GFP Control Research Articles

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

Abstract WP91: Chemogenetic Activation of Cholinergic Neurons in the Mouse Nucleus Basalis During Stroke Recovery

The cholinergic projection neurons of the nucleus basalis (NB) mediate learning and cortical plasticity. Artificial activation of NB during tone presentation has been shown to increase the representation of paired frequencies in the auditory cortex, and when NB is stimulated during motor behavior it increases task-relevant representations of the cortical motor map. NB activation during successful movements also increases performance after training. We tested the hypothesis that increased activity of NB neurons during rehabilitative motions can improve the extent of stroke recovery. We injected a cre-dependent Gq DREADD (Designer Receptors Activated by Designer Drugs) or control GFP virus bilaterally into the nucleus basalis of transgenic C57/BL6 mice expressing cre in cholinergic neurons. Photothrombotic stroke was then targeted to sensorimotor representations of the forelimb, and performance in the automated reach task used to assess functional deficits and recovery. From weeks 2-6 after stroke, mice received and injection of the DREADD-activating drug clozapine-N-oxide before rehabilitative sessions, and final drug-free performance was assessed at week 7. We see a biologically significant trend toward greater improvement at week 7 after stroke in the average performance of DREADD-injected mice compared to the control group. Methods for improved replication and increasing effect size will be discussed.

Delivery of Sonic Hedgehog Gene Repressed Irradiation-induced Cellular Senescence in Salivary Glands by Promoting DNA Repair and Reducing Oxidative Stress.

Rationale: Irreversible hypofunction of salivary glands or xerostomia is common in head and neck cancer survivors treated with radiotherapy even when various new techniques are applied to minimize the irradiation (IR) damage. This condition severely impairs the quality of life of patients and can only be temporarily relieved with current treatments. We found recently that transient expression of Sonic Hedgehog (Shh) in salivary glands after IR rescued salivary function, but the underlying mechanisms are not totally clear.Methods: We generated a mouse model of IR-induced hyposalivation, and delivered adenoviral vectors carrying Shh or control GFP gene into submandibular glands (SMGs) via retrograde ductal instillation 3 days after IR. The cellular senescence was evaluated by senescence-associated beta-galactosidase assay and the expression of senescence markers. The underlying mechanisms were explored by examining DNA damage, oxidative stress, and the expression of related genes by qRT-PCR, Western blot and immunofluorescent staining.Results: Shh gene transfer repressed IR-induced cellular senescence by promoting DNA repair and decreasing oxidative stress, which is mediated through upregulating expression of genes related to DNA repair such as survivin and miR-21 and repressing expression of pro-senescence gene Gdf15 likely downstream of miR-21.Conclusion: Repressing cellular senescence contributes to the rescue of IR-induced hyposalivation by transient activation of Hh signaling, which is related to enhanced DNA repair and decreased oxidative stress in SMGs.

Intragland Shh gene delivery mitigated irradiation-induced hyposalivation in a miniature pig model

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found recently in mouse models that transient activation of Hedgehog pathway following irradiation rescued salivary gland function by preserving salivary stem/progenitor cells, parasympathetic innervation and microvessels. Due to huge differences between salivary glands of rodents and humans, to examine the translational potential of this approach, we evaluated effects of Shh gene transfer in a miniature pig model of irradiation-induced hyposalivation.Methods: The right parotid of each pig was irradiated with a single dose of 20 Gray. Shh and control GFP genes were delivered into irradiated parotid glands by noninvasive retrograde ductal instillation of corresponding adenoviral vectors 4 or 16 weeks after irradiation. Parotid saliva was collected every two weeks. Parotid glands were collected 5 or 20 weeks after irradiation for histology, Western blot and qRT-PCR assays.Results: Shh gene delivery 4 weeks after irradiation significantly improved stimulated saliva secretion and local blood supply up to 20 weeks, preserved saliva-producing acinar cells, parasympathetic innervation and microvessels as found in mouse models, and also activated autophagy and inhibited fibrogenesis in irradiated glands.Conclusion: These data indicate the translational potential of transient activation of Hedgehog pathway to preserve salivary function following irradiation.

