The FASEB Journal current issue

Free Radicals Can Kill You: Lavoisier's Oxygen Revolution [Editorial]

Weissmann, G. — Sun, 28 Feb 2010 21:00:58 PST

Secretory granules in inositol 1,4,5-trisphosphate-dependent Ca2+ signaling in the cytoplasm of neuroendocrine cells [Reviews]

Yoo, S. H. — Sun, 28 Feb 2010 21:00:58 PST

Of all the intracellular organelles, secretory granules contain by far the highest calcium concentration; secretory granules of typical neuroendocrine chromaffin cells contain ~40 mM Ca²⁺ and occupy ~20% cell volume, accounting for >60% of total cellular calcium. They also contain the majority of cellular inositol 1,4,5-trisphosphate receptors (IP₃Rs) in addition to the presence of >2 mM of chromogranins A and B that function as high-capacity, low-affinity Ca²⁺ storage proteins. Chromogranins A and B also interact with the IP₃Rs and activate the IP₃R/Ca²⁺ channels. In experiments with both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells, in which the number of secretory granules present was changed by either suppression or induction of secretory granule formation, secretory granules were demonstrated to account for >70% of the IP₃-induced Ca²⁺ releases in the cytoplasm. Moreover, the IP₃ sensitivity of secretory granule IP₃R/Ca²⁺ channels is at least ~6- to 7-fold more sensitive than those of the endoplasmic reticulum, thus enabling secretory granules to release Ca²⁺ ahead of the endoplasmic reticulum. Further, there is a direct correlation between the number of secretory granules and the IP₃ sensitivity of cytoplasmic IP₃R/Ca²⁺ channels and the increased ratio of IP₃-induced cytoplasmic Ca²⁺ release, highlighting the importance of secretory granules in the IP₃-dependent Ca²⁺ signaling. Given that secretory granules are present in all secretory cells, these results presage critical roles of secretory granules in the control of cytoplasmic Ca²⁺ concentrations in other secretory cells.—Yoo, S. H. Secretory granules in inositol 1,4,5-trisphosphate-dependent Ca²⁺ signaling in the cytoplasm of neuroendocrine cells.

Calreticulin: non-endoplasmic reticulum functions in physiology and disease [Reviews]

Sun, 28 Feb 2010 21:00:58 PST

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.—Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.

Fail-safe termination elements: a common feature of the eukaryotic genome? [Hypothesis]

Nabavi, S., Nazar, R. N. — Sun, 28 Feb 2010 21:00:58 PST

A recent scan of the human genome (1) <$REFLINK> identified ~11 million hairpins. Some have been linked to known sequences, such as viruses, transposable elements, and, more recently, regulating or microRNAs, but the significance, if any, of most sequences that can be predicted to form hairpins remains unknown. Here we show that hairpins that are cleaved by RNase III- like nucleases can induce termination, even with normally polyadenylated transcripts, and that a cleaved hairpin downstream of a normal termination signal can induce fail-safe termination. Because such cleavage sites appear common to intergenic regions, the results raise the possibility that similar fail-safe termination elements are widely distributed in the eukaryotic genome to prevent read-through transcription from disrupting downstream promoter elements or opposing transcription.—Nabavi, S., Nazar, R. N. Fail-safe termination elements: a common feature of the eukaryotic genome?

ILT2/HLA-G interaction impairs NK-cell functions through the inhibition of the late but not the early events of the NK-cell activating synapse [Research Communications]

Favier, B., LeMaoult, J., Lesport, E., Carosella, E. D. — Sun, 28 Feb 2010 21:00:58 PST

Natural killer (NK) cells play a crucial role in the antitumoral responses through cytolytic function and cytokine production. Expression of HLA-G at the surface of tumoral cells confers a protection against NK-cell cytolysis through its interaction with the ILT2 inhibitory receptor. Even though the role of this interaction on the inhibition of NK-cell cytotoxicity is well established, its effect on the molecular events occurring at the NK/target-cell synapse is not well characterized. We found that the interaction of the inhibitory receptor ILT2 with HLA-G inhibited the polarization of NK-cell lytic granules and the microtubule organizing center (MTOC) as well as the accumulation of filamentous actin (F-actin) at the area of contact. However, it did not affect the recruitment of the activatory receptor CD2 at the NK/target-cell interface. Even though CD2 was accumulated to the NK-cell synapse, the interaction of ILT2 with HLA-G efficiently inhibited intracellular calcium mobilization and IFN- polarized production of NK cells. These results indicate that while the ILT2/HLA-G interaction leads to the inhibition of NK-cell functions, it displays differential effects on cytoskeleton reorganization and CD2 localization at the NK-cell synapse.—Favier, B., LeMaoult, J., Lesport, E., Carosella, E. D. ILT2/HLA-G interaction impairs NK-cell functions through the inhibition of the late but not the early events of the NK-cell-activating synapse.

Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors [Research Communications]

Sun, 28 Feb 2010 21:00:58 PST

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1⁺ multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1⁺ progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.—Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1⁺ cardiovascular progenitors.

CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Sepsis is a severe infection-induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g., TNF-, has failed in clinical trials. The aim of this study was to investigate the effects of inhibiting CD14, a key upstream innate immunity molecule, on the early inflammatory and hemostatic responses in a pig model of gram-negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli lipopolysaccharide (LPS) (n=25 and n=9 animals, respectively). The animals were allocated into treatment (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF-, IL-1β, IL-6, and IL-8, but not the anti-inflammatory cytokine IL-10, were efficiently inhibited by anti-CD14. Furthermore, anti-CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase-9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin-antithrombin III complexes and plasminogen activator inhibitor-1 were significantly attenuated. Anti-CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14 inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation and reverses the procoagulant state but does not interfere with LPS levels or bacterial counts in E. coli-induced sepsis.—Thorgersen, E. B., Hellerud, B. C., Nielsen, E. W., Barratt-Due, A., Fure, H., Lindstad, J. K., Pharo, A., Fosse, E., Tønnessen, T. I., Johansen, H. T., Castellheim, A., Mollnes, T. E. CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs.

A versatile system for the neuronal subtype specific expression of lentiviral vectors [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Lentiviral expression vectors are powerful tools for gene therapy and long-term gene expression/repression in the mammalian brain. However, no specificity of transduction has been reported so far in the central nervous system. Here we have developed a novel system to achieve a neuronal subtype specific expression in either dopaminergic (DA) or GABAergic neurons. We employed a delivery strategy by which the transgene is not expressed until its activation by Cre recombinase. We successfully tested the system in vitro and then used this novel lentivector, containing loxP sites, in 2 different transgenic mouse lines expressing Cre either in DA or in GABAergic neurons. In both lines the reporter gene was detected exclusively in Cre-positive cells, demonstrating that with this experimental approach we were able to achieve completely specific expression of transgenes delivered by lentiviral vectors. This universal system can be applied to all neural subtypes making use of the growing number of specific Cre driver lines.— Tolu, S., Avale, M. E., Nakatani, H., Pons, S., Parnaudeau, S., Tronche, F., Vogt, A., Monyer, H., Vogel, R., de Chaumont, F., Olivo-Marin, J.-C., Changeux, J.-P., Maskos, U. A versatile system for the neuronal subtype specific expression of lentiviral vectors.

Tlr2 is critical for diet-induced metabolic syndrome in a murine model [Research Communications]

Himes, R. W., Smith, C. W. — Sun, 28 Feb 2010 21:00:59 PST

Obesity and its associated comorbidities, termed metabolic syndrome, are increasingly prevalent, and they pose a serious threat to the health of individuals and populations. Gene-environment interactions have been scrutinized since the kinetics of the increased prevalence of obesity would argue against a purely genetic etiology. Toll-like receptors (TLRs), widely expressed and highly conserved transmembrane receptors, are at the intersection of diet and metabolism, and may therefore be important determinants of weight gain and its sequellae. We sought specifically to determine the role of Tlr2 in the development of obesity and metabolic syndrome utilizing two dietary models that approximate contemporary diet compositions. Using C57BL/6 Hsd mice (wild type, WT) and mice with a targeted mutation in Tlr2 (Tlr2^–/–), we showed that mice lacking TLR2 are substantially protected from diet-induced adiposity, insulin resistance, hypercholesterolemia, and hepatic steatosis. In adipose tissue, Tlr2 deletion was associated with attenuation of adipocyte hypertrophy, as well as diminished macrophage infiltration and inflammatory cytokine expression.—Himes, R. W., Smith, C. W. Tlr2 is critical for diet-induced metabolic syndrome in a murine model.

Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-β₁, and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A_2BR. Based on this fact, we further purified CCFCs from A_2BR-deficient mice and demonstrated that A_2BR is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-β functions downstream of the A_2BR to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A_2BR signaling and offer a potential target for prevention and treatment of penile fibrosis, a dangerous complication seen in priapism.—Wen, J., Jiang, X., Dai, Y., Zhang, Y., Tang, Y., Sun, H., Mi, T., Phatarpekar, P. V., Kellems, R. E., Blackburn, M. R., Xia, Y. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A_2B adenosine receptor signaling.

Plasticity and recovery of skeletal muscle satellite cells during limb regeneration [Research Communications]

Morrison, J. I., Borg, P., Simon, A. — Sun, 28 Feb 2010 21:00:59 PST

Salamander limb regeneration depends on local progenitors whose progeny are recruited to the new limb. We previously identified a Pax7⁺ cell population in skeletal muscle whose progeny have the potential to contribute to the regenerating limb. However, the plasticity of individual Pax7⁺ cells, as well as their recovery within the new limb, was unclear. Here, we show that Pax7⁺ cells remain present after multiple rounds of limb amputation/regeneration. Pax7⁺ cells are found exclusively within skeletal muscle in the regenerating limb and proliferate where the myofibers are growing. Pax7 is rapidly down-regulated in the blastema, and analyses of clonal derivatives show that Pax7⁺ cell progeny are not restricted to skeletal muscle during limb regeneration. Our data suggest that the newt regeneration blastema is not entirely a composite of lineage-restricted progenitors. The results demonstrate that except for a transient and subsequently blunted increase, skeletal muscle satellite cells constitute a stable pool of reserve cells for multiple limb regeneration events.—Morrison, J. I., Borg, P., Simon, A. Plasticity and recovery of skeletal muscle satellite cells during limb regeneration.

De novo synthesis of {beta}-catenin via H-Ras and MEK regulates airway smooth muscle growth [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

β-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of β-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in increased β-catenin protein stability. Here, we report on a novel pathway that regulates the expression and nuclear presence of β-catenin. In proliferating human airway smooth muscle cells, we observed increased expression of β-catenin, which was required for proliferation. Interestingly, increased β-catenin expression was accompanied by an increase in β-catenin mRNA and was independent of β-catenin liberation from the plasma membrane, suggesting a role for de novo synthesis. This was confirmed using actinomycin D and cycloheximide, which abrogated the induction and nuclear localization of β-catenin protein. GSK-3 inhibition using SB216763 failed to regulate β-catenin mRNA. However, expression of dominant negative H-Ras or pharmacological inhibition of MEK reduced serum and TGF-β-induced β-catenin mRNA and protein. Collectively, these data indicate that β-catenin is an important signaling intermediate in airway smooth muscle growth and that its cellular accumulation and nuclear localization require de novo protein synthesis effected, in part, via H-Ras and MEK.—Gosens, R., Baarsma, H. A., Heijink, I. H., Oenema, T. A., Halayko, A. J., Meurs, H., Schmidt, M. De novo synthesis of β-catenin via H-Ras and MEK regulates airway smooth muscle growth.

Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells [Research Communications]

