SOD1 Phosphorylation by mTORC1 Couples Nutrient Sensing and Redox Regulation.

Nutrients are not only organic compounds fueling bioenergetics and biosynthesis, but also key chemical signals controlling growth and metabolism. Nutrients enormously impact the production of reactive oxygen species (ROS), which play essential roles in normal physiology and diseases. How nutrient signaling is integrated with redox regulation is an interesting, but not fully understood, question. Herein, we report that superoxide dismutase 1 (SOD1) is a conserved component of the mechanistic target of rapamycin complex 1 (mTORC1) nutrient signaling. mTORC1 regulates SOD1 activity through reversible phosphorylation at S39 in yeast and T40 in humans in response to nutrients, which moderates ROS level and prevents oxidative DNA damage. We further show that SOD1 activation enhances cancer cell survival and tumor formation in the ischemic tumor microenvironment and protects against the chemotherapeutic agent cisplatin. Collectively, these findings identify a conserved mechanism by which eukaryotes dynamically regulate redox homeostasis in response to changing nutrient conditions.

Postsurgical Latent Pain Sensitization Is Driven by Descending Serotonergic Facilitation and Masked by µ-Opioid Receptor Constitutive Activity in the Rostral Ventromedial Medulla.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3-4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished on chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11 939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6ß-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6ß-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.SIGNIFICANCE STATEMENT Surgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here, we show that either chemogenetic inhibition of serotonergic neuron activity in the RVM or pharmacological inhibition of 5-HT3 receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MORCA in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

Contribution of µ Opioid Receptor-expressing Dorsal Horn Interneurons to Neuropathic Pain-like Behavior in Mice.

BACKGROUND: Intersectional genetics have yielded tremendous advances in our understanding of molecularly identified subpopulations and circuits within the dorsal horn in neuropathic pain. The authors tested the hypothesis that spinal µ opioid receptor-expressing neurons (Oprm1-expressing neurons) contribute to behavioral hypersensitivity and neuronal sensitization in the spared nerve injury model in mice. METHODS: The authors coupled the use of Oprm1Cre transgenic reporter mice with whole cell patch clamp electrophysiology in lumbar spinal cord slices to evaluate the neuronal activity of Oprm1-expressing neurons in the spared nerve injury model of neuropathic pain. The authors used a chemogenetic approach to activate or inhibit Oprm1-expressing neurons, followed by the assessment of behavioral signs of neuropathic pain. RESULTS: The authors reveal that spared nerve injury yielded a robust neuroplasticity of Oprm1-expressing neurons. Spared nerve injury reduced Oprm1 gene expression in the dorsal horn as well as the responsiveness of Oprm1-expressing neurons to the selective µ agonist (D-Ala2, N-MePhe4, Gly-ol)-enkephalin (DAMGO). Spared nerve injury sensitized Oprm1-expressing neurons, as reflected by an increase in their intrinsic excitability (rheobase, sham 38.62 ± 25.87 pA [n = 29]; spared nerve injury, 18.33 ± 10.29 pA [n = 29], P = 0.0026) and spontaneous synaptic activity (spontaneous excitatory postsynaptic current frequency in delayed firing neurons: sham, 0.81 ± 0.67 Hz [n = 14]; spared nerve injury, 1.74 ± 1.68 Hz [n = 10], P = 0.0466), and light brush-induced coexpression of the immediate early gene product, Fos in laminae I to II (%Fos/tdTomato+: sham, 0.42 ± 0.57% [n = 3]; spared nerve injury, 28.26 ± 1.92% [n = 3], P = 0.0001). Chemogenetic activation of Oprm1-expressing neurons produced mechanical hypersensitivity in uninjured mice (saline, 2.91 ± 1.08 g [n = 6]; clozapine N-oxide, 0.65 ± 0.34 g [n = 6], P = 0.0006), while chemogenetic inhibition reduced behavioral signs of mechanical hypersensitivity (saline, 0.38 ± 0.37 g [n = 6]; clozapine N-oxide, 1.05 ± 0.42 g [n = 6], P = 0.0052) and cold hypersensitivity (saline, 6.89 ± 0.88 s [n = 5] vs. clozapine N-oxide, 2.31 ± 0.52 s [n = 5], P = 0.0017). CONCLUSIONS: The authors conclude that nerve injury sensitizes pronociceptive µ opioid receptor-expressing neurons in mouse dorsal horn. Nonopioid strategies to inhibit these interneurons might yield new treatments for neuropathic pain.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

Short-term in vitro glutamine restriction differentially impacts the chromosomal stability of transformed and non-transformed cells.

