Overexpression Mouse Model Research Articles

Syntaxin 4 (STX4) Enrichment in Skeletal Muscle Remediates Insulin Resistance via and Improving Mitochondrial Function

Type 2 diabetes afflicts 10% of the US population and is the result of both peripheral insulin resistance (skeletal muscle and adipose tissue) as well as insulin secretion defects (pancreas). Insulin resistance, also commonly termed ‘pre‐diabetes’ is estimated to impact more than 30% of our population, and is caused by the inability of skeletal muscle to clear excess blood glucose from the circulation. Human skeletal muscle accounts for over 80% of glucose clearance in the body, a process important in maintaining glucose homeostasis. Currently the only therapeutics that directly improve muscle glucose uptake are diet and exercise. However, with only 3% of the population leading a “healthy lifestyle”, including exercise, directed therapeutics are required to restore skeletal muscle function in pre‐diabetics. Several studies have identified correlations between insulin resistance and reduced levels of a SNARE protein named Syntaxin 4 (STX4) in human skeletal muscle. STX4 interacts with other SNARE proteins to facilitate the fusion of glucose transporter 4 (GLUT4) vesicles with the plasma membrane, to then channel extracellular glucose to the interior of the muscle cell, ultimately clearing glucose from the circulation.To further define the function of STX4 in muscle, we developed a doxycycline Tet‐on inducible skeletal muscle specific STX4 overexpressing mouse model, under the muscle creatine kinase promoter (skm‐STX4 mice). Chow fed female mice overexpressing STX4 exhibited enhanced insulin sensitivity and improved glucose tolerance. Serum insulin levels from these mice were also reduced compared to littermate controls, concordant with their improved glucose tolerance. The data recapitulated the global overexpression model that we had previously published, suggesting that skeletal muscle is the primary driver in modulating glucose homeostasis. To establish that enriching STX4 can remediate insulin resistance, male skm‐STX4 mice were fed a 45% high fat diet (HF; to mimic a typical western diet), until they became insulin resistant. Upon establishing pre‐diabetes in the mice, STX4 enrichment was induced in the skeletal muscle (HFD+STX4 mice). Remarkably HFD+STX4 mice had the insulin sensitivity of a chow‐fed mouse, resolving the HFD‐linked insulin resistance in full. Moreover, the HFD+STX4 mice had significantly improved voluntary movement and increased respiratory exchange ratio, indicating increased reliance on carbohydrate rather than fat oxidation. The maximal oxygen consumption rate was also increased in primary myotubes from these HFD+ STX4 mice.Taken together, these results suggest that STX4 enrichment carries the potential as a therapeutic to remedy obesity‐linked insulin resistance, potentially via improving mitochondrial function. These results suggest that STX4 could be a viable therapeutic target for treatment of pre‐diabetes.Support or Funding InformationResearch was supported by the NIH (DK067912, DK102233, and DK112917) to D.C.T and by the H.N. & Frances C. Berger Foundation, and Helen & Payson Chu Fellowships to K.E.MThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Short-term preoperative protein restriction attenuates vein graft disease via induction of cystathionine γ-lyase.

Therapies to prevent vein graft disease, a major problem in cardiovascular and lower extremity bypass surgeries, are currently lacking. Short-term preoperative protein restriction holds promise as an effective preconditioning method against surgical stress in rodent models, but whether it can improve vein graft patency after bypass surgery is undetermined. Here, we hypothesized that short-term protein restriction would limit vein graft disease via up-regulation of cystathionine γ-lyase and increased endogenous production of the cytoprotective gaseous signalling molecule hydrogen sulfide. Low-density lipoprotein receptor knockout mice were preconditioned for 1 week on a high-fat high-cholesterol (HFHC) diet with or without protein prior to left common carotid interposition vein graft surgery with caval veins from donor mice on corresponding diets. Both groups were returned to a complete HFHC diet post-operatively, and vein grafts analysed 4 or 28 days later. A novel global transgenic cystathionine γ-lyase overexpressing mouse model was also employed to study effects of genetic overexpression on graft patency. Protein restriction decreased vein graft intimal/media+adventitia area and thickness ratios and intimal smooth muscle cell infiltration 28 days post-operatively, and neutrophil transmigration 4 days post-operatively. Protein restriction increased cystathionine γ-lyase protein expression in aortic and caval vein endothelial cells (ECs) and frequency of lung EC producing hydrogen sulfide. The cystathionine γ-lyase inhibitor propargylglycine abrogated protein restriction-mediated protection from graft failure and the increase in hydrogen sulfide-producing ECs, while cystathionine γ-lyase transgenic mice displayed increased hydrogen sulfide production capacity and were protected from vein graft disease independent of diet. One week of protein restriction attenuates vein graft disease via increased cystathionine γ-lyase expression and hydrogen sulfide production, and decreased early inflammation. Dietary or pharmacological interventions to increase cystathionine γ-lyase or hydrogen sulfide may thus serve as new and practical strategies to improve vein graft durability.

