Tissue-specific Knockout Mice Research Articles

SAT-348 Overexpression of the Notch Intracellular Domain Results in Abnormal Adrenal Development.

In the adrenal gland, stem and progenitor cells reside in the capsule and the outer part of the cortex, respectively. The newly formed cortical cells from stem/progenitor cells move inward to the corticomedullary boundary during development. Throughout this developmental process, adrenocortical cells express specific marker genes at different stages which lead to concentric zones of the adrenal cortex (zonation). In mice, the majority of the functional cortex are definitive zones whereas cortical cells in the corticomedullary boundary form the X-zone, which is considered the aged cell population in the cortex and undergoes regression over time. Disruption of signaling pathways such as Wnt, Hedgehog and TGF-beta can cause abnormalities of the developmental process of the adrenal cortex, which may lead to malfunction or malignancy of the adrenal gland. The role of Notch, another key signaling pathway, in adrenal development has not yet been fully studied. Notch signaling pathway involves a variety of gene regulatory mechanisms that control cell proliferation, differentiation, and apoptosis. Although key molecules in Notch signaling pathways are expressed both in the adrenal cortex and in the medulla, the inhibition of canonical Notch signaling only leads to minor/limited effects on the function or the structure of the adrenal gland. We confirmed these observations using Sf1-Cre mediated tissue-specific knockout mouse model that lacks Hes1, one of the most common Notch target genes, in cortical cells in the adrenal gland. Immunostaining with proliferation markers (PCNA & Ki67) and marker genes of different cell populations in the adrenal gland (e.g., β-catenin, 20αHSD, tyrosine hydroxylase) showed normal zonation and proliferation patterns. The unaffected adrenal glands in Hes1 conditional knockout mice suggests Notch signaling is not a dominant developmental pathway in the adrenal cortex. Next, to study how overactivation of Notch signaling affects adrenocortical cells, we used Sf1-Cre mice to overexpress Notch intracellular domain (NICD) in cortical cells in the adrenal gland. The over-expression of NICD in the adrenal cortex disrupted adrenal cortex zonation and led to a small, disorganized adrenal. These Sf1-Cre mediated NICD over-overexpressed adrenals had scattered and disorganized medulla irregularly distributed in the margin of the gland. Immunostaining showed clusters of 3βHSD low-expressing cells underneath the capsule partially surrounding the 3βHSD high-expressing cortex. Moreover, X-zone cells (labeled by 20αHSD) were significantly reduced in NICD over-overexpressed adrenals in two weeks old male and female mice. Our data suggest that overactivation of Notch signaling in the adrenal cortex not only disrupts the development of the definitive cortical zones but also affects the aging and the differentiation of fetal cortical cells in mice.

Intestinal Epithelial AMPK Regulates Paracellular Electrolyte Flux and Electrogenic Ion Transport Responses in Mouse Colon

Ion transport within the colonic epithelium is crucial to maintain colonic homeostasis. During pathological states such as intestinal inflammation, alteration of ion transport processes contributes to the development of diarrhea and constipation. These pathways are tightly regulated by expression and localization to ensure fluid balance. AMP‐Activated Protein Kinase (AMPK) is a highly conserved energy‐sensing kinase that has previously been shown to regulate ion channels and transporters such as CFTR and NKCC1, in both intestinal epithelium and other epithelia. The aim of this study was to specifically test the impact of AMPK activity on ion flux across intestinal epithelia by utilizing a tissue‐specific knockout mouse model lacking both AMPK‐α catalytic isoforms.MethodsIntestinal epithelial cell‐specific knockout of AMPK activity (AMPKαΔIEC KO) was achieved by crossing Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl) with Villin‐Cre mice. For Ussing chamber experiments, large intestinal segments were stripped of the serosal layer and the mucosal layer was mounted in Ussing chambers containing Kreb's Ringer's buffer. Chloride secretion was stimulated using Forskolin (300μM) applied bilaterally, followed by Carbachol (10μM) applied basolaterally. For immunostaining, whole intestinal tissue was isolated and fixed in 4% paraformaldehyde overnight, washed with 1X PBS, and stored in 30% sucrose in 1X until OCT embedding. 5μm frozen sections of intestinal tissues were sliced and mounted onto glass slides.ResultsWe previously demonstrated AMPKαΔIEC KO mice do not have a barrier defect indicated by macromolecular intestinal permeability, histological analysis, and response to dextran sulfate sodium (DSS) compared to AMPKαfl/fl mice. We next determined if AMPK alters electrolye permeability in different regions of the large intestine. Indeed, loss of AMPK decreased transepithelial electrical resistance (TEER) in mouse proximal colon (35.21 ± 3.3 Ω·cm2, n = 6–7, p = 0.0007). Conversely, AMPKαΔIEC KO mice exhibited an increase in basal TEER in the distal colon (62.5 ± 4.5 Ω·cm2, n = 2–3, p = 0.2683), although this was not statistically significant. We next tested if AMPKαΔIEC KO mice exhibited changes in electrogenic ion transport by intestinal region. Preliminary evidence in the cecum indicated increased electrogenic ion transport responses to carbachol and Forskolin in AMPKαΔIEC KO mice (n = 1–3) while only forskolin‐dependent ion transport was increased in the distal colon (n = 2–3). In the proximal colon, Ca2+‐dependent ion transport was increased two‐fold (n = 6–7, p = 0.1131) while there was no alteration in cAMP‐dependent ion transport (n = 6–7). To determine if these region‐specific effects were associated with changes in transporter expression, colonic sections were stained for the Na+‐K+‐Cl− Cotransporter 1 (NKCC1) as a key contributor to electrogenic ion secretion. NKCC1 expression in AMPKαΔIEC KO mice was similar to that of AMPKαfl/fl mice (n = 2–3).ConclusionsAMP‐activated protein kinase mediates region‐specific maintenance of the intestinal barrier and regulation of electrogenic ion transport in mice.Support or Funding InformationNIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Unique Role of Carboxylesterase 3 (Ces3) in β-Adrenergic Signaling-Stimulated Thermogenesis.

Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography-tandem mass spectrometry. To investigate the function of Ces3 in the β-adrenergic signaling-activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)-treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1α genes via peroxisome proliferator-activated receptor γ. We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue-specific Ces3 heterozygous knockout (Adn-Cre-Ces3flx/wt) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.

Milk biosynthesis requires the Golgi cation exchanger TMEM165

Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H+ is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H+/Ca2+/Mn2+ exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca2+ and Mn2+ to the Golgi complex in exchange for H+ to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.

SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity

The essential metal manganese becomes neurotoxic at elevated levels. Yet, the mechanisms by which brain manganese homeostasis is regulated are unclear. Loss-of-function mutations in SLC30A10, a cell surface-localized manganese efflux transporter in the brain and liver, induce familial manganese neurotoxicity. To elucidate the role of SLC30A10 in regulating brain manganese, we compared the phenotypes of whole-body and tissue-specific Slc30a10 knockout mice. Surprisingly, unlike whole-body knockouts, brain manganese levels were unaltered in pan-neuronal/glial Slc30a10 knockouts under basal physiological conditions. Further, although transport into bile is a major route of manganese excretion, manganese levels in the brain, blood, and liver of liver-specific Slc30a10 knockouts were only minimally elevated, suggesting that another organ compensated for loss-of-function in the liver. Additional assays revealed that SLC30A10 was also expressed in the gastrointestinal tract. In differentiated enterocytes, SLC30A10 localized to the apical/luminal domain and transported intracellular manganese to the lumen. Importantly, endoderm-specific knockouts, lacking SLC30A10 in the liver and gastrointestinal tract, had markedly elevated manganese levels in the brain, blood, and liver. Thus, under basal physiological conditions, brain manganese is regulated by activity of SLC30A10 in the liver and gastrointestinal tract, and not the brain or just the liver. Notably, however, brain manganese levels of endoderm-specific knockouts were lower than whole-body knockouts, and only whole-body knockouts exhibited manganese-induced neurobehavioral defects. Moreover, after elevated exposure, pan-neuronal/glial knockouts had higher manganese levels in the basal ganglia and thalamus than controls. Therefore, when manganese levels increase, activity of SLC30A10 in the brain protects against neurotoxicity.

CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery.

In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.

RNA-Binding Protein HuR Regulates Rac1 Nucleocytoplasmic Shuttling Through Nucleophosmin in the Intestinal Epithelium.