Glutamine (Gln) is a non-essential amino acid central for generating building blocks and cellular energy in tumours and rapidly proliferating non-transformed cells. However, the influence of Gln on regulating chromosomal stability of transformed and non-transformed cells remain poorly understand. We hypothesised that Gln is required for maintaining a homeostatic level of chromosomal stability. To this end, transformed cells HeLa and A375 and non-transformed cells NCM460 and HUVEC cells were intervened with varying concentrations of Gln (10, 1, 0.1 and 0.01 mM), with or without cisplatin (0.1 µg/ml), for 24 h. The cytokinesis-block micronucleus (MN) assay was used to determine chromosomal instability (CIN), the extent of which is reflected by the frequency of MN, nucleoplasmic bridge (NPB) and nuclear bud (NB). We demonstrated an unexpected decrease in the spontaneous rate of MN, but not NPB and NB, after Gln restriction in HeLa and A375 cells. Gln restriction reduced cisplatin-induced MN, but not NPB and NB, in HeLa and A375 cells. We further revealed that Gln restriction suppressed the proliferation of HeLa cells with high CIN induced by nocodazole, partially explaining why Gln restriction decreased the frequency of spontaneous and cisplatin-induced MN in transformed cells. In contrast, Gln restriction increased MN and NB, but not NPB, in NCM460 cells. In HUVEC cells, Gln restriction increased MN, NPB and NB. Meanwhile, Gln restriction sensitised NCM460 cells to cisplatin-induced genotoxicity. A similar but more pronounced pattern was observed in HUVEC cells. Collectively, these results suggest that the in vitro influences of Gln metabolism on CIN depend on cellular contexts: Transformed cells require high Gln to fine tune their CIN in an optimal rate to maximise genomic heterogeneity and fitness, whereas non-transformed cells need high Gln to prevent CIN.

CREG1 Interacts with Sec8 to Promote Cardiomyogenic Differentiation and Cell-Cell Adhesion.

Understanding the regulation of cell-cell interactions during the formation of compact myocardial structures is important for achieving true cardiac regeneration through enhancing the integration of stem cell-derived cardiomyocytes into the recipient myocardium. In this study, we found that cellular repressor of E1A-stimulated genes 1 (CREG1) is highly expressed in both embryonic and adult hearts. Gain- and loss-of-function analyses demonstrated that CREG1 is required for differentiation of mouse embryonic stem (ES) cell into cardiomyocytes and the formation of cohesive myocardium-like structures in a cell-autonomous fashion. Furthermore, CREG1 directly interacts with Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Site-directed mutagenesis and rescue of CREG1 knockout ES cells showed that CREG1 binding to Sec8 is required for cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8, and N-cadherin colocalize at intercalated discs in vivo and are enriched at cell-cell junctions in cultured cardiomyocytes. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis. Stem Cells 2016;34:2648-2660.

miR-3940-5p reduces amyloid ß production via selectively targeting PSEN1.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aß) in brain. Mounting evidence has revealed critical roles of microRNAs (miRNAs) in AD pathogenesis; however, the miRNAs directly targeting presenilin1 (PSEN1), which encodes the catalytic core subunit of γ-secretase that limits the production of Aß from amyloid precursor protein (APP), are extremely understudied. The present study aimed to identify miRNAs targeting PSEN1 and its effect on Aß production. This study first predicted 5 candidate miRNAs that may target PSEN1,through websites such as TargetScan, miRDB, and miRwalk. Subsequently, the targeting specificity of the candidate miRNAs towards PS1 was validated using dual-luciferase reporter assays. To investigate the regulatory effect of miR-3940-5p on gene expression based on its targeting of PS1, miR-3940-5p mimics or inhibitors were transiently transfected into SH-SY5Y cells. Changes in PSEN1 transcription and translation in the tested cells were detected using RT-qPCR and Western Blot, respectively. Finally, to explore whether miR-3940-5p affects Aß production, SH-SY5Y APPswe cells overexpressing the Swedish mutant type of APP were transiently transfected with miR-3940-5p mimics, and the expression level of Aß was detected using ELISA. The results are as follows: The dual-luciferase reporter assays validated the targeting specificity of miR-3940-5p for PSEN1. Overexpression of miR-3940-5p significantly reduced the mRNA and protein levels of PSEN1 in SH-SY5Y cells. Conversely, inhibition of miR-3940-5p led to an increase in PSEN1 mRNA levels. Transfection of miR-3940-5p mimics into SH-SY5Y-APPswe cells resulted in a significant reduction in Aß42 and Aß40. Lentiviral-mediated overexpression of miR-3940-5p significantly decreased the expression of PSEN1 and did not significantly affect the expression of other predicted target genes. Furthermore, stable overexpression of miR-3940-5p in SH-SY5Y-APPswe cells mediated by lentivirus significantly reduced the expression of PSEN1 and the production of Aß42 and Aß40. Therefore, our study demonstrates for the first time the functional importance of miR-3940-5p in antagonizing Aß production through specific and direct targeting of PSEN1.