Pleiotropic Effects of PPARD Accelerate Colorectal Tumorigenesis, Progression, and Invasion.

APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580 ) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer-invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness. SIGNIFICANCE: These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

Mouse Models of Overexpression Reveal Distinct Oncogenic Roles for Different Type I Protein Arginine Methyltransferases.

Protein arginine methyltransferases (PRMT) are generally not mutated in diseased states, but they are overexpressed in a number of cancers, including breast cancer. To address the possible roles of PRMT overexpression in mammary gland tumorigenesis, we generated Cre-activated PRMT1, CARM1, and PRMT6 overexpression mouse models. These three enzymes are the primary type I PRMTs and are responsible for the majority of the asymmetric arginine methylation deposited in the cells. Using either a keratin 5-Cre recombinase (K5-Cre) cross or an MMTV-NIC mouse, we investigated the impact of PRMT overexpression alone or in the context of a HER2-driven model of breast cancer, respectively. The overexpression of all three PRMTs induced hyper-branching of the mammary glands and increased Ki-67 staining. When combined with the MMTV-NIC model, these in vivo experiments provided the first genetic evidence implicating elevated levels of these three PRMTs in mammary gland tumorigenesis, albeit with variable degrees of tumor promotion and latency. In addition, these mouse models provided valuable tools for exploring the biological roles and molecular mechanisms of PRMT overexpression in the mammary gland. For example, transcriptome analysis of purified mammary epithelial cells isolated from bigenic NIC-PRMT1 Tg and NIC-PRMT6 Tg mice revealed a deregulated PI3K-AKT pathway. In the future, these PRMT Tg lines can be leveraged to investigate the roles of arginine methylation in other tissues and tumor model systems using different tissue-specific Cre crosses, and they can also be used for testing the in vivo efficacy of small molecule inhibitors that target these PRMT. SIGNIFICANCE: These findings establish Cre-activated mouse models of three different arginine methyltransferases, PRMT1, CARM1, and PRMT6, which are overexpressed in human cancers, providing a valuable tool for the study of PRMT function in tumorigenesis.See related commentary by Watson and Bitler, p. 3.

Suppression of YAP/TAZ-Notch1-NICD axis by bromodomain and extraterminal protein inhibition impairs liver regeneration.

Background and aims: Biological mechanisms that control liver regeneration remain poorly defined. However, these mechanisms are remarkable issues in the clinic that affect management of hepatic loss caused by liver surgery, traumatic injury, chronic infection, or liver poisoning. Increasing evidence has shown that various growth factors, cytokines, and metabolic signaling pathways affect the liver regenerative process. Our aim is to study the effect of bromodomain and extraterminal (BET) protein inhibition on liver regeneration and its mechanism.Methods: We studied the role of BET protein inhibitor, JQ1, in liver regeneration in a mouse model after 70% partial hepatectomy (PH). We evaluated yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) and Notch signaling pathways, which were affected by BET protein inhibitor in mouse hepatic tissues and primary hepatocytes in vivo and AML12 cell lines in vitro. We evaluated the relationship of YAP/TAZ and Notch signaling pathway using YAP/TAZ pathway inhibitor in liver regeneration in vivo. Moreover, we analyzed the relationship of YAP/TAZ and Notch signaling pathways via overexpression or RNA silencing of Yap in AML12 cells. Furthermore, we used Yap overexpression mouse model to examine whether it can rescue liver regeneration damage caused by inhibition of BET proteins.Results: In this study, we report that BET protein inhibitor JQ1 molecule impairs the early phase of liver regeneration in a mouse model after 70% PH. Mechanistically, YAP/TAZ and Notch1-NICD pathways were suppressed by BET protein inhibitor in mouse hepatic tissues and primary hepatocytes in vivo and mouse AML12 cell lines in vitro. By using YAP/TAZ pathway inhibitor, we confirmed that the liver regeneration and the activation of Notch pathway were impaired by the inhibition of YAP/TAZ pathway in vivo. Furthermore, the study showed that Yap knockdown by shRNA in normal mouse hepatic cell line downregulated Notch1 signal transduction, whereas Yap overexpression promoted Notch1-NICD signals. Specific overexpression of Yap in mouse liver could rescue the effect of BET protein inhibition on liver regeneration injury.Conclusion: These results revealed the crucial role of the YAP/TAZ-Notch1-NICD axis in liver regeneration. Therefore, BET protein inhibitors must be used in caution in the treatment of hepatic diseases by reason of its suppressive roles in liver regeneration.