Background & AimsThe mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated via well-controlled mechanisms. The RNA-binding protein HuR is essential for maintaining gut epithelial integrity, and targeted deletion of HuR in intestinal epithelial cells (IECs) disrupts mucosal regeneration and delays repair after injury. Here, we defined the role of HuR in regulating subcellular distribution of small guanosine triphosphatase Rac1 and investigated the implication of nucleophosmin (NPM) as a molecular chaperone in this process.MethodsStudies were conducted in intestinal epithelial tissue-specific HuR knockout (IE-HuR-/-) mice and cultured IEC-6 cells, derived from rat small intestinal crypts. Functions of HuR and NPM in vitro were investigated via their gene silencing and overexpression.ResultsThe abundance of cytoplasmic Rac1 in the small intestinal mucosa increased significantly in IE-HuR-/- mice, although HuR deletion did not alter total Rac1 levels. HuR silencing in cultured IECs also increased the cytoplasmic Rac1 levels, without an effect on whole-cell Rac1 content. In addition, HuR deficiency in the intestinal epithelium decreased the levels of NPM in IE-HuR-/- mice and cultured IECs. NPM physically interacted with Rac1 and formed the NPM/Rac1 complex. NPM silencing decreased the NPM/Rac1 association and inhibited nuclear accumulation of Rac1, along with an increase in cytoplasmic abundances of Rac1. In contrast, ectopically expressed NPM enhanced Rac1 nuclear translocation and restored Rac1 subcellular localization to near normal in HuR-deficient cells.ConclusionsThese results indicate that HuR regulates Rac1 nucleocytoplasmic shuttling in the intestinal epithelium by altering NPM expression.

Identification of Novel Therapeutic Targets for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are fatal diseases; however, their pathogenesis still remains to be elucidated. We have recently screened novel pathogenic molecules and have performed drug discovery targeting those molecules. Pulmonary artery smooth muscle cells (PASMCs) in patients with PAH (PAH-PASMCs) have high proliferative properties like cancer cells, which leads to thickening and narrowing of distal pulmonary arteries. Thus, we conducted a comprehensive analysis of PAH-PASMCs and lung tissues to search for novel pathogenic proteins. We validated the pathogenic role of the selected proteins by using tissue-specific knockout mice. To confirm its clinical significance, we used patient-derived blood samples to evaluate the potential as a biomarker for diagnosis and prognosis. Finally, we conducted a high throughput screening and found inhibitors for the pathogenic proteins.

Adipose Tissue, Inter-Organ Communication, and the Path to Type 2 Diabetes: The 2016 Banting Medal for Scientific Achievement Lecture.

My scientific career has focused on understanding the mechanisms underlying insulin resistance with the goal of developing new strategies to prevent and treat type 2 diabetes. My early studies focused on understanding how insulin promotes glucose transport into adipocytes, a classic model of highly insulin-responsive target cells. When we found changes in adipocyte glucose transport in altered metabolic states, we were highly motivated to understand the consequences of this on whole-body glucose homeostasis. In the late 1980s, when GLUT4, the major insulin-regulated glucose transporter, was identified, my lab observed that it was downregulated in adipocytes but not in skeletal muscle in insulin-resistant states, such as obesity and type 2 diabetes, in humans and rodents. We investigated the role of GLUT4 in adipose tissue and muscle in whole-body insulin sensitivity, making tissue-specific GLUT4-overexpressing and GLUT4 knockout mice. These studies led to the discovery that adipocytes, and specifically glucose transport into adipocytes, regulate whole-body glucose homeostasis. As adipocytes take up relatively little glucose, we investigated the underlying mechanisms. In the 1990s, we performed DNA microarrays on adipose tissue from adipose-specific GLUT4-overexpressing and GLUT4 knockout mice to find reciprocally regulated genes, and we identified several molecules that were not previously known to regulate systemic insulin sensitivity and/or energy balance. More recently, with Alan Saghatelian’s lab, we discovered a novel class of lipids with antidiabetes and anti-inflammatory effects. We also investigated the effects of the adipose-secreted hormone, leptin, on insulin sensitivity. We found that the AMP-activated protein kinase (AMPK) pathway mediates leptin’s effects on fatty acid oxidation in muscle and also plays a role in leptin’s anorexigenic effects in the hypothalamus. These studies transformed AMPK from a “fuel gauge” that regulates energy supply at the cellular level to a sensing and signaling pathway that regulates organismal energy balance. Overall, these studies have expanded our understanding of the multifaceted role of adipose tissue in metabolic health and how adipose dysfunction increases the risk for type 2 diabetes.