Telomere length and micronuclei trajectories in APP/PS1 mouse model of Alzheimer's disease: Correlating with cognitive impairment and brain amyloidosis in a sexually dimorphic manner.

Although studies have demonstrated that genome instability is accumulated in patients with Alzheimer's disease (AD), the specific types of genome instability linked to AD pathogenesis remain poorly understood. Here, we report the first characterization of the age- and sex-related trajectories of telomere length (TL) and micronuclei in APP/PS1 mice model and wild-type (WT) controls (C57BL/6). TL was measured in brain (prefrontal cortex, cerebellum, pituitary gland, and hippocampus), colon and skin, and MN was measured in bone marrow in 6- to 14-month-old mice. Variation in TL was attributable to tissue type, age, genotype and, to a lesser extent, sex. Compared to WT, APP/PS1 had a significantly shorter baseline TL across all examined tissues. TL was inversely associated with age in both genotypes and TL shortening was accelerated in brain of APP/PS1. Age-related increase of micronuclei was observed in both genotypes but was accelerated in APP/PS1. We integrated TL and micronuclei data with data on cognition performance and brain amyloidosis. TL and micronuclei were linearly correlated with cognition performance or Aß40 and Aß42 levels in both genotypes but to a greater extent in APP/PS1. These associations in APP/PS1 mice were dominantly driven by females. Together, our findings provide foundational knowledge to infer the TL and micronuclei trajectories in APP/PS1 mice during disease progression, and strongly support that TL attrition and micronucleation are tightly associated with AD pathogenesis in a female-biased manner.

Activation of Wnt/ß-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance.

AIMS: The present study investigated the exact proportion, the extent of in vitro proliferation potential, and oxaliplatin chemoresistance of EpCAMhigh/CD44+ cancer stem cells in colorectal cancer. Its underlying mechanism was also explored. BACKGROUND: Colorectal cancer stem cells (CSC) play crucial roles in tumorigenicity and chemoresistance. Multiple studies have shown that JAK/STAT, NOTCH, and Wnt/-catenin pathways, associated with tumour recurrence and metastasis, contribute to the proliferation and maintenance of CSCs. CSCs become resistant to chemo-radiotherapies by improving DNA damage repair, changing cell cycle checkpoints, and scavenging reactive oxygen species, resulting in a bad patient prognosis. OBJECTIVE: This work was carried out to determine the precise fraction, the degree of in vitro proliferation capability, and the level of oxaliplatin chemoresistance exhibited by EpCAMhigh/CD44+ cancer stem cells in colorectal cancer. The research was also done to investigate its underlying process. METHODS: Fluorescence-activated cell sorting (FACS) was applied to isolate the EpCAMhigh/CD44+ populations from three human colorectal cancer cell lines (HCT116, HT29, and LoVo), and we quantified the average proportion of the EpCAMhigh/CD44+ cells in every cell lines. The comparison of their proliferation ability and the chemoresistance to oxaliplatin with the parental cells was estimated by CCK8 assay. The activated signaling pathway was tested by Western Blotting. RESULTS: EpCAMhigh/CD44+ subpopulation comprises about 4.98±1.24% of the total human colorectal cancer cell lines, and the EpCAMhigh/CD44+ cells exhibited a highly better proliferation ability and stronger oxaliplatin chemoresistance than the parental cells. The wnt/ß-catenin signaling pathway is activated in EpCAMhigh/CD44+ HCT116 cells. CONCLUSION: Activation of Wnt/ß-Catenin signaling in EpCAMhigh/CD44+ cells endow colorectal cancer with tumor proliferation and oxaliplatin chemoresistance.