A clinical-grade gene therapy vector for pharmacoresistant epilepsy successfully overexpresses NPY in a human neuronal cell line

PurposeEpilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial. MethodsA human neuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods ResultsWe found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls. ConclusionThis first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.

Overlap of SEC23A and SEC23B Function Suggests a Novel Therapeutic Approach for Congenital Dyserythropoietic Anemia Type II

Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disease characterized by anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from loss of function mutations in SEC23B encoding a core component of coat complex protein II (COPII) vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Despite the identification of the genetic cause of CDAII, the pathophysiology of the disease remains unknown.Morpholino-induced SEC23B deficiency in zebrafish (ZF) has been previously reported to result in an erythroid phenotype mimicking CDAII (Shwartz et al, Nature genetics 2009), suggesting conservation of the underlying CDAII mechanism from fish to humans. Thus, we were puzzled to observe the absence of anemia or other CDAII characteristics in mice with erythroid specific (EpoR -Cre) and pan-hematopoietic (Vav1 -Cre) SEC23B deficiency (Khoriaty et al, MBC and Khoriaty et al, Sci Rep).To re-examine the ZF phenotype, we injected the morpholino targeting Sec23b into one-cell stage ZF embryos demonstrating no increase in circulating bi-nucleated erythroid cells, in contrast to the previous report. Given the variable knock-down that can result from morpholinos, we next generated ZF heterozygous for a 53 bp deletion (Sec23b+/-) using CRISPR/Cas9 genome editing. Intercrosses between Sec23b+/- ZF demonstrated lethality of Sec23b-/- ZF between days 17-21. However, the percentage of circulating bi-nucleated erythrocytes observed at day 16 was indistinguishable between Sec23b-/- ZF and wildtype (WT) clutch mate controls.Mammals and fish express two paralogs for SEC23, SEC23A and SEC23B, encoding highly related (~85%) proteins. To investigate the different functions of SEC23A and SEC23B, we defined the SEC23A and SEC23B interactomes using “BioID” (proximity dependent biotinylation) in HEK293 cells expressing BirA*-tagged SEC23A, SEC23B, or GFP control. Surprisingly, SEC23A and SEC23B exhibit indistinguishable interactomes. We also demonstrated that both mouse and human SEC23 paralogs can complement SEC23 deficiency in yeast. Similarly, rescue of the Sec23b-/- lethal phenotype in ZF by a Sec23a transgene demonstrated at least partial functional overlap of SEC23A/SEC23B function in vertebrates.To extend these observations to mammals, we genetically engineered the murine Sec23a cDNA into the endogenous mouse genomic locus of Sec23b . We demonstrated that SEC23B-deficient mice (previously shown to die perinatally from pancreatic degeneration) are rescued by SEC23A, exhibiting normal survival and pancreas histology, with no abnormalities apparent on detailed hematologic and anatomic examination.The expression of SEC23A and SEC23B mRNAs in human and mouse BMs were examined by qRT-PCR. SEC23B is the predominantly expressed paralog in human BM, with greater levels of SEC23A and reduced SEC23B in mouse BM. We therefore hypothesized that mice with erythroid deficiency of SEC23A alone or combined SEC23A/SEC23B deficiency might exhibit an erythroid defect. We first generated mice with erythroid-specific SEC23A deficiency, with the latter mice exhibiting no anemia or other CDAII characteristic. In contrast, mice with combined erythroid SEC23A and SEC23B deficiency die at ~E12.5, exhibiting reduced size and appear white in color compared to their WT litter mate controls, consistent with requirement of SEC23 in the erythroid compartment.Taken together, these data suggest complete (or near complete) overlap in function between SEC23A and SEC23B, and suggest that therapies that increase the expression of either SEC23 paralog might prove effective in treating CDAII. This paradigm might also apply to other disorders due to mutations in paralogous genes. Finally, our findings also suggest that a switch in paralog expression could account for other disparate disease phenotypes observed between animal models and humans. DisclosuresNo relevant conflicts of interest to declare.