Female adipose tissue-specific Bscl2 knockout mice develop only moderate metabolic dysfunction when housed at thermoneutrality and fed a high-fat diet

Mutations affecting the BSCL2 gene cause the most severe form of congenital generalised lipodystrophy. Affected individuals almost completely lack adipose tissue and suffer from severe diabetes and metabolic complications. Likewise, mice lacking Bscl2 in all tissues have dramatically reduced adipose mass, glucose intolerance and hyperinsulinaemia. However, male adipose tissue-specific Bscl2 knockout mice fail to develop the metabolic dysfunction observed in Bscl2 null mice and BSCL2 deficient patients, despite a similar generalised lack of adipose tissues. Clinical reports indicate gender differences frequently exist in cases of lipodystrophy, with female patients more adversely affected than male patients. We therefore generated and characterised female mice lacking Bscl2 specifically in adipose tissue (Ad-B2(−/−)). We show that female Ad-B2(−/−) mice also develop early-onset lipodystrophy when fed a chow diet and are maintained under standard housing conditions (21 °C) or thermoneutrality (30 °C). Despite this, female Ad-B2(−/−) mice fail to develop severe metabolic dysfunction. Only when female Ad-B2(−/−) mice are maintained at thermoneutrality and fed a high-fat diet do subtle alterations to metabolic homeostasis manifest. This is despite a striking inability to expand adipose mass. Our findings provide further evidence that loss of Bscl2 in non-adipose tissues may contribute to the severity of metabolic dysfunction in this condition.

Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing

Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

2028 - Loss of FBXO9 Accelerates the Onset of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients with the inv(16)/t(16;16) subtype typically have a good prognosis. Despite this positive prediction, 25–58% of patients presenting with AMLs of this subtype relapse with a leukemia unresponsive to current therapies. Additionally, the majority of patients are diagnosed over the age of 60 a patient population unable to withstand harsh chemotherapy and thus unable to effectively prevent progression of the disease. In order to more effectively combat AML and provide new therapies for treatment-resistant relapse, we must increase our understanding of the mechanisms promoting AML. We identified FBXO9, the substrate recognition component of the Skp1-Cul1-F-box (SCF) E3 ligase complex, as an important regulator of normal and malignant hematopoiesis. Analysis by qRT-PCR revealed high Fbxo9 expression in hematopoietic stem and progenitor cells (HSPC) and silencing of Fbxo9 leads to a decrease in HSPCs and reduced colony formation. Furthermore, we have found reduced expression in AML patients, particularly in the inv(16)/t(16;16) subtype. To facilitate study of FBXO9 in HSPC self-renewal and transformation, we engineered the first tissue-specific conditional Fbxo9 knockout mouse model. Our mouse studies revealed that loss of Fbxo9 concurrent with expression of the inv(16) fusion protein CBFB-SMMHC leads to the rapid onset and progression of AML. Alone, CBFB-SMMHC expression results in a block in myeloid differentiation but is insufficient to give rise to AML. When combined with loss of Fbxo9, the resulting AML has a very aggressive phenotype with high frequency of immature blasts in the blood and bone marrow. To further elucidate the molecular mechanisms regulated by Fbxo9, we performed a TMT-labeled mass spectrometry analysis of tumor cells and identified differentially expressed proteins. These analyses, in combination with ubiquitin motif search, provide potential novel Fbxo9 substrates and begin to outline the proteomic landscape of AML-inv(16). Taken together, our studies identify FBXO9 as a novel tumor suppressor of AML and provide a means of identifying potential targets for the development of more effective therapies.

PO-206 Modelling T cell acute lymphoblastic leukaemia using CRISPR/Cas9 mediated genome editing in Xenopus tropicalis

IntroductionThe helicase BRIP1 is critically required for genomic maintenance, and BRIP1 helicase deficient germline variants are associated with Fanconi Anaemia (bi-allelic) and various cancers (mono-allelic), in particular ovarian and breast cancer. Cellular based assays and biochemical studies have elucidated the function of BRIP1 in replication stress responses, including interaction partners and enzymatic substrate specificity. However, functional characterisation of BRIP1 for these processes in vivo are largely unexplored, including the importance of BRIP1 as a tumour suppressor for breast cancer, and therapy responses. This project aims to elucidate the functional roles of BRIP1 as a tumour suppressor for breast cancer and in therapy responses.Material and methodsWe have developed breast tissue-specific knockout mice for BRIP1, and will monitor the development of breast tumours. In addition, we have developed mice deficient for BRIP1 helicase activity (K52R) to further investigate the functional role of BRIP1 as a tumour suppressor. In addition, we will use the mouse models for intervention studies, testing known and novel therapies for BRIP1 deficient breast cancer. Moreover, we will investigate BRIP1 germline variants identified in high-risk breast cancer families by large scale sequencing efforts. This comprehensive list of patient derived mutations will be introduced endogenously in a primary epithelial cell line using a CRISPR-CAS9 approach. To monitor the effect on BRIP1 activity, these cell lines will be tested in cancer relevant functional assays for which BRIP1 deficient cells are known to have a phenotype.Results and discussionsThis project is at an early stage, and we will present our preliminary data from our BRIP1 mouse models, and from our in vitro CRISPR assay for testing BRIP1 gene variants of uncertain significance (VUS).ConclusionThis project is expected to yield novel insight into the regulation of critical genomic stability pathways and provide important knowledge for developing improved germ line variant risk prediction and treatment opportunities for patients.