Cdc42 controls vascular network assembly through protein kinase Cι during embryonic vasculogenesis.

OBJECTIVE: The goal of this study was to determine the role of Cdc42 in embryonic vasculogenesis and the underlying mechanisms. METHODS AND RESULTS: By using genetically modified mouse embryonic stem (ES) cells, we demonstrate that ablation of the Rho GTPase Cdc42 blocks vascular network assembly during embryoid body (EB) vasculogenesis without affecting endothelial lineage differentiation. Reexpression of Cdc42 in mutant EBs rescues the mutant phenotype, establishing an essential role for Cdc42 in vasculogenesis. Chimeric analysis revealed that the vascular phenotype is caused by inactivation of Cdc42 in endothelial cells rather than surrounding cells. Endothelial cells isolated from Cdc42-null EBs are defective in directional migration and network assembly. In addition, activation of atypical protein kinase Cι (PKCι) is abolished in Cdc42-null endothelial cells, and PKCι ablation phenocopies the vascular abnormalities of the Cdc42-null EBs. Moreover, the inhibitory phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9 depends on Cdc42 and PKCι, and expression of kinase-dead GSK-3ß in Cdc42-null EBs promotes the formation of linear endothelial segments without branches. These results suggest that PKCι and GSK-3ß are downstream effectors of Cdc42 during vascular morphogenesis. CONCLUSIONS: Cdc42 controls vascular network assembly but not endothelial lineage differentiation by activating PKCι during embryonic vasculogenesis.

Genetic and functional evidence for gp130/IL6ST-induced transient receptor potential ankyrin 1 upregulation in uninjured but not injured neurons in a mouse model of neuropathic pain.

ABSTRACT: Peripheral nerve injuries result in pronounced alterations in dorsal root ganglia, which can lead to the development of neuropathic pain. Although the polymodal mechanosensitive transient receptor potential ankyrin 1 (TRPA1) ion channel is emerging as a relevant target for potential analgesic therapies, preclinical studies do not provide unequivocal mechanistic insight into its relevance for neuropathic pain pathogenesis. By using a transgenic mouse model with a conditional depletion of the interleukin-6 (IL-6) signal transducer gp130 in Nav1.8 expressing neurons (SNS-gp130-/-), we provide a mechanistic regulatory link between IL-6/gp130 and TRPA1 in the spared nerve injury (SNI) model. Spared nerve injury mice developed profound mechanical hypersensitivity as indicated by decreased withdrawal thresholds in the von Frey behavioral test in vivo, as well as a significant increase in mechanosensitivity of unmyelinated nociceptive primary afferents in ex vivo skin-nerve recordings. In contrast to wild type and control gp130fl/fl animals, SNS-gp130-/- mice did not develop mechanical hypersensitivity after SNI and exhibited low levels of Trpa1 mRNA in sensory neurons, which were partially restored by adenoviral gp130 re-expression in vitro. Importantly, uninjured but not injured neurons developed increased responsiveness to the TRPA1 agonist cinnamaldehyde, and neurons derived from SNS-gp130-/- mice after SNI were significantly less responsive to cinnamaldehyde. Our study shows for the first time that TRPA1 upregulation is attributed specifically to uninjured neurons in the SNI model, and this depended on the IL-6 signal transducer gp130. We provide a solution to the enigma of TRPA1 regulation after nerve injury and stress its significance as an important target for neuropathic pain disorders.

CREG1 promotes lysosomal biogenesis and function.

CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis.Abbreviations: AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB.

A comprehensive study of the genotoxic and anti-genotoxic effects of homocysteine in HUVECs and mouse bone marrow cells.