Ectopic expression of TWIST1 upregulates the stemness marker OCT4 in the esophageal squamous cell carcinoma cell line KYSE30

BackgroundThe transcription factor TWIST1 plays an important role in the epithelial–mesenchymal transition (EMT) process and in the migration, invasion and metastasis of cancer cells. OCT4, which is a homeobox transcription factor, has an important role in the self-renewal potential of cancer cells. Our aim here is to elucidate impact of ectopic expression of TWIST1 on OCT4 gene expression in esophageal squamous cell carcinoma (ESCC).MethodsThe ESCC line was KYSE30. GP293T cells were transfected with purf-IRES-GFP and pGP plasmids to produce recombinant viral particles. A semi-confluent KYSE30 culture was transduced with the prepared retroviral particles. mRNA extraction and cDNA synthesis were performed from normal KYSE30 cells and those ectopically expressing TWIST1. Expressional analysis of TWIST1 and OCT4 were performed with relative comparative real-time PCR.ResultsEctopic expression of TWIST1 in KYSE30 cells was related to its significant overexpression: nearly nine-fold higher in GFP-hTWIST1 KYSE-30 cells than in control GFP cells. This induced expression of TWIST1 caused significant upregulation of OCT4 in GFP-hTWIST1 KYSE-30 cells: nearly eight-fold higher. In silico analysis predicted the correlation of TWIST1 and OCT4 through ETS2.ConclusionsOverexpressed TWIST1 can be correlated with upregulation of the cancer stem cell marker OCT4 and the protein may play critical regulatory role in OCT4 gene expression. Since OCT4 is involved in the self-renewal process, the results may suggest a new linkage between TWIST1 and OCT4 in the cell biology of ESCC, highlighting the probable role of TWIST1 in inducing self-renewal.

Dorsal raphé nucleus glucocorticoid receptors inhibit tph2 gene expression in male C57BL/6J mice.

The serotonergic dorsal raphé nucleus (DRN) expresses glucocorticoid receptors (GR), and systemic glucocorticoids have been shown to regulate expression and activity of tryptophan hydroxylase isoform 2, the rate-limiting enzyme for serotonin synthesis in brain. We have used intra-DRN injection of pseudotyped adeno-associated virus AAV2/9 transducing either green fluorescent protein (GFP control) or Cre recombinase (DRN GR deletion) in floxed GR mice to determine if DRN GR directly regulate DRN mRNA levels of tryptophan hydroxylase 2 (tph2). In a separate set of similarly-treated floxed GR mice, we also measured limbic forebrain region concentrations of serotonin (5-hydroxytryptamine; 5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA). DRN GR deletion increased tph2 mRNA levels in the dorsal, lateral wing, and caudal parts of the DRN without altering tissue concentrations of 5-HT, 5-HIAA, or the 5-HIAA/5-HT ratio in limbic forebrain regions. We conclude that DRN GR inhibit DRN tph2 gene expression in mice without marked effects on serotonin metabolism, at least under basal conditions at the circadian nadir. These data provide the first evidence of localized control of DRN tph2 mRNA expression by DRN GR in mice.

Expression and regulation of ATL9, an E3 ubiquitin ligase involved in plant defense.

Plants are continually exposed to a variety of pathogenic organisms, including bacteria, fungi and viruses. In response to these assaults, plants have developed various defense pathways to protect themselves from pathogen invasion. An understanding of the expression and regulation of genes involved in defense signaling is essential to controlling plant disease. ATL9, an Arabidopsis RING zinc finger protein, is an E3 ubiquitin ligase that is induced by chitin and involved in basal resistance to the biotrophic fungal pathogen, Golovinomyces cichoracearum (G. cichoracearum). To better understand the expression and regulation of ATL9, we studied its expression pattern and the functions of its different protein domains. Using pATL9:GUS transgenic Arabidopsis lines we found that ATL9 is expressed in numerous tissues at various developmental stages and that GUS activity was induced rapidly upon wounding. Using a GFP control protein, we showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Using a series of deletion mutants, we found that the PEST domain and the RING domain have effects on ATL9 degradation. Further infection assays with G. cichoracearum showed that both the RING domain and the TM domains are important for ATL9’s resistance phenotype. Interestingly, the PEST domain was also shown to be significant for resistance to fungal pathogens. This study demonstrates that the PEST domain is directly coupled to plant defense regulation and the importance of protein degradation in plant immunity.