Leukemia Risk Gene ARID5B is a Crucial Regulator of B-Cell Development

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Genome wide association studies by us and others have identified germline genetic variants at the ARID5B locus that strongly influence susceptibility to ALL (Nat Genet 2009, J Clin Oncol 2012). Despite compelling results from these genetic association studies, the molecular mechanisms by which ARID5B contributes to ALL pathogenesis remains largely unknown. ARID5B is a member of the ARID transcription family characterized by a conservative AT rich binding domain, but the physiological functions of ARID5B are poorly understood. Therefore we sought to develop mouse models to comprehensively characterize the roles of ARID5B in normal and malignant hematopoiesis. To this end, we first established a Vav-1 specific Arid5b overexpression (AOE) mouse model. Mice were designed with a tetO element knocked in before the start codon of Arid5b to create an Arid5b tetO mouse. Arid5b tetO mice were then crossed with Vav-1-tTA mice and, overexpression of Arid5b confirmed in the hematopoietic system of progenies with the desired genotype.At 6-8 weeks old complete blood counts (CBC) analysis revealed that AOE mice had a large decrease in white blood cells (WBCs) and a slight reduction in red blood cells (RBCs) and hemoglobin (Hb). We also noted a significant reduction in the bone marrow cellularity of our Arid5b overexpressing mice. To further characterize our AOE mouse model we used flow cytometry to quantify the proportions of mature as well as various stem and progenitor cell populations during hematopoiesis. We found that AOE mice showed an increase in the MegE biased MPP2 (LSK flt3−/CD150+/CD48+) population and a decrease in the lymphoid biased MPP4 (LSK (Flt3+/CD150−/CD48+/−) population. Overexpression of Arid5b resulted in a significant reduction in the proportion of all bone marrow B cell populations (Hardy Fraction A-F) (Figure 1A) and B220+ cells in the spleen. In vitro methylcellulose colony forming assays further revealed a loss of functional pre B lymphoid progenitor in AOE bone marrow. To determine if the hematopoietic defects seen in younger AOE mice was due to a delay in hematopoiesis we aged AOE mice. We found that as we aged AOE mice there was still a reduction in bone marrow cellularity and all bone marrow B cell populations. Surprisingly, as mice were aged we found that AOE mice were prone to sudden death and displayed a dramatic reduction in overall survival when compared to wildtype littermates with a mean survival age of 8 months (Figure 1B). At the time of death, we found that AOE mice presented with severely enlarged spleens. To predict the death of AOE mice we performed ultrasounds to track spleen volume. Based on ultrasounds performed on AOE mice and wildtype littermates, AOE mice spleens become larger than those of their littermates as early as 6 months old, however spleen volume did not predict sudden death. In aged AOE mice, while the spleens were grossly enlarged and contained larger amounts of red pulp and increased Ter119+ cells, there was still a reduction in the proportion of B cells and no increase in the proportions of other white blood cells (Figure 1D). Older AOE mice exhibited anemia, hypergammaglobulinemia, and splenomegaly. By 30 weeks, defects in B cell proportions were seen in the lymph nodes of AOE mice via immunohistochemistry (Figure 1C). Compared to wildtype littermates, AOE mice displayed reduced white blood cells, red blood cells, and platelets (Figure 1D). The anemia was associated with higher reticulocyte numbers and increased serum erythropoietin concentration. The life span of erythrocytes from AOE mice was less than that of wildtype littermates. Together, these results indicated that Arid5b plays an important role in B lymphopoiesis and erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 16822: Endothelial c-Myc Protects the Heart From Pro-Inflammatory Changes During Aging and Atherogenic Stress

Background: Inflammation plays an important role in cardiac remodeling and occurs in response to risk factors such as aging and high-fat diet (HFD). We have recently reported that c-Myc knockdown in endothelial cells triggers a senescence-associated pro-inflammatory phenotype. We found that c-Myc expression declines in human coronary artery endothelial cells (HCAECs) during replicative senescence and aging. These findings suggest that threshold levels of c-Myc in endothelial cells are required to protect the heart. Here we investigated the impact of endothelial c-Myc in the heart during aging and exposure to HFD using a transgenic mouse model with endothelial-specific c-Myc overexpression. Methods and Results: Gene expression profiling and pathway analysis of young (2-month) and old (10-month) mice showed that endothelial c-Myc overexpression has a protective effect, increasing the expression of genes that prevent cardiac hypertrophy such as Rgs2, Braf and Ppargc1a and reducing inflammatory mediators such as S100a9 and Ptx3. We validated the expression of inflammatory markers IL-1β, Cxcl1 and TGF-β2 by qPCR and found that in aged group, their expression was downregulated in transgenic mice by 2.46 (p<0.002), 2.92 (p<0.001) and 1.85 (p<0.001) fold, respectively, relative to control mice. We further explored this novel protective role of c-Myc in the context of HFD using pre-designed Taqman stress array. Results showed significant downregulation of genes involved in oxidative stress (Noxo1, -1.72-fold, p<0.01), autophagy (Tfeb, -1.72-fold, p=0.03) and DNA repair (Bub1b, -1.37-fold, p=0.02) in transgenic mice under control diet. In response to HFD, most stress genes were downregulated in control mice while a specific group was upregulated in transgenic animals, including genes involved in autophagy (Tfeb, 1.39-fold, p=0.06) and antioxidant pathways (Txnrd2, 1.58-fold, p=0.03; Msra, 1.72-fold, p=0.04 and Msrb3, 1.47-fold, p=0.05). Conclusion: Our c-Myc overexpression mouse model exhibits a distinct gene expression pattern that is potentially cardiac protective in response to aging and HFD. Endothelial c-Myc may be essential to protect cardiac tissues under stress conditions.