MiR-34a Aggravates Obesity-Induced Adipose Inflammation and Metabolic Dysfunction via Blocking Polarization of Anti-inflammatory M2 Macrophage

Adipose tissue-resident macrophages are an important contributor to systemic chronic inflammation and cardiometabolic abnormalities associated with obesity. However, the etiological factors that alter the number and polarization of adipose-resident macrophages remain poorly characterized. As our previous clinical study has identified microRNA (miR)-34a as one of the most significantly elevated genes in visceral fat of obese patients, we investigated the roles of miR-34a in obesity-related adipose tissue inflammation and metabolic disorders by generated adipose tissue-specific miR-34a knockout (KO) mice. After feeding with high fat diet (HFD), adipose miR-34a KO mice exhibited significant attenuation of HFD-induced glucose intolerance, insulin resistance, hyperlipidemia, steatohepatitis and systemic inflammation compared to HFD-fed wild type (WT) controls. These benefits of miR-34a deficiency were mainly attributed to the reduced number of total macrophages and polarization of adipose macrophages towards the pro-inflammatory M1 phenotype. Mechanistically, we found that miR-34a directly repressed the expression of the transcription factor Klf4, thereby enhancing macrophage polarization to the M1 phenotype. Silencing of Klf4 in the adipose-specific miR-34a KO mice suppressed M2 macrophage polarization. In visceral fat from obese patients, the expression of Klf4 was significantly decreased and negatively correlated with miR-34a level. These data demonstrated that increased miR-34a in adipose tissues exacerbates obesity-induced adipose tissue inflammation by suppressing the expression of Klf4, thereby leading to increased accumulation of pro-inflammatory M1 macrophages. Disclosure Y. Pan: None. H. Hui: None. R. Hoo: None. T. Feng: None. K.S. Lam: None. A. Xu: None.

Epithelial and Endothelial Pannexin1 Channels Mediate AKI.

Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation.Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI.Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro, Panx1-deficient proximal tubule cells released less and retained more ATP under hypoxic stress.Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI.

Skeletal muscle-specific Cre recombinase expression, controlled by the human α-skeletal actin promoter, improves glucose tolerance in mice fed a high-fat diet.

Cre-loxP systems are frequently used in mouse genetics as research tools for studying tissue-specific functions of numerous genes/proteins. However, the expression of Cre recombinase in a tissue-specific manner often produces undesirable changes in mouse biology that can confound data interpretation when using these tools to generate tissue-specific gene knockout mice. Our objective was to characterise the actions of Cre recombinase in skeletal muscle, and we anticipated that skeletal muscle-specific Cre recombinase expression driven by the human α-skeletal actin (HSA) promoter would influence glucose homeostasis. Eight-week-old HSA-Cre expressing mice and their wild-type littermates were fed a low- or high-fat diet for 12weeks. Glucose homeostasis (glucose/insulin tolerance testing) and whole-body energy metabolism (indirect calorimetry) were assessed. We also measured circulating insulin levels and the muscle expression of key regulators of energy metabolism. Whereas tamoxifen-treated HSA-Cre mice fed a low-fat diet exhibited no alterations in glucose homeostasis, we observed marked improvements in glucose tolerance in tamoxifen-treated, but not corn-oil-treated, HSA-Cre mice fed a high-fat diet vs their wild-type littermates. Moreover, Cre dissociation from heat shock protein 90 and translocation to the nucleus was only seen following tamoxifen treatment. These improvements in glucose tolerance were not due to improvements in insulin sensitivity/signalling or enhanced energy metabolism, but appeared to stem from increases in circulating insulin. The intrinsic glycaemia phenotype in the HSA-Cre mouse necessitates the use of HSA-Cre controls, treated with tamoxifen, when using Cre-loxP models to investigate skeletal muscle-specific gene/protein function and glucose homeostasis.