Abstract P209: Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling Generates Reactive Oxygen Species in Mouse Endothelial Cells via NFkB Signaling

Obesity is associated with vascular endothelial dysfunction and reactive oxygen species (ROS) signaling. We have previously demonstrated a novel role for the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in the regulation of cardiovascular reactivity. In this study, we assessed the ability of mTORC1 signaling to generate ROS in mouse endothelial cells (MEC) and investigated the role of NF κ B signaling as a potential transcriptional regulator. We used an adenoviral construct of a constitutively active (CA) S6-kinase (Ad-CAS6K) to enhance mTORC1 signaling in MEC. Infection of MEC (48hrs) with the Ad-CAS6K resulted in increased mRNA levels of NADPH oxidase 1 (3.4±0.7 fold) and 2 (3.6±0.6 fold) and decreased superoxide dismutase 2 expression (0.5±0.2 fold) compared to an adenoviral GFP (Ad-GFP) control (p<0.05). Next, we used dihydroethidium staining as a measure of ROS signaling in MEC. Infection of MEC with the Ad-CAS6K enhanced ROS signaling compared to the Ad-GFP (1.5±0.1 fold; p<0.05). Conversely, infection with a dominant negative S6K construct (Ad-DNS6K) decreased ROS generation (0.6±0.1; p<0.05). Notably, infection of aortic rings of wildtype mice with the Ad-CAS6K resulted in reduced acetylcholine induced vasorelaxation compared to Ad-GFP (Max. relaxation: 67±5 vs. 81±3%; p<0.05) with no change in relaxation evoked by sodium nitroprusside (Max. relaxation: 90±1% vs. 90±2%). The reduced endothelial-mediated vasorelaxation was rescued with Tempol (Max. relaxation: 74±2%; p<0.05) indicating ROS signaling as a mechanism of mTORC1 signaling induced endothelial dysfunction. Using immunoprecipitation assays, we uncovered a physical interaction between the mTOR subunit and the IKKβ subunit of the NF κ B complex. We also found that blockade of the IKKβ subunit with BMS-345541 (300nM) prevented the increased ROS generation in MEC in response to the Ad-CAS6K (1.4±0.1 vs 0.8±0.1 fold; p<0.05) implicating NF κ B in underlying mTORC1 signaling induced ROS generation. Our data demonstrate that enhanced mTORC1 signaling in endothelial cells impair endothelial function via ROS production and NF κ B signaling complex and may play a critical role in the endothelial dysfunction associated with various conditions including obesity.

Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.

Abstract 301: CITED4 Gene Therapy Alters Maladaptive Cardiac Remodeling After Ischemia-reperfusion Injury

Introduction: Cardiac hypertrophy is an adaptive response to increased physiologic or pathologic hemodynamic stress. Previous work from our laboratory suggested that the CEBPβ/ CITED4 pathway plays an important role in exercise-induced cardiac hypertrophy. Consistent with this model, our laboratory recently found that inducible cardiac expression of CITED4 in adult mice increases in heart weight and cardiomyocyte size with normal systolic function and a gene expression profile consistent with physiologic growth. After ischemia-reperfusion injury (IRI), induced CITED4 mice show significant functional recovery and evidence for decreased adverse remodeling. Hypothesis: Here, we assessed the hypothesis that CITED4 gene therapy delivered in a clinically relevant time frame after IRI in a mouse model, will also lead to improved systolic function and favorable cardiac remodeling. Methods and Results: Cardiomyocyte-specific CITED4 gene delivery via intravenous AAV9 (CITED4 and GFP control) injections in young wild type (WT) mice led to a steady 4-fold increase in cardiac CITED4 expression. After four weeks, CITED4 treated animals developed physiologic cardiac hypertrophy with increased heart weights (heart weight to tibia length controls 6.54±0.17g/mm vs. CITED4 7.31±0.12g/mm), as well as increased left ventricular mass index and wall thickness with unchanged systolic function evaluated by echocardiography. CITED4 gene therapy in the setting of IRI, delivered 20min. after reperfusion, promoted decreased maladaptive remodeling with improved systolic function (%FS controls 37.5±3.6 vs. C4KO 47.9±1.6), a smaller scar size (% fibrotic area controls 9.1±1.9 vs. C4KO 2.4±0.5) and a favorable gene expression profile eight weeks after IRI. After injury, CITED4 gene therapy led to a 6-fold overexpression already after one week post-IRI, responsible for less apoptosis, fibrosis and inflammation when compared to control mice. Conclusion: Taken together, our data identify CITED4 as a regulator of physiologic cardiac growth that protects against adverse remodeling after ischemic injury in a clinically relevant therapeutic intervention after IRI. CITED4 may represent a novel therapeutic target to mitigate adverse ventricular remodeling.