HGF alleviates renal interstitial fibrosis via inhibiting the TGF-β1/SMAD pathway.

To study the role of HGF (stem cell growth factor) in renal interstitial fibrosis and to explore its underlying mechanism. A unilateral ureteral obstruction (UUO) mouse model was first constructed, and kidney samples of mice were then collected. Fibrosis-related indicators in UUO mice kidney were detected by Western blot. The mRNA and protein levels of HGF in UUO mice were detected by quantitative Real-time-polymerase chain reaction (qRT-PCR) and Western blot, respectively. The HGF overexpression mouse model was established by using UUO mice. For in vitro experiments, fibrosis-related indicators and the expression of HGF were detected in transforming growth factor-β1 (TGF-β1)-induced NRK-52E cells. Finally, a p-SMAD3 knockdown mouse model was established to confirm whether p-SMAD3 was involved in HGF-regulated renal interstitial fibrosis. The expression levels of HGF and α-SMA (α-smooth muscle actin) were both significantly increased in UUO mice, while E-cadherin expression was significantly decreased, which were consistent with results of in vitro experiments. Overexpression of HGF remarkably decreased the protein and mRNA levels of α-SMA in fibrotic NRK-52E cells. After overexpression of HGF in UUO mice, α-SMA was remarkably downregulated, whereas E-cadherin was significantly upregulated. Further, results also demonstrated that HGF was upregulated and α-SMA was downregulated after p-SMAD3 knockdown in UUO mice. HGF is highly expressed during renal interstitial fibrosis, which may suppress renal interstitial fibrosis by inhibiting the TGF-β1/SMAD signaling pathway.

MiR-132/212 is induced by stress and its dysregulation triggers anxiety-related behavior

miR-132 and miR-212 are structurally-related microRNAs that are expressed from the same non-coding transcript. Accumulating evidence has shown that the dysregulation of these microRNAs contributes to aberrant neuronal plasticity and gene expression in the mammalian brain. Consistent with this, altered expression of miR-132 is associated with a number of affect-related psychiatric disorders. Here, we tested the functional contribution of the miR-132/212 locus to the development of stress-related and anxiety-like behaviors. Initially, we tested whether expression from the miR-132/212 locus is altered by stress-inducing paradigms. Using a 5-h acute-stress model, we show that both miR-132 and miR-212 are increased more than two-fold in the WT murine hippocampus and amygdala, whereas after a 15 day chronic-stress paradigm, expression of both miR-132 and miR-212 are upregulated more than two-fold within the amygdala but not in the hippocampus. Next, we used a tetracycline-inducible miR-132 overexpression mouse model and a miR-132/212 conditional knockout (cKO) mouse model to examine whether dysregulation of miR-132/212 expression alters basal anxiety-like behaviors. Interestingly, in both the miR-132 overexpression and cKO lines, significant increases in anxiety-like behaviors were detected. Importantly, suppression of transgenic miR-132 expression (via doxycycline administration) mitigated the anxiety-related behaviors. Further, expression of Sirt1 and Pten—two miR-132 target genes that have been implicated in the regulation of anxiety—were differentially regulated in the hippocampus and amygdala of miR-132/212 conditional knockout and miR-132 transgenic mice. Collectively, these data raise the prospect that miR-132 and miR-212 may play a key role in the modulation of stress responsivity and anxiety.

MicroRNA-206 Downregulates Connexin43 in Cardiomyocytes to Induce Cardiac Arrhythmias in a Transgenic Mouse Model

MicroRNAs (miRNAs) are critical modulators of various physiological and pathological processes, but their role in cardiac arrhythmias remains yet to be completely understood. Connexin43 (Cx43) is an important cardiac gap junction protein and a potential target of miR-206, and downregulation of Cx43 induces ventricular tachyarrhythmias. We investigated the effects of miR-206 overexpression on the adult mouse heart and in cardiac arrhythmias. Luciferase activity assay was employed to validate Cx43 as a direct target of miR-206. Expression of Cx43 was measured in cardiac muscle cell line HL-1 securely expressing miR-206. An inducible miR-206 overexpression mouse model was established to evaluate the in vivo effect of miR-206 on Cx43 expression and cardiac rhythm. MiR-206 directly recognised 3'-untranslated region of Cx43 mRNA to inhibit its expression in HL-1 cells. Induction of miR-206 in the adult mouse heart suppressed Cx43 expression, particularly in the atria and ventricle. Importantly, miR-206 overexpression also induced abnormal heart-rate and PR interval, and shortened life-span in the experimental mice. In cardiomyocytes, miR-206 is a upstream regulator of Cx43, and its overexpression downregulates Cx43 to induce abnormal heart-rate and PR interval.