Elevated Homocysteine (Hcy) is associated with increased risk of vascular disease, but whether it induces genotoxicity to vascular endothelial cells remains unknown. Here, we conducted a comprehensive study of the genotoxicity, and unexpected anti-genotoxicity, of Hcy by cytokinesis-blocked micronucleus assay in HUVECs and erythrocyte micronucleus test in mouse bone marrow cells. Our experiments led to several important findings. First, while supraphysiological Hcy (SP-Hcy) exhibited remarkable genotoxicity, physiologically-relevant Hcy (PR-Hcy) reduced the basal genotoxicity. Second, among the metabolites of Hcy, cysteine phenocopied the anti-genotoxicity of PR-Hcy and, methionine, S-adenosylhomocysteine and H2S phenocopied the genotoxicity of SP-Hcy. Third, the genotoxicity of SP-Hcy was mitigated by vitamin B6, Fe2+ and Cu2+, but was exacerbated by N-acetylcysteine. Fourth, under pre-, co- or post-treatment protocol, both SP-Hcy and PR-Hcy attenuated the genotoxicity of cisplatin, mitomycin-C, nocodazole or deoxycholate. Finally, 100 and 250 mg/kg Hcy ameliorated cisplatin-induced genotoxicity in bone marrow cells of CF-1 and Kunming mice. Our results suggest that genotoxicity may be one mechanism through which Hcy confers an increased risk for vascular disease, but more importantly, they challenge the long-standing paradigm that Hcy is always harmful to human health. Our study calls for a more systematic effort in understanding the molecular mechanisms underlying the anti-genotoxicity of Hcy.

PTEN dephosphorylates Abi1 to promote epithelial morphogenesis.

The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.

PTEN suppresses epithelial-mesenchymal transition and cancer stem cell activity by downregulating Abi1.

The epithelial-mesenchymal transition (EMT) is an embryonic program frequently reactivated during cancer progression and is implicated in cancer invasion and metastasis. Cancer cells can also acquire stem cell properties to self-renew and give rise to new tumors through the EMT. Inactivation of the tumor suppressor PTEN has been shown to induce the EMT, but the underlying molecular mechanisms are less understood. In this study, we reconstituted PTEN-deficient breast cancer cells with wild-type and mutant PTEN, demonstrating that restoration of PTEN expression converted cancer cells with mesenchymal traits to an epithelial phenotype and inhibited cancer stem cell (CSC) activity. The protein rather than the lipid phosphatase activity of PTEN accounts for the reversal of the EMT. PTEN dephosphorylates and downregulates Abi1 in breast cancer cells. Gain- and loss-of-function analysis indicates that upregulation of Abi1 mediates PTEN loss-induced EMT and CSC activity. These results suggest that PTEN may suppress breast cancer invasion and metastasis via dephosphorylating and downregulating Abi1.

Cloning and spatio-temporal expression of porcine CDK5 and CDK5R1(p35) genes.

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase homologue attributed to the mitotic cyclin-dependent kinase family. Both the kinase activity and the biological effects of CDK5 in central nervous system are mainly dependent on association with its regulatory subunit 1 known as CDK5R1 (p35). In the present study, the full-length coding regions of CDK5 and CDK5R1 were cloned from pigs. Radiation hybrid mapping localized porcine CDK5 to chromosome 18q12-13, whereas CDK5R1 was electro-localized to chromosome 12q12. Real-time quantitative RT-PCR (qRT-PCR) showed that CDK5 mRNA is ubiquitously present in all porcine tissues examined, with relatively high levels in cerebral cortex, cerebellum, testicle and lung. We also examined the expression profile of porcine CDK5/CDK5R1 in various tissues at different developmental stages. The results indicated that CDK5 mRNA reaches the highest level in cerebral cortex at two months of age and in cerebellum and liver at 4 months of age, respectively, whereas the peak level of CDK5R1 was observed in both cerebral cortex and cerebellum at two months of age, indicating the pivotal role of CDK5/CDK5R1 during the development of porcine brain.

Nerve growth factor promotes formation of lumen-like structures in vitro through inducing apoptosis in human umbilical vein endothelial cells.

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl- current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl- current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl- current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl- currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

[Effect of CagA(+) helicobacter pylori strain on the expression of connexin 43 and cell proliferation in BGC-823 cells].