Abstract 92: Cardiac Bridging Integrator 1 Gene Transfer Improves Left Ventricular Lusitropy in Mice With Continuous Infusion of Isoproterenol

Background: Cardiac bridging integrator 1(cBIN1) is a membrane scaffolding protein, which organizes t-tubule dyad microdomains critical for normal calcium transient development. BIN1 is transcriptionally reduced in heart failure, and reduced BIN1 diminishes calcium transients, limiting excitation-contraction coupling and impairing contractility. Furthermore, cBIN1-microdomains organize with acute β-adrenergic activation. It remains unclear whether over-expression of cBIN1 improves myocardial function in mice with chronic sympathetic overdrive. Methods: Adult male C57BL/6 mice received retro-orbital injections of adeno-associate virus 9 (AAV9) transducing either control GFP, sBIN1 (small BIN1), BIN1+13, BIN1+17, or cBIN1 (BIN1+13+17). Three weeks after virus injection, mice were subjected to subcutaneous implantation of micro osmotic pumps continuously releasing isoproterenol at 30mg /kg/day for four weeks. Left ventricular (LV) geometry and function were evaluated by echocardiography before and weekly after isoproterenol infusion. Results: In the control GFP group, isoproterenol infusion significantly increased LV mass and relative wall thickness (RWT), consistent with concentric hypertrophy. Over-expression of any of the four BIN1 isoforms attenuated left ventricular hypertrophy (n=5 per group). However, only cBIN1 over-expressing mice became more efficient. Their hearts had improved left ventricular relaxation, and were able to preserve ejection fraction yet with increased end systolic and diastolic volumes. As a result of improved relaxation, cBIN1 over-expression increased stroke volume and cardiac output without their hearts becoming hyperdynamic. Thus cBIN1 conveys a general BIN1 protection against isoproterenol induced hypertrophy together with a cBIN1 specific improvement in lusitropy. Conclusions: In mice with chronic sympathetic overdrive, over-expression of cBIN1 increases LV compliance, preserves heart lusitropy, and improves cardiac function. This athletic heart like phenotype induced by cBin1 gene therapy indicates that exogenous cBIN1 can reverse the diastolic dysfunction associated with sympathetic overdrive and heart failure progression.

Abstract 297: GJA1-20k Protects the Heart From Ischemic Injury by Inducing Mitochondrial Metabolic Quiescence