PL-020 Establishment of skeletal muscle-specific PGC-1α overexpression model via in vivo local transfection

Objective To establish a skeletal muscle-specific PGC-1α overexpression mouse model via in vivo local transfection.
 Methods For the PGC-1α in vivo transfection study, the Male FVB/N mice were randomly divided into 2 groups: subject to green fluorescent protein (GFP) transfection (Con-GFP), and subject to PGC-1α in vivo transfection (Con-PGC-1α). Plasmid DNA solution (2.5 mg/ml GFP or 2.7 mg/ml Flag-PGC-1α) were injected into the proximal (6 ml) and distal (6 ml) ends of the muscle belly. Following the injections, electric pulses were applied through 2 stainless steel pin electrodes laid on top of the proximal and distal myotendinous junctions. Then skeletal muscle and myocardium were isolated, and PGC-1α, mtTFA, NF-κB, MnSOD, FNDC5 protein expression were measured with Western blot.
 Results n skeletal muscle, compared with con-GFP group, the expression of PGC-1α (+125%, p<0.01), mtTFA (+210%, p<0.01), and FNDC5 (+47%, p<0.05) were significantly increased in con-PGC-1α group. However, NF-κB and MnSOD protein level had no change in con-PGC-1α group. In myocardium, compared with con-GFP group, the expression of mtTFA (+130%, p<0.01) and FNDC5 (+55%, p<0.05) were significantly increased in con-PGC-1α group.
 Conclusions Skeletal muscle-specific PGC-1α overexpression model via in vivo local transfection was established, which was supported by elevated expression of PGC-1α and its downstream FNDC5 and mtTFA. Furthermore, skeletal muscle-specific PGC-1α overexpression induced increase in myocardial mitochondrial biogenesis, while relative mechanism remains to be determined.

Commentary on Giralt et al.: PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is the most common cause of dementia and the 6th leading cause of death. Although research has revealed significant information about AD, much is yet to be discovered about the precise biological changes that cause AD and how the disease could be prevented, slowed, or stopped. Accumulating evidence suggests the involvement of the non-receptor proline-rich tyrosine kinase 2 (Pyk2) in AD, but the downstream signaling events triggered by this protein and their implications on the pathology of the disease were unclear until recently. A recent paper by Giralt et al. used genetically depleted and overexpression mouse models to elucidate the role of Pyk2 in AD. Here, we discuss the findings presented in this paper in light of previous information and hypotheses, and suggest interpretations and explanations for this surprising and unexpected phenotype.

REST overexpression in mice causes deficits in spontaneous locomotion

Overexpression of REST has been implicated in brain tumors, ischemic insults, epilepsy, and movement disorders such as Huntington’s disease. However, owing to the lack of a conditional REST overexpression animal model, the mechanism of action of REST overexpression in these disorders has not been established in vivo. We created a REST overexpression mouse model using the human REST (hREST) gene. Our results using these mice confirm that hREST expression parallels endogenous REST expression in embryonic mouse brains. Further analyses indicate that REST represses the dopamine receptor 2 (Drd2) gene, which encodes a critical nigrostriatal receptor involved in regulating movement, in vivo. Overexpression of REST using Drd2-Cre in adult mice results in increased REST and decreased DRD2 expression in the striatum, a major site of DRD2 expression, and phenocopies the spontaneous locomotion deficits seen upon global DRD2 deletion or specific DRD2 deletion from indirect-pathway medium spiny neurons. Thus, our studies using this mouse model not only reveal a new function of REST in regulating spontaneous locomotion but also suggest that REST overexpression in DRD2-expressing cells results in spontaneous locomotion deficits.

Obstruction of ventricular Ca2+ -dependent arrhythmogenicity by inositol 1,4,5-trisphosphate-triggered sarcoplasmic reticulum Ca2+ release.