Zhu, M.-L., Kyprianou, N. — Sun, 28 Feb 2010 21:00:59 PST

Androgens are functionally required for the normal growth of the prostate gland and in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development and in cancer cell metastasis induced by factors within the microenvironment, such as transforming growth factor-β (TGF-β). This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells. The EMT pattern was evaluated on the basis of expression of the epithelial markers E-cadherin/β-catenin, and the mesenchymal markers N-cadherin, as well as cytoskeleton reorganization in response to 5-dihydrotestosterone (DHT; 1 nM) and/or TGF-β (5 ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and lead to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. These findings indicate the ability of androgens to induce EMT by potentially bypassing the functional involvement of TGF-β, thus contributing to metastatic behavior of prostate cancer cells.—Zhum, M.-L., Kyprianou, N. Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.—Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Activation of the cannabinoid 2 receptor (CB₂) reduces ischemic injury in several organs. However, the mechanisms underlying this protective action are unclear. In a mouse model of ischemic stroke, we show that the CB₂ agonist JWH-133 (1 mg · kg^–1 · d^–1) decreases the infarct size measured 3 d after onset of ischemia. The neuroprotective effect of JWH-133 was lost in CB₂-deficient mice, confirming the specificity of JWH-133. Analysis of bone marrow chimeric mice revealed that bone marrow-derived cells mediate the CB₂ effect on ischemic brain injury. CB₂ activation reduced the number of neutrophils in the ischemic brain as shown by FACS analysis and by measuring the levels of the neutrophil marker enzyme myeloperoxidase. Indeed, we found in vitro that CB₂ activation inhibits adherence of neutrophils to brain endothelial cells. JWH-133 (1 µM) also interfered with the migration of neutrophils induced by the endogenous chemokine CXCL2 (30 ng/ml) through activation of the MAP kinase p38. This effect on neutrophils is likely responsible for the neuroprotection mediated by JWH-133 because JWH-133 was no longer protective when neutrophils were depleted. In conclusion, our data demonstrate that by activating p38 in neutrophils, CB₂ agonists inhibit neutrophil recruitment to the brain and protect against ischemic brain injury.—Murikinati, S., Jüttler, E., Keinert, T., Ridder, D. A., Muhammad, S., Waibler, Z., Ledent, C., Zimmer, A., Kalinke, U., Schwaninger, M. Activation of cannabinoid 2 receptors protects against cerebral ischemia by inhibiting neutrophil recruitment.

Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments [Research Communications]

Na, N., Chandel, N. S., Litvan, J., Ridge, K. M. — Sun, 28 Feb 2010 21:00:59 PST

Hypoxia can cause stress and structural changes to the epithelial cytoskeleton. The intermediate filament (IF) network is known to reorganize in response to stress. We examined whether rats exposed to hypoxia had altered keratin IF expression in their alveolar epithelial type II (ATII) cells. There was a significant decrease in keratin protein levels in hypoxic ATII cells compared with those in ATII cells isolated from normoxic rats. To define the mechanisms regulating this process we studied changes to the keratin IF network in A549 cells (an alveolar epithelial cell line) exposed to 1.5% oxygen. We observed a time-dependent disassembly-degradation of keratin 8 and 18 proteins, which was associated with an increase in reactive oxygen species (ROS). Hypoxia-treated A549 cells deficient in mitochondrial DNA or A549 cells treated with a small interfering RNA against the Rieske iron-sulfur protein of mitochondrial complex III did not have increased levels of ROS nor was the keratin IF network disassembled and degraded. The superoxide dismutase (SOD)/catalase mimetic (EUK-134) prevented the hypoxia-mediated keratin IF degradation as did the overexpression of SOD1 but not of SOD2. Accordingly, we provide evidence that hypoxia promotes the disassembly and degradation of the keratin IF network via mitochondrial complex III-generated reactive oxygen species.—Na, N., Chandel, N. S., Litvan, J., Ridge, K. M. Mitochondrial reactive oxygen species are required for hypoxia-induced degradation of keratin intermediate filaments.

Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-{gamma}/JAK/STAT1 signaling is critical for the expression of PTN in macrophages [Research Communications]

Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. — Sun, 28 Feb 2010 21:00:59 PST

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)- was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN- in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at –2086 to –2078 bp is essential for IFN--regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN- stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN- and that PTN is involved in the adaptive immunity.—Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-/JAK/STAT1 signaling is critical for the expression of PTN in macrophages

Ethanol elevates physiological all-trans-retinoic acid levels in select loci through altering retinoid metabolism in multiple loci: a potential mechanism of ethanol toxicity [Research Communications]

Kane, M. A., Folias, A. E., Wang, C., Napoli, J. L. — Sun, 28 Feb 2010 21:00:59 PST