The mRNA of the GJA1 gene that encodes gap junction protein connexin 43 (Cx43) undergoes alternative translation, producing N-terminal truncated smaller protein isoforms. Unlike the well-characterized full-length Cx43 (GJA1-43k), little is known about the function and regulation of the smaller isoforms. Here, we report that global ischemia/reperfusion injury in Langendorff-perfused mouse hearts upregulates endogenous GJA1-20k, the most abundant isoform of alternative translation. Biochemical fractionation indicates that the induced GJA1-20k is preferentially enriched in cardiomyocyte mitochondria. When introduced in vitro through adenovirus-medicated gene expression, exogenous GJA1-20k, but not full length protein GJA1-43k, localizes to mitochondria and improves the survival and viability of adult cardiomyocytes when subjected to oxidative stress. In vivo gene transfer of GJA1-20k through retro-orbital injection of AAV9 virus results in lower mitochondria-dependent basal oxygen consumption as well as maximal respiratory capacity in cardiomyocytes, unlike GFP or GJA1-43k controls. Thus GJA1-20k, but not GJA1-43k, protects the heart against ischemic injury induced by permanent LAD ligation. As compared to GFP control group, myocardial infarct size in GJA1-20k treated hearts is reduced by 30% at 72 hours post LAD ligation. These results indicate that endogenous GJA1-20k is induced upon stress and has strong tropism to mitochondria. Increased GJA1-20k induces mitochondrial metabolic quiescence and affords cardioprotection in vitro and in vivo. Alternatively translated GJA1-20k acts as a novel mitochondrial stress protein and demonstrates therapeutic potential against ischemic injury.

Sphingosine kinase 1 expression enhances colon tumor growth

BackgroundAccumulating evidence suggests that sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate pathway plays a pivotal role in colon carcinogenesis.MethodsTo further support the evidence, we investigated the effects of SphK1 using three separate animal models: SphK1 knockout mice, SphK1 overexpressing transgenic mice, and SphK1 overexpression in human colon cancer xenografts. Using azoxymethane (AOM, colon carcinogen), we analyzed colon tumor development in SphK1 KO and SphK1 overexpression in intestinal epithelial cells regulated by a tet-on system. Then, we analyzed subcutaneous tumor growth using xenografts of HT-29 human colon cancer cell. Finally, immunohistochemical analyses for SphK1 and COX-2 were performed on human colon cancer tissue microarray.ResultsSphK1 KO mice, compared to wild-type mice, demonstrated a significant inhibition in colon cancer development induced by AOM (58.6% vs. 96.4%, respectively, P < 0.005). Tumor multiplicity (1.00 vs. 1.64 per colon, respectively, P < 0.05) and tumor volume (14.82 mm3 vs. 29.10 mm3, P < 0.05) were both significantly reduced in SphK1 KO mice compared to wild-type mice. Next, SphK1 overexpression in HT-29 enhanced tumor growth as compared to GFP control in nude mice (229.5 mm3 vs. 90.9 mm3, respectively, P < 0.05). Furthermore, overexpression of SphK1 in intestinal epithelial cells significantly enhances AOM-induced colon tumor formation (P < 0.05). Lastly, SphK1 and COX-2 intensity tended to reduce overall survival of late stage colon cancer patients.ConclusionsSphK1 expression regulates the early stage of colon carcinogenesis and tumor growth, thus inhibition of SphK1 may be an effective strategy for colon cancer chemoprevention.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7+ neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7+ and HNK-1+ cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1+ neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU+ neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Selective Nrf2 Gene Deletion in the Rostral Ventrolateral Medulla Evokes Hypertension and Sympathoexcitation in Mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcriptional regulator of redox homeostasis that impacts antioxidant gene expression. Central oxidative stress and reduced antioxidant enzyme expression in the rostral ventrolateral medulla (RVLM) contributed to sympathoexcitation in chronic heart failure. In the current study, we hypothesized that deletion of Nrf2 in the RVLM would increase sympathetic drive and blood pressure. Experiments were performed in Nrf2-floxed mice treated with microinjection of lentiviral-Cre-GFP or lentiviral-GFP into the RVLM. Two weeks after viral administration, Nrf2 message, protein, oxidative stress, cardiovascular function, and sympathetic outflow were evaluated. We found that (1) Nrf2 mRNA and protein in the RVLM were significantly lower in Cre mice compared with control GFP mice. Nrf2-targeted antioxidant enzymes were downregulated, whereas reactive oxygen species were elevated. (2) Blood pressure measurements indicated that Cre mice displayed a significant increase in blood pressure (mean arterial pressure, 123.7±3.8 versus 100.2±2.2 mm Hg; P<0.05, n=6), elevated urinary norepinephrine (NE) concentration (456.4±16.9 versus 356.5±19.9 ng/mL; P<0.05, n=6), and decreased spontaneous baroreflex gain (up sequences, 1.66±0.17 versus 3.61±0.22 ms/mm Hg; P<0.05, n=6; down sequences, 1.89±0.12 versus 2.98±0.19 ms/mm Hg; P<0.05, n=6). (3) Cre mice displayed elevated baseline renal sympathetic nerve activity and impaired inducible baroreflex function. These data suggest that Nrf2 gene deletion in the RVLM elevates blood pressure, increases sympathetic outflow, and impairs baroreflex function potentially by impaired antioxidant enzyme expression.