Augmented inositol 1,4,5-trisphosphate (IP3 ) receptor (IP3 R2) expression has been linked to a variety of cardiac pathologies. Although cardiac IP3 R2 function has been in the focus of research for some time, a detailed understanding of its potential role in ventricular myocyte excitation-contraction coupling under pathophysiological conditions remains elusive. The present study focuses on mechanisms of IP3 R2-mediated sarcoplasmic reticulum (SR)-Ca2+ release in ventricular excitation-contraction coupling under IP3 R2-overexpressing conditions by studying intracellular Ca2+ events. We report that, upon IP3 R2 overexpression in ventricular myocytes, IP3 -induced Ca2+ release (IP3 ICR) modulates the SR-Ca2+ content via "eventless" SR-Ca2+ release, affecting the global SR-Ca2+ leak. Thus, IP3 R2 activation could act as a SR-Ca2+ gateway mechanism to escape ominous SR-Ca2+ overload. Our approach unmasks a so far unrecognized mechanism by which "eventless" IP3 ICR plays a protective role against ventricular Ca2+ -dependent arrhythmogenicity. Augmented inositol 1,4,5-trisphosphate (IP3 ) receptor (IP3 R2) function has been linked to a variety of cardiac pathologies including cardiac arrhythmias. The functional role of IP3 -induced Ca2+ release (IP3 ICR) within ventricular excitation-contraction coupling (ECC) remains elusive. As part of pathophysiological cellular remodelling, IP3 R2s are overexpressed and have been repeatedly linked to enhanced Ca2+ -dependent arrhythmogenicity. In this study we test the hypothesis that an opposite scenario might be plausible in which IP3 ICR is part of an ECC protecting mechanism, resulting in a Ca2+ -dependent anti-arrhythmogenic response on the cellular scale. IP3 R2 activation was triggered via endothelin-1 or IP3 -salt application in single ventricular myocytes from a cardiac-specific IP3 R type2 overexpressing mouse model. Upon IP3 R2 overexpression, IP3 R activation reduced Ca2+ -wave occurrence (46 vs. 21.72%; P<0.001) while its block increased SR-Ca2+ content (∼29.4% 2-aminoethoxydiphenyl borate, ∼16.4% xestospongin C; P<0.001), suggesting an active role of IP3 ICR in SR-Ca2+ content regulation and anti-arrhythmogenic function. Pharmacological separation of ryanodine receptor RyR2 and IP3 R2 functions and two-dimensional Ca2+ event analysis failed to identify local IP3 ICR events (Ca2+ puffs). SR-Ca2+ leak measurements revealed that under pathophysiological conditions, "eventless" SR-Ca2+ efflux via enhanced IP3 ICR maintains the SR-Ca2+ content below Ca2+ spark threshold, preventing aberrant SR-Ca2+ release and resulting in a protective mechanism against SR-Ca2+ overload and arrhythmias. Our results support a so far unrecognized modulatory mechanism in ventricular myocytes working in an anti-arrhythmogenic fashion.

Abstract 539: MCUB Regulates the Macromolecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Ischemic Cardiac Injury

The mitochondrial calcium uniporter channel (mtCU) resides in the inner mitochondrial membrane and is a multi-subunit complex required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. RNA-seq and PCR analysis of mtCU component expression revealed that MCUB expression increases after myocardial infarction. To investigate how MCUB , a paralog of the MCU gene, contributes to mtCU regulation we created a MCUB -/- cell line (HeLa) using CRISPR-Cas9n. MCUB deletion increased histamine-mediated m [Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO). Biophysical characterization found MCUB deletion increased mtCU capacitance and rate of m [Ca 2+ ] uptake. FPLC fractionation of mtCU revealed loss of MCUB increased MCU incorporation into the supercomplex, suggesting MCUB replacement of MCU and overall increase in functional mtCU supercomplexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (mito genetic calcium reporter) and adult cardiomyocytes were isolated to record [ m Ca 2+ ] transients during pacing. Increasing the MCUB/MCU ratio decreased [ m Ca 2+ ] peak amplitude by ~30% and significantly reduced the [ m Ca 2+ ] uptake rate. FPLC assessment of the mtCU revealed MCUB was undetectable in the supercomplex fraction (~700kD) of Cre controls, but enriched in MCUB -Tg hearts. Examining the oxygen consumption rate of cardiomyocytes isolated from MCUB- Tg hearts found decreased maximal respiration and spare reserve capacity, which correlated with severe impairment in isoproterenol-induced contractile reserve. Mitochondria from MCUB -Tg mouse hearts were resistant to Ca 2+ induced mitochondrial swelling suggesting MCUB protects against permeability transition. MCUB -Tg mice subjected to myocardial ischemia reperfusion injury had a ~50% decrease in infarct size suggesting increased MCUB expression prevents m Ca 2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive in mitochondrial energetic responsiveness.