All-trans-retinoic acid (atRA) supports embryonic development, central nervous system function, and the immune response. atRA initiates neurogenesis and dendritic growth in the hippocampus and is required for spatial memory; superphysiological atRA inhibits neurogenesis, causes teratology and/or embryo toxicity, and alters cognitive function and behavior. Because abnormal atRA shares pathological conditions with alcoholism, inhibition of retinol (vitamin A) activation into atRA has been credited widely as a mechanism of ethanol toxicity. Here, we analyze the effects of ethanol on retinoid concentrations in vivo during normal vitamin A nutriture, using sensitive and analytically robust assays. Ethanol either increased or had no effect on atRA, regardless of changes in retinol and retinyl esters. Acute ethanol (3.5 g/kg) increased atRA in adult hippocampus (1.6-fold), liver (2.4-fold), and testis (1.5-fold). Feeding dams a liquid diet with 6.5% ethanol from embryonic day 13 (e13) to e19 increased atRA in fetal hippocampus (up to 20-fold) and cortex (up to 50-fold), depending on blood alcohol content. One-month feeding of the 6.5% ethanol diet increased atRA in adult hippocampus (20-fold), cortex (2-fold), testis (2-fold), and serum (10-fold). Tissue-specific increases in retinoid dehydrogenase mRNAs and activities, extrahepatic retinol concentrations, and atRA catabolism combined to produce site-specific effects. Because a sustained increase in atRA has deleterious effects on the central nervous system and embryo development, these data suggest that superphysiological atRA contributes to ethanol pathological conditions, including cognitive dysfunction and fetal alcohol syndrome.—Kane, M. A., Folias, A. E., Wang, C., Napoli, J. L. Ethanol elevates physiological all-trans-retinoic acid levels in select loci through altering retinoid metabolism in multiple loci: a potential mechanism of ethanol toxicity.

Loss of CAK phosphorylation of RAR{alpha} mediates transcriptional control of retinoid-induced cancer cell differentiation [Research Communications]

Wang, A., Alimova, I. N., Luo, P., Jong, A., Triche, T. J., Wu, L. — Sun, 28 Feb 2010 21:00:59 PST

Although the role of the classic retinoic acid (RA)-induced genomic pathway in cancer cell differentiation is well recognized, the underlying mechanisms remain to be dissected. Retinoic acid receptor (RAR) is a transcription factor activated by RA, and its serine 77 (RARS77) is the main residue phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. We report here that in both human myeloid leukemia and mouse embryonic teratocarcinoma stem cells, either RA-suppressed CAK phosphorylation of RAR or mutation of RARS77 to alanine (RARS77A) coordinates CAK-dependent G₁ arrest with cancer cell differentiation by transactivating RA-target genes. Both hypophosphorylated RAR and RARS77A reduce binding to retinoic acid-responsive elements (RARE) in the promoters of RA-target genes while stimulating gene transcription. The enhanced transactivation and reduced RAR-chromatin interaction are accompanied by RAR dissociation from the transcriptional repressor N-CoR and are association with the coactivator NCoA-3. Such effects of decreased CAK phosphorylation of RARS77 on mediating RA-dependent transcriptional control of cancer cell differentiation are examined correspondingly in both RA-resistant myeloid leukemia and embryonic teratocarcinoma stem RAR^–/– cells. These studies demonstrate, for the first time, that RA couples G₁ arrest to transcriptional control of cancer cell differentiation by suppressing CAK phosphorylation of RAR to release transcriptional repression.—Wang, A., Alimova, I. N., Luo, P. Jong, A., Triche, T. J., Wu, L. Loss of CAK phosphorylation of RAR mediates transcriptional control of retinoid-induced cancer cell differentiation.

Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser]^Sec gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV⁺ neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se-dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR⁺ interneurons remained normal in Gpx4-mutant mice, the number of PV⁺ interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.—Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.

Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

The functional significance of neuronal death for pathogenesis of epilepsy and the underlying molecular mechanisms thereof remain incompletely understood. The p53 transcription factor has been implicated in seizure damage, but its target genes and the influence of cell death under its control on epilepsy development are unknown. In the present study, we report that status epilepticus (SE) triggered by intra-amygdala kainic acid in mice causes rapid p53 accumulation and subsequent hippocampal damage. Expression of p53-up-regulated mediator of apoptosis (Puma), a proapoptotic Bcl-2 homology domain 3-only protein under p53 control, was increased within a few hours of SE. Induction of Puma was blocked by pharmacologic inhibition of p53, and hippocampal damage was also reduced. Puma induction was also blocked in p53-deficient mice subject to SE. Compared to Puma-expressing mice, Puma-deficient mice had significantly smaller hippocampal lesions after SE. Long-term, continuous telemetric EEG monitoring revealed a ~60% reduction in the frequency of epileptic seizures in the Puma-deficient mice compared to Puma-expressing mice. These are the first data showing genetic deletion of a proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long-term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.—Engel, T., Murphy, B. M., Hatazaki, S., Jimenez-Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.

Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Melanocortin-3 receptors (Mc3rs) in the central nervous system are involved in expression of anticipatory rhythms and synchronizing clocks maintaining circadian rhythms during restricted feeding (RF) [mice housed under a 12-h light-dark cycle with lights on between zeitgeber time (ZT) 0 to ZT12 fed 60% of normal calories between ZT7 and ZT11]. Because the systems governing circadian rhythms are important for adaptation to RF, we investigated whether Mc3rs are required for metabolic adaption to RF. Mc3r^–/– mice subjected to RF exhibited normal weight loss; however, they developed hyperinsulinemia, glucose intolerance, increased expression of lipogenic genes, and increased ketogenesis relative to controls. Rhythmic expression of transcription factors regulating liver clock activity and energy metabolism (Bmal1, Rev-erb, Pgc1, Foxo1, Hnf4, and Pck1) was severely compromised in Mc3r^–/– mice during RF. Inhibition of neural melanocortin receptors by agouti-related peptide also attenuated rhythmicity in the hepatic expression of these genes during RF. Collectively, these data suggest that neural Mc3rs are important for adapting metabolism and maintaining rhythms of liver metabolism during periods when feeding is restricted to the light cycle.—Sutton, G. M., Begriche, K., Kumar, K. G., Gimble, J. M., Perez-Tilve, D., Nogueiras, R., McMillan, R. P., Hulver, M. W., Tschöp, M. H., Butler, A. A. Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle.

Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function [Research Communications]

Minhas, N., Xue, M., Fukudome, K., Jackson, C. J. — Sun, 28 Feb 2010 21:00:59 PST

Activated protein C (APC) is an anticoagulant, approved as a treatment for severe sepsis, that can prevent apoptosis, inflammation, and vascular leakage. The aim of this study was to investigate whether APC protects endothelial barrier function through the angiopoietin (Ang)/Tie2 axis. APC significantly up-regulated gene and protein expression of Tie2 and Ang1 in a dose (0.01–10 µg/ml)- and time (0.5–24 h)-dependent manner in human umbilical vein endothelial cells (HUVECs). Interestingly, it markedly inhibited Ang2 with an IC₅₀ of ~0.1 µg/ml. HUVEC permeability, measured using Evans blue dye transfer, was significantly reduced in the presence of APC, and, in concordance, the tight junction associated protein zona occludens (ZO)-1 was up-regulated and localized peripherally around cells, compared with controls. Smooth muscle cell migration toward APC-stimulated HUVECs was elevated compared with unstimulated cells. Blocking antibodies and small interfering (si) RNA treatment, compared with isotype (IgG) or scrambled siRNA controls, showed that APC requires 3 receptors, the endothelial protein C receptor, protease-activated receptor 1, and Tie2 to perform all these barrier stabilization functions. In summary, this study demonstrates that APC has novel effects on the Ang/Tie2 axis, which enhance endothelial barrier function and are likely to contribute to its therapeutic effect in sepsis and other diseases associated with vascular leakage.—Minhas, N., Xue, M., Fukudome, K., Jackson, C. J. Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1, Guy’s 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 µg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 µg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.—Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

Transcriptional regulation of the small GTPase RhoB gene by TGF{beta}-induced signaling pathways [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