Atrogin‐1 Transgenic (AT1 Tg+) Mice Have Age‐Dependent Cardiac Dysfunction With Atrogin‐1 Mediated Glucocorticoid Receptor (GR) Expression Down Regulation

Previous studies have identified the muscle‐specific ubiquitin ligase Atrogin‐1 (MAFbx) as a critical regulator of pathologic and physiologic cardiac hypertrophy. Mechanistically, Atrogin‐1 regulates these processes by ubiquitinating transcription factors (NFAT and FOXO1/3, respectively) to inhibit these processes. However, their role in regulating transcription factors in aging has not previously been described.MethodsTransgenic mice with increased cardiomyocyte‐specific (alpha‐MHC promoter driven) Atrogin‐1 were phenotypically characterized at baseline and up to one year by conscious echocardiography. In parallel, HL‐1 cells were cultured with increasing adenovirus expressing myc‐Atrogin‐1 (Ad.Atrogin1) or GFP control (Ad.GFP) at a multiplicity of infection (MOI) of 0, 10, 20, 50, 100 and 200. Data were subject to statistical analysis with two‐tailed t‐test and/or one‐way ANOVA using GraphPad 7.0 for Mac.ResultsPost‐transduction, HL‐1 cells were lysed and protein was collected. Immunoblots were then performed to identify potential Atrogin‐1 substrates. Conscious echocardiographic analysis of Atrogin‐1 Tg hearts revealed not differences in phenotype up to four months of age. However, by 12 months, Atrogin‐1 hearts exhibited significantly decreased ejection fraction, fractional shortening and left ventricular (LV) mass (p&gt;0.05). This aging phenotype was reminiscent of recent studies where the cardiac GR was deleted, so we next investigated if Atrogin‐1 potentially targeted GR for degradation in the HL‐1 cardiomyocyte‐derived cell line. In HL‐1 cells with increasing Ad.Atrogin‐1 MOI, GR protein expression was significantly decreased in a dose‐dependent manner compared to controls (Ad.GFP) (p&lt;0.05).ConclusionThese results suggest that chronically increasing cardiomyocyte Atrogin‐1 can lead to an age‐dependent decrease in systolic function, mimicking the alterations in GR signaling reported in humans. In the heart, the muscle‐specific ubiquitin ligase Atrogin‐1 may down‐regulate cardiomyocyte GR by mediating its polyubiquitination and degradation by the 26S proteasome. Future studies will delineate the molecular details in which Atrogin‐1 regulated GR levels post‐translationally so that Atrogin‐1's role as a therapeutic target may be evaluated further in aging.Support or Funding InformationSupported by the Leducq Foundation.

Regulation of ATF1 and ATF2 transcripts by sequences in their 3' untranslated region in cleavage-stage cattle embryos.

The sequence of a 3' untranslated region (3'UTR) of mRNA governs the timing of its polyadenylation and translation in mammalian oocytes and early embryos. The objective of this study was to assess the influence of cis-elements in the 3'UTR of the developmentally important ATF1 and ATF2 transcripts on their timely translation during first cleavages in bovine embryos. Eight different reporter mRNAs (coding sequence of green fluorescent protein [GFP] fused to the 3'UTR of short or long isoforms of cattle ATF1 or -2, with or without polyadenylation) or a control GFP mRNA were microinjected separately into presumptive bovine zygotes at 18 hr post-insemination (hpi), followed by epifluorescence assessment for GFP translation between 24 and 80 hpi (expressed as percentage of GFP-positive embryos calculated from the total number of individuals). The presence of either polyadenine or 3'UTR sequence in deadenylated constructs is required for GFP translation (implying the need for polyadenylation), and all exogenous mRNAs that met either criteria were translated as soon as 24 hpi-except for long-deadenylated ATF2-UTR, whose translation began at 36 hpi. Overall, GFP was more visibly translated in competent (cleaving) embryos, particularly in long ATF1/2 constructs. The current data shows a timely GFP translation in bovine embryos depending on sequences in the 3'UTR of ATF1/2, and indicates a difference between short and long isoforms. In addition, cleaving embryos displayed increased translational capacity of the tested constructs. Functional confirmation of the identification cis-sequences in the 3'UTR of ATF1/2 will contribute to the understanding of maternal mRNA translation regulation during early cattle development.