Abstract 4626: A TRIM24-like mutational signature predicts worse relapse-free survival (RFS) and overall survival (OS) in patients (pts) with metaplastic breast cancer (MpBC)

Abstract Background: TRIM24 is a multifunctional, epigenetic PHD/Bromodomain protein that negatively regulates p53, via an E3-ubiquitin ligase RING domain, and co-regulates transcription. Over-expression of TRIM24 in murine mammary epithelium results in the development of carcinosarcomas that bear close histologic resemblance to human MpBCs and are characterized by a distinct genomic signature. Here, we investigate the association between the presence of a TRIM24-like mutational signature and clinical outcomes in a cohort of human MpBCs. Methods: 20 archived samples of human MpBC with annotated clinical data underwent whole exome sequencing. In parallel, we sequenced 7 spontaneously arising carcinosarcomas from a TRIM24 overexpressing mouse model. To compare the mutations with those seen in the human MpBCs, we grouped them into pathways and created a signature of pathway activation seen in TRIM24-driven tumors. Median RFS and OS were estimated using the Kaplan-Meier method and compared between pts with and without a TRIM24-like mutational signature. Results: Baseline clinical characteristics are summarized in Table 1. Of the 20 MpBC pts, 5 had a TRIM24-like mutational signature and the remaining 15 pts did not. There were no statistically significant differences in baseline characteristics between the 2 groups. Median RFS for pts with a TRIM24-like mutational signature was 9 months (95% confidence interval [CI]: 8-undefined months). Median RFS for pts without a TRIM24-like mutational signature has not been reached (log-rank, p=0.0149). Median OS for pts with and without a TRIM24-like mutational signature was 17 months (95% CI: 10-undefined months) and 147 months (95% CI: 22-undefined months), respectively (log-rank, p=0.0402). Conclusion: In this cohort, pts with MpBCs that exhibit a TRIM24-like mutational signature had worse RFS and OS. This finding should be confirmed in larger, prospective studies. Table 1: Baseline Clinicopathological Characteristics by TRIM24 StatusTRIM24-like (n=5)Non TRIM24-like (n=15)p valueAge at diagnosis - Median (years, interquartile range)55 (50-61)56 (47-66)0.965Clinical Tumor SizeMean (cm, standard deviation)4.2 (1.5)3.4 (1.6)0.383Clinical Nodal StatusNegative - n (%)3 (60)11 (73)0.570Positive - n (%)2 (40)3 (20)Unknown - n (%)01 (7)ER/PR StatusNegative - n (%)5 (100)14 (93)1.000Positive - n (%)01 (7)HER2 StatusNegative - n (%)5 (100)15 (100)NAPositive - n (%)00HistologySpindle Cell - n (%)4 (80)7 (47)0.319Matrix-producing - n (%)1 (20)8 (53) Citation Format: Clinton Yam, Aundrietta Duncan, Jeffrey T. Chang, Michael Z. Gilcrease, Vrutant V. Shah, Michelle Barton, Stacy L. Moulder. A TRIM24-like mutational signature predicts worse relapse-free survival (RFS) and overall survival (OS) in patients (pts) with metaplastic breast cancer (MpBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4626.

PO-086 BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous cell carcinoma

IntroductionLung cancer accounts for the highest rate of cancer related diagnosis and mortality worldwide. Small cell lung cancer and non-small cell lung cancer (NSCLC) make up the major subtypes of lung cancer. NSCLC patients only have a 15% five-year survival rate. The major subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUSC accounts for more than 4 00 000 deaths each year and unlike LUAD there are limited targeted therapies. Therefore, a great deal of work still needs to be done to understand the drivers for this cancer.Material and methods shRNA was utilised to modulate the levels of BCL11A and SOX2 in LUSC cell lines. In vitro 3D colony assays and xenograft mouse models were employed to understand the role of BCL11A in driving LUSC tumorigenesis. To explore the role of BCL11A in vivo, a Cre-inducible BCL11A overexpression mouse model was used. To further investigate BCL11A in LUSC, a mouse tracheal basal cells organoid assay was employed. CHIP-seq, immunoprecipitation and immunoblotting assays were designed to dissect the mechanism by which BCL11A elicits its function in LUSC. Drug dose response assays were used to test the efficacy of SETD8 inhibitors and cisplatin on an array of lung cancer cell lines.Results and discussionsAnalysis of TCGA has revealed BCL11A to be upregulated in LUSC but not LUAD. Subsequently reducing BCL11A levels in LUSC cell lines results in diminished xenograft tumour growth. Inversely, its overexpression in vivo led to lung airway hyperplasia and the development of reserve cell hyperplastic lesions which is a precursor to squamous metaplasia. Moreover, deleting Bcl11a in mouse tracheal basal cells abolished the development of tracheosphere organoids while its overexpression led to solid tracheospheres expressing markers of squamous cells.At the molecular level we found BCL11A to be a target of SOX2 and we show that it is required for the oncogenic role of SOX2 in LUSC. Furthermore, we showed that BCL11A and SOX2 interact at the protein level and that together they co-regulated the expression of several transcription factors. We demonstrate that pharmacological inhibition of SETD8, a gene co-regulated by BCL11A and SOX2, alone or in combination with cisplatin treatment, shows significant selectivity to LUSC in comparison to LUAD cells.ConclusionCollectively, these results indicate that the disruption of the BCL11A-SOX2 transcriptional program provides a future framework for the development of targeted therapeutic intervention for LUSC patients.