The purpose of the present study was to investigate the mechanism of transcriptional induction of the small GTPase RhoB gene by the transforming growth factor β (TGFβ) signaling pathway and the role of this regulation in TGFβ-induced cell migration. To achieve our goals, we utilized a combination of siRNA-mediated gene silencing, adenovirus-mediated gene transfer receptor and MAPK inhibition, transactivation assays, and DNA-protein interaction assays in human HaCaT keratinocytes. We found that the RhoB gene is a direct transcriptional target of TGFβ. We show that TGFβ activates an early MEK/ERK pathway and that this activation is required for the recruitment of Smad3 to a novel, nonclassical, Smad binding element in the proximal RhoB promoter, in a p53-dependent manner. This element is overlapping with a CCAAT box that constitutively binds nuclear factor Y. Mutagenesis of this site abolished the Smad-mediated transactivation of the RhoB promoter. Finally, silencing of RhoB gene expression via siRNA or utilization of a dominant negative form of RhoB significantly inhibited TGFβ-induced migration of HaCaT keratinocytes and DU145 prostate cancer cells. Our findings establish RhoB as a direct transcriptional target of TGFβ in human keratinocytes and identify an important role of RhoB in TGFβ-induced cell migration.—Vasilaki, E., Papadimitriou, E., Tajadura, V., Ridley, A. J., Stournaras, C., Kardassis, D. Transcriptional regulation of the small GTPase RhoB gene by TGFβ-induced signaling pathways.

Docosahexaenoic acid metabolome in neural tumors: identification of cytotoxic intermediates [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Docosahexaenoic acid (DHA) protects neural cells from stress-induced apoptosis. On the contrary, DHA exerts anticancer effects, and we have shown that DHA induces apoptosis in neuroblastoma, an embryonal tumor of the sympathetic nervous system. We now investigate the DHA metabolome in neuroblastoma using a targeted lipidomic approach in order to elucidate the mechanisms behind the DHA-induced cytotoxicity. LC-MS/MS analysis was used to identify DHA-derived lipid mediators in neuroblastoma cells. Presence of the 15-lipoxygenase enzyme was investigated using immunoblotting, and cytotoxic potency of DHA and DHA-derived compounds was compared using the MTT cell viability assay. Neuroblastoma cells metabolized DHA to 17-hydroxydocosahexaenoic acid (17-HDHA) via 17-hydroperoxydocosahexaenoic acid (17-HpDHA) through 15-lipoxygenase and autoxidation. In contrast to normal neural cells, neuroblastoma cells did not produce the anti-inflammatory and protective lipid mediators, resolvins and protectins. 17-HpDHA had significant cytotoxic potency, with an IC₅₀ of 3–6 µM at 72 h, compared to 12–15 µM for DHA. -Tocopherol protected cells from 17-HpDHA-induced cytotoxicity. DHA inhibited secretion of prostaglandin-E₂ and augmented the cytotoxic potency of the cyclooxygenase-2-inhibitor celecoxib. The cytotoxic effect of DHA in neuroblastoma is mediated through production of hydroperoxy fatty acids that accumulate to toxic intracellular levels with restricted production of its products, resolvins and protectins.—Gleissman, H., Yang, R., Martinod, K., Lindskog, M., Serhan, C. N., Johnsen, J. I., Kogner, P. Docosahexaenoic acid metabolome in neural tumors: identification of cytotoxic intermediates.

The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding [Research Communications]

Hoashi, T., Tamaki, K., Hearing, V. J. — Sun, 28 Feb 2010 21:00:59 PST

Ectodomain shedding is a proteolytic mechanism by which a transmembrane protein is converted into a secreted form. Pmel17/gp100 is a melanocyte-specific membrane-bound glycoprotein that has amyloid characteristics and forms fibrillar structures in melanosomes after a complex sequence of post-translational processing and trafficking events, including cleavage by a furin-like proprotein convertase (PC). A secreted form of Pmel17 (termed sPmel17) was also thought to be released due to cleavage by a PC. We used multidisciplinary approaches to demonstrate that sPmel17 is released by ectodomain shedding at the juxtamembrane and/or intramembrane motif and to show that this is independent of cleavage by a PC. We further show that sPmel17 consists of 2 fragments linked by disulfide bonds and that the shedding is inhibited at low temperature but not by metalloproteinase inhibitors. Moreover, treatment with a phorbol ester or a calmodulin inhibitor induces Pmel17 shedding. We also refine the reactivity of HMB50 and NKI/beteb, 2 monoclonal antibodies commonly used as melanoma-specific markers. The fact that those antibodies require physically separated domains of Pmel17 sheds interesting light on its 3-dimensional conformation. We conclude that sPmel17 is released by regulated proteolytic ectodomain shedding.—Hoashi, T., Tamaki, K., Hearing, V. J. The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding.