OBJECTIVE: To determine the effect of CagA(+) Helicobacter pylori(H.pylori)strain and anti-H.pylori drugs on the expression of connexin 43(Cx43) and cell proliferation of BGC-823 cells in vitro,and to investigate the relation between the changes of Cx43 expression, cell proliferation of BGC-823 cells and CagA(+)H.pylori. METHODS: BGC-823 cells were co-cultured with CagA(+) H.pylori strain(NCTC J99) or CagA(-) H.pylori strain(NCTC 12908)at bacteria/cells ratio of 20:1,100:1 and 500:1 for 24 hours and 48 hours respectively. anti-H.pylori drugs was given in the group co-cultured at bacteria/cells ratio of 100:1 after 16 hours. In the control group, BGC-823 cells were cultured for 24 hours and 48 hours respectively,but without H.pylori or antij H.pylori drugs. Immunocytochemical SABC method and the image analysis of the computer were applied to detect the changes of Cx43 expression in BGC-823 cells. The cell proliferation was examined by methyl tetrazolium (MTT) method. RESULTS: (1)The expression of Cx43 in the control group after cultivation for 48 hours was higher than that for 24 hours (P< 0.05). The expression of Cx43 in the groups co-cultured with CagA(+) H.pylori strain after cultivation for 48 hours was lower than that co-cultured for only 24 hours, and that of the groups co-cultured with CagA(+) H.pylori strain was lower than that of the control group for both 24 hours and 48 hours (P< 0.05). The expression of Cx43 in the groups at bacteria/cells ratio of 500:1 was lower than that at bacteria/cells ratio of 20:1 and 100:1 for both 24 and 48 hours (P< 0.05),and that at bacteria/cells ratio of 100:1 was lower than that at bacteria/cells ratio of 20:1 for 48 hours (P< 0.05).However, there was no significant difference in Cx43 expression between 24 and 48 hours in the groups co-cultured with CagA(-) H.pylori strain (P>0.05). Cx43 expression in the groups co-cultured with CagA(-) H.pylori strain at the ratio of 100:1 and 500:1 was lower than that in the control group, and Cx43 expression at the ratio of 500:1 was lower than that at the ratio of 20:1 for 24 hours and 48 hours. Cx43 expression increased after the intervention with anti-H.pylori drugs for 48 hours. (2) In the groups co-cultured with CagA(+)H.pylori strain, the optical density value of MTT indicated that the cell proliferation at the bacteria/cells ratio of 100:1 was higher than that in the control group, but no significant difference was found in other two groups co-cultured for 24 hours. After co-culturing for 48 hours, the cell proliferation at the bacteria/cells ratio of 20:1 and 100:1 was significantly accelerated, while the cell proliferation at 500:1 was inhibited. In the groups co-cultured with CagA(-) H.pylori strain,there was no change in the cell proliferation. Intervention with anti-H.pylori drugs could suppress the cell proliferation. CONCLUSION: CagA(+) H.pylori can down-regulate the expression of Cx43 in BGC-823 cells,which is related to the reaction time and the density of H.pylori. Low density of CagA(+)H.pylori suspensions can accelerate the proliferation of BGC-823 cells, while high density can suppress the cell proliferation. The CagA(-) H.pylori has no effect on the cell proliferation. Intervention with anti-H.pylori drugs can up-regulate the expression of Cx43,and suppress the cell proliferation of BGC-823 cells.

Integrin and dystroglycan compensate each other to mediate laminin-dependent basement membrane assembly and epiblast polarization.

During early embryogenesis, endodermal γ1-laminin expression is required for basement membrane (BM) assembly, promoting conversion of non-polar pluripotent cells into polarized epiblast. The influence of laminin-111 (Lm111) and its integrin and dystroglycan (DG) receptors on epiblast in embryoid bodies (EBs), a model for differentiation of the embryonic plate, was further investigated. Lm111 added to the medium of EBs initiated conversion of inner nonpolar cell to the polarized epiblast epithelium with an exterior-to-central basal-to-apical orientation. Microinjection of Lm111 into EB interiors resulted in an interior BM with complete inversion of cell polarity. Lm111 assembled a BM on integrin-ß1 null EBs with induction of polarization at reduced efficiency. ß-Integrin compensation was not detected in these nulls with integrin adaptor proteins failing to assemble. A dimer of laminin LG domains 4-5 (LZE3) engineered to strongly bind to α-dystroglycan almost completely inhibited laminin accumulation on integrin ß1-null EBs, reducing BM and ablating cell polarization. When Lm111 was incubated with integrin-ß1/dystroglycan double-knockout EBs, laminin failed to accumulate on the EBs, the EBs did not differentiate, and the EBs underwent apoptosis. Collectively the findings support the hypotheses that the locus of laminin cell surface assembly can determine the axis of epithelial polarity. This requires integrin- and/or dystroglycan-dependent binding to laminin LG domains with the highest efficiency achieved when both receptors are present. Finally, EBs that cannot assemble a matrix undergo apoptosis.