VEGF-A-Expressing Adipose Tissue Shows Rapid Beiging, Enhanced Survival after Transplantation

Adipocyte-derived VEGF-A plays a crucial role in angiogenesis and contributes to adipocyte function and systemic metabolism, such as insulin resistance, chronic inflammation and beigeing of subcutaneous adipose tissue. Utilizing a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpressing mouse model, we investigated the dynamics of local VEGF-A effects on tissue beiging of adipose tissue transplants. VEGF-A overexpression in adipocytes triggers angiogenesis. We also observe a rapid appearance of beige fat cells in subcutaneous white adipose tissue (sWATs) within as early as 2 days post induction of VEGF-A. In contrast to conventional cold-induced beiging, VEGF-A-induced beiging is independent of IL-4. We subjected metabolically healthy VEGF-A overexpressing adipose tissue to autologous transplantation. Transfer of subcutaneous adipose tissues taken from VEGF-A overexpressing mice into diet-induced obese mice resulted in systemic metabolic benefits, associated with improved survival of adipocytes and a concomitant reduced inflammatory response. These effects of VEGF-A are tissue autonomous, inducing WAT beigeing and angiogenesis within the transplanted tissue. Our findings indicate that manipulation of adipocyte functions with a bona fide angiogenic factor, such as VEGF-A, significantly improves the survival and volume retention of fat grafts and can convey metabolically favorable properties on the recipient on the basis of beiging. Disclosure M. Kim: None. Y.A. An: None. H. Bang: None. P.E. Scherer: None. J. Park: None.

PO-090 Expression of the cocaine- and amphetamine-regulated transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the oestrogen receptor

IntroductionLung cancer accounts for the highest rate of cancer related diagnosis and mortality worldwide. Small cell lung cancer and non-small cell lung cancer (NSCLC) make up the major subtypes of lung cancer. NSCLC patients only have a 15% five-year survival rate. The major subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUSC accounts for more than 4 00 000 deaths each year and unlike LUAD there are limited targeted therapies. Therefore, a great deal of work still needs to be done to understand the drivers for this cancer.Material and methods shRNA was utilised to modulate the levels of BCL11A and SOX2 in LUSC cell lines. In vitro 3D colony assays and xenograft mouse models were employed to understand the role of BCL11A in driving LUSC tumorigenesis. To explore the role of BCL11A in vivo, a Cre-inducible BCL11A overexpression mouse model was used. To further investigate BCL11A in LUSC, a mouse tracheal basal cells organoid assay was employed. CHIP-seq, immunoprecipitation and immunoblotting assays were designed to dissect the mechanism by which BCL11A elicits its function in LUSC. Drug dose response assays were used to test the efficacy of SETD8 inhibitors and cisplatin on an array of lung cancer cell lines.Results and discussionsAnalysis of TCGA has revealed BCL11A to be upregulated in LUSC but not LUAD. Subsequently reducing BCL11A levels in LUSC cell lines results in diminished xenograft tumour growth. Inversely, its overexpression in vivo led to lung airway hyperplasia and the development of reserve cell hyperplastic lesions which is a precursor to squamous metaplasia. Moreover, deleting Bcl11a in mouse tracheal basal cells abolished the development of tracheosphere organoids while its overexpression led to solid tracheospheres expressing markers of squamous cells.At the molecular level we found BCL11A to be a target of SOX2 and we show that it is required for the oncogenic role of SOX2 in LUSC. Furthermore, we showed that BCL11A and SOX2 interact at the protein level and that together they co-regulated the expression of several transcription factors. We demonstrate that pharmacological inhibition of SETD8, a gene co-regulated by BCL11A and SOX2, alone or in combination with cisplatin treatment, shows significant selectivity to LUSC in comparison to LUAD cells.ConclusionCollectively, these results indicate that the disruption of the BCL11A-SOX2 transcriptional program provides a future framework for the development of targeted therapeutic intervention for LUSC patients.

Inverse correlation between vascular endothelial growth factor back-filtration and capillary filtration pressures

Vascular endothelial growth factor A (VEGF) is an essential growth factor during glomerular development and postnatal homeostasis. VEGF is secreted in high amounts by podocytes into the primary urine, back-filtered across the glomerular capillary wall to act on endothelial cells. So far it has been assumed that VEGF back-filtration is driven at a constant rate exclusively by diffusion. In the present work, glomerular VEGF back-filtration was investigated in vivo using a novel extended model based on endothelial fenestrations as surrogate marker for local VEGF concentrations. Single nephron glomerular filtration rate (SNGFR) and/or local filtration flux were manipulated by partial renal mass ablation, tubular ablation, and in transgenic mouse models of systemic or podocytic VEGF overexpression or reduction. Our study shows positive correlations between VEGF back-filtration and SNGFR as well as effective filtration rate under physiological conditions along individual glomerular capillaries in rodents and humans. Our results suggest that an additional force drives VEGF back-filtration, potentially regulated by SNGFR.