Paraoxonase 1 protects against protein N-homocysteinylation in humans [Research Communications]

Perla-Kajan, J., Jakubowski, H. — Sun, 28 Feb 2010 21:00:59 PST

Genetic or nutritional disorders in homocysteine (Hcy) or folate metabolism elevate plasma Hcy-thiolactone and lead to vascular and/or brain pathologies. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-Hcy-protein with autoimmunogenic and prothrombotic properties. Paraoxonase (PON1), carried on high-density lipoproteins (HDLs) in the blood, hydrolyzes Hcy-thiolactone and protects against the accumulation of N-Hcy-protein in vitro. To determine its role in vivo, we studied how natural variation in Hcy-thiolactonase activity of PON1 affects plasma N-Hcy-protein levels in cystathionine β-synthase-deficient patients (n=28). We found that plasma N-Hcy-protein was negatively correlated with serum Hcy-thiolactonase activity (r=–0.43, P=0.01), i.e., the higher the Hcy-thiolactonase activity, the lower N-Hcy protein levels. This relation was faithfully replicated in vitro in experiments with radiolabeled Hcy-thiolactone. We also found that enzymatic activities of the PON1 protein measured with artificial substrates correlated less strongly (r=–0.36, P=0.025 for paraoxonase activity) or did not correlate at all (phenylacetate hydrolase and TBLase activities) with plasma N-Hcy protein. These findings provide evidence that the Hcy-thiolactonase activity of PON1 is a determinant of plasma N-Hcy-protein levels and that Hcy-thiolactonase/PON1 protects proteins against N-homocysteinylation in vivo, a novel mechanism likely to contribute to atheroprotective roles of HDL in humans.—Perla-Kaján, J., Jakubowski, H. Paraoxonase 1 protects against protein N-homocysteinylation in humans.

Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma [Research Communications]

Jarmalavicius, S., Trefzer, U., Walden, P. — Sun, 28 Feb 2010 21:00:59 PST

The aim of the study was to identify as potential therapeutic targets specific molecular alterations in tumor cells recognized by the immune system. To identify such targets, we analyzed the human leukocyte antigen (HLA) peptidomes of human melanoma cells by 2-dimensional nano-HPLC/mass spectrometry and tested the immunological significance of the peptides by ex vivo ELISpot assays with lymphocytes from melanoma patients. The peptide SQNPRFYHK was identified as derived from the regulator of the nuclear corepressor complex (NCoR) G-protein pathway suppressor 2 (GPS-2) and to be differentially unmethylated, monomethylated or asymmetrically dimethylated at the arginine. The methylation state was specifically recognized by the immune system in that only the monomethylated variant induced T-cell responses and significantly stronger responses in patients than in healthy controls. The methylations were confirmed with synthetic analogues and in vitro radiolabeling assays with recombinant GPS-2 and synthetic peptides. The immunity of the 3 variants of GPS-2 was tested in T-cell assays with T lymphocytes of melanoma patients compared with healthy donors. The results show for the first time that GPS-2 is differentially methylated at a site that lacks known methylation motifs and that the methylation state is detected by the immune system.—Jarmalavicius, S., Trefzer, U., Walden, P. Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma.

Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1 [Research Communications]

Sun, 28 Feb 2010 21:00:59 PST

Hacs1, a SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1^–/– mice by deletion of the SH3 and SAM domains. Hacs1^–/– mice were viable and fertile and had normal bone marrow B-cell development and normal splenic T- and B-cell populations. However, adult Hacs1^–/– mice had increased peritoneal B1a cells (IgM⁺CD5⁺). On immunization with T-cell-independent antigen TNP-Ficoll, Hacs1^–/– mice had increased production of anti-TNP IgM and IgG3. Purified splenic B cells from Hacs1^–/– mice showed increased cell proliferation on BCR (B-cell receptor) stimulation. We further demonstrate that the Hacs1^–/– B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T-helper type 1 (T_h1) and T-helper type 2 (T_h2) humoral responses were enhanced in Hacs1^–/– mice. In vitro bone marrow-derived Hacs1^–/– dendritic cells showed increased IL-12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.—Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.-Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

Reviewer Acknowledgments [Reviewer Acknowledgments]

Sun, 28 Feb 2010 21:00:59 PST