubiquitination while the wild-type TF-tGFP was oligoubiquitinated. The TF-associated oligoubiquitin chain was estimated to contain up to 4 ubiquitin units, with the linkage formed between Lys63 of one ubiquitin unit, and the C-terminus of the next unit. The Lys255 → Arg substitution of TF-tGFP prolonged the phosphorylation of Ser253 within TF, compared to the wild-type TF-tGFP, lengthened the presence of TF-tGFP at the cell surface and extended the duration of TF-tGFP release from cells following PAR2 activation. A biotinylated 19-mer peptide corresponding to the C-terminus of TF (TFc) was used as substrate to show that the ubiquitination of TF was mediated by the Ube2D family of E2-enzymes and involved Mdm2. Moreover, double-phosphorylation of TFc was prerequisite for ubiquitination, with subsequent dephosphorylation of Ser253 by phosphatase PP2A. In conclusion, oligoubiquitination of Lys255 within TF permits PP2A to bind and dephosphorylate Ser253 and occurs to terminate TF release and contain its activity.Display Omitted
Keywords: Tissue factor; Ubiquitination; Phosphorylation; Ubiquitin-conjugating enzymes; Protein phosphatase 2 A; Microvesicles;