Abstract P5-05-04: Vitamin K dependent gamma-carboxylation in breast cancer

Abstract The secreted protein periostin (PN) is strongly linked to breast cancer aggressiveness and serum PN is a promising marker of disease activity. However, the regulation and function of PN in relation to the cancer phenotype has yet to be clearly defined. PN is one of the rare proteins (&amp;lt;15 in mammalian systems) that is subject to the post-translational addition of γ-carboxylated glutamatic acid (GLA) residues. However, no studies have addressed whether γ-carboxylation of PN has functional relevance in breast cancer, despite evidence that the proteins that catalyze γ-carboxylation are expressed in normal mammary gland. Using publically available datasets we found that GGCX, VKORC1 and VKORC1L1 (genes that mediate γ-carboxylation) are overexpressed in 24% of breast cancers. Furthermore, survival of patients whose tumors overexpress these genes is significantly worse than that of patients whose tumors do not overexpress these genes. Follow-up studies utilized the Human Protein Atlas to examine expression of GGCX, VKORC1 and VKORC1L1 proteins in normal breast and invasive ductal carcinomas (IDC). Normal ductal epithelial cells showed intense staining for GGCX, patchy moderate staining for VKORC1 and diffuse low signal for VKORC1L1. All cases of IDC exhibited strong staining for GGCX, 80% had moderate-intense staining for VKORC1 and 77% had diffuse but low staining for VKORC1L1. Staining for all 3 proteins was localized only in tumor cells indicating that stromal cells are unlikely to contribute to protein γ-carboxylation. Next we explored expression of the γ-carboxylation pathway genes using in vitro models of epithelial-mesenchymal transition (EMT) and tumor progression. The EMT model consists of human mammary epithelial cells immortalized with telomerase and SV40 (HMLE cells) that constitutively express GFP (control), TWIST, SNAIL or TGFβ. Relative to control cells, GGCX, VKORC1 and VKORC1L1 were up-regulated in HMLE-TWIST cells, GGCX and VKORC1 were up-regulated in HMLE-SNAIL cells and VKORC1 was up-regulated in HMLE-TGFβ cells. These data suggest that γ-carboxylation may be triggered early in breast cancer oncogenesis. In the tumor progression model, we found up-regulation of GGCX and VKORC1 in HMLE+RAS cells relative to HMLE cells. Introduction of SV40 alone did not alter expression of these genes. In HMLE-TWIST and HMLE-RAS cells with the highest expression of GGCX, VKORC1 and VKORC1L1, PN was up-regulated 200-300-fold, supporting the concept that PN may be a relevant target for γ-carboxylation during EMT and tumor progression. In summary, available genomic and proteomic data suggest that the vitamin K dependent pathway genes GGCX, VKORC1 and VKORC1L1 are present in normal mammary gland but up-regulated in a subset of invasive breast cancers that are characterized by poor overall survival. Genes in this pathway are also up-regulated in mammary epithelial cells expressing triggers of EMT (particularly TWIST) or oncogenes such as RAS. Since the only known function of these genes is in γ-carboxylation, studies to confirm PN as a GGCX substrate and to identify additional γ-carboxylated proteins and their functions in breast cancer are clearly warranted. Our limited understanding of this ancient, conserved pathway may be masking an important therapeutic opportunity. Citation Format: Welsh J, Beaudin S, Tenniswood M. Vitamin K dependent gamma-carboxylation in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-04.