PO-205 Functional characterisation of BRIP1/FANCJ in tumour suppression and therapy response

Introduction The helicase BRIP1 is critically required for genomic maintenance, and BRIP1 helicase deficient germline variants are associated with Fanconi Anaemia (bi-allelic) and various cancers (mono-allelic), in particular ovarian and breast cancer. Cellular based assays and biochemical studies have elucidated the function of BRIP1 in replication stress responses, including interaction partners and enzymatic substrate specificity. However, functional characterisation of BRIP1 for these processes in vivo are largely unexplored, including the importance of BRIP1 as a tumour suppressor for breast cancer, and therapy responses. This project aims to elucidate the functional roles of BRIP1 as a tumour suppressor for breast cancer and in therapy responses. Material and methods We have developed breast tissue-specific knockout mice for BRIP1, and will monitor the development of breast tumours. In addition, we have developed mice deficient for BRIP1 helicase activity (K52R) to further investigate the functional role of BRIP1 as a tumour suppressor. In addition, we will use the mouse models for intervention studies, testing known and novel therapies for BRIP1 deficient breast cancer. Moreover, we will investigate BRIP1 germline variants identified in high-risk breast cancer families by large scale sequencing efforts. This comprehensive list of patient derived mutations will be introduced endogenously in a primary epithelial cell line using a CRISPR-CAS9 approach. To monitor the effect on BRIP1 activity, these cell lines will be tested in cancer relevant functional assays for which BRIP1 deficient cells are known to have a phenotype. Results and discussions This project is at an early stage, and we will present our preliminary data from our BRIP1 mouse models, and from our in vitro CRISPR assay for testing BRIP1 gene variants of uncertain significance (VUS). Conclusion This project is expected to yield novel insight into the regulation of critical genomic stability pathways and provide important knowledge for developing improved germ line variant risk prediction and treatment opportunities for patients.

PID1 in adipocytes modulates whole-body glucose homeostasis

The novel obesity-associated protein Phosphotyrosine Interaction Domain containing 1 (PID1) inhibits insulin-PI3K/Akt signaling pathway and insulin-stimulated glucose uptake in vitro. In this study, we generated fat tissue-specific aP2-PID1 transgenic (aP2-PID1tg) mice and PID1 knockout (PID1−/−) mice to explore how PID1 affects glucose metabolism in vivo. We observed insulin resistance and impaired insulin-PI3K/Akt signaling in aP2-PID1tg mice. Consistent with these data, the PID1−/− mice displayed improved glucose tolerance and insulin sensitivity under chow diet, with increased Akt phosphorylation in white adipose tissue (WAT). We further demonstrated that PID1 could interact with low density lipoprotein receptor-related protein 1 (LRP1) but not the insulin receptor (IR) in adipocytes, and its overexpression could lead to decreased GLUT4 level. Our results thus indentify PID1 as a critical regulator of glucose metabolism in adipocytes.

Generation and Analysis of Striated Muscle Selective LINC Complex Protein Mutant Mice.

The linker of nucleoskeleton and cytoskeleton (LINC) complex mediates intracellular cross talk between the nucleus and the cytoplasm. In striated muscle, the LINC complex provides structural support to the myocyte nucleus and plays an essential role in regulating gene expression and mechanotransduction. A wide range of cardiac and skeletal myopathies have been linked to mutations in LINC complex proteins. Studies utilizing tissue-specific knockout and mutant mouse models have revealed important insights into the roles of the LINC complex in striated muscle. In this chapter, we describe several feasible approaches for generating striated muscle-specific gene knockout and mutant mouse models to study LINC complex protein function in cardiac and skeletal muscle. The experimental procedures used for phenotyping and analysis of LINC complex knockout mice are also described.

Study of the Endogenous CRAC Channel Using shRNA-Mediated Gene Silencing.

The Ca2+ release-activated Ca2+ (CRAC) current is a major signaling event in non-excitable cells whereby Ca2+ store depletion activates Ca2+ entry across the plasma membrane from the extracellular space. Stromal interaction molecule 1 (STIM1) and Orai1 proteins are the key molecular players of the CRAC channel. Previous studies have linked activity of this channel to many physiological functions, and dysregulation of the CRAC channel has been associated with various diseases. In the absence of inducible tissue-specific knockout mice, in vivo knockdown studies examining the endogenous function of CRAC channel proteins, STIM1 and Orai1, are a challenge. In this chapter, we describe a lentiviral delivery system of shRNA-mediated gene silencing that has proven successful in studying the endogenous CRAC channel in vivo.