EKO Mice Research Articles

The transcriptome profiling of diseased mouse aortas discloses a dysregulation of the sympathetic neurotransmission in atherosclerosis

Previous reports suggest an association between the development of atherosclerosis and alterations in the aortic sympathetic nervous system, but there is no agreement on whether atherosclerotic plaques are accompanied by increased or decreased sympathetic innervation in the arterial wall.In the present study, the aortic transcriptional profile of mice with different predisposition to atherosclerosis was investigated to clarify how the expression of genes involved in sympathetic neurotransmission varied.Eight-week-old C57Bl/6J control mice, Apoe knockout mice (EKO), EKO mice overexpressing human apoA-I (EKO/hA-I) and double Apoe/Apoa1 knockout mice (DKO) mice were fed either a standard rodent diet or a Western-type diet for 22 weeks. Atherosclerosis was quantified, and the aortic transcriptome was analyzed by RNAseq. Western-type diet administration deeply modified the aortic transcriptome. In the genetically modified atherosclerosis-prone mouse lines, an upregulated expression of genes associated with the immunomodulatory response was observed, paralleled by a downregulated expression of the genes related to sympathetic nervous system. Functional enrichment analysis indicated that the presence of advanced atherosclerosis was accompanied by reduced neuronal generation, modulation of synapse chemical transmission, and catecholamine biosynthesis, supporting a relationship between atherosclerosis, dyslipidemia, and sympathetic neurotransmission.

Endothelial peroxiredoxin-4 is indispensable for blood–brain barrier integrity and long-term functional recovery after ischemic stroke

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

The EZH2–PRC2–H3K27me3 axis governs the endometrial cell cycle and differentiation for blastocyst invasion

Infertility occurs in 15% of couples worldwide. Recurrent implantation failure (RIF) is one of the major problems in in vitro fertilization and embryo transfer (IVF–ET) programs, and how to manage patients with RIF to achieve successful pregnancy outcomes remains unresolved. Here, a uterine polycomb repressive complex 2 (PRC2)-regulated gene network was found to control embryo implantation. Our RNA-seq analyses of the human peri-implantation endometrium obtained from patients with RIF and fertile controls revealed that PRC2 components, including its core enzyme enhancer of zeste homolog 2 (EZH2)-catalyzing H3K27 trimethylation (H3K27me3) and their target genes are dysregulated in the RIF group. Although fertility of uterine epithelium-specific knockout mice of Ezh2 (eKO mice) was normal, Ezh2-deleted mice in the uterine epithelium and stroma (uKO mice) exhibited severe subfertility, suggesting that stromal Ezh2 plays a key role in female fertility. The RNA-seq and ChIP-seq analyses revealed that H3K27me3-related dynamic gene silencing is canceled, and the gene expression of cell-cycle regulators is dysregulated in Ezh2-deleted uteri, causing severe epithelial and stromal differentiation defects and failed embryo invasion. Thus, our findings indicate that the EZH2–PRC2–H3K27me3 axis is critical to preparing the endometrium for the blastocyst invasion into the stroma in mice and humans.

Abstract 455: Lats1/2 Expression Reduced By The Pathological Flow Triggers Intraplaque Hemorrhage And Distal Pulmonary Thrombosis By Promoting EC Apoptosis And Proliferation In Concert

Atherosclerotic and high-risk plaques are predominantly localized to vessel wall regions with disturbed blood flow (d-flow), leading to the theory that d- and low-flow-driven endothelial cells (EC) dysfunction play a role in this process. It has become clear that high flow (h-flow; high shear stress due to increased flow rate) is also critical to developing high-risk plaques including both rupture and erosion. We aim to determine how the high-risk plaque is formed by pathological flow. The pathological flow (both h- and d-flow) resulted in decreased LATS1/2 expression, but physiological laminar flow did not. Within 14 days after inducing LATS1/2 deletion in tamoxifen-inducible EC-specific LATS1 homo /LATS2 homo knock-out mice (LATS1 homo /LATS2 homo -EKO), all the mice (27/27) had died of severe systemic edema, accompanied by increased EC apoptosis and vascular permeability. We generated EC-specific LATS1 he t /LATS2 homo -EKO mice under hypercholesterolemia (H-chol) with pAAV/D377Y-mPCSK9 injections and a high-fat diet (HFD). After 8weeks of HFD, we detected plaque erosion-like lesions at the h- and d-flow area in the aortic arch and carotids, which revealed strong fibrin/fibrinogen-positive organized thrombus formation without a sizeable necrotic core. Interestingly, we also found a significant increase in distal pulmonary thrombosis and EC proliferation in LATS1 he t /LATS2 homo -EKO mice. H-chol increased the incidents of aortic but not lung lesions. We also performed partial left carotid artery ligation (PLCL) and found that d-flow induced by PLCL developed severe atherosclerosis formation with unique intraplaque hemorrhage in H-chol LATS1 he t /LATS2 homo -EKO mice, which was not observed in wild-type mice. In LATS1 he t /LATS2 homo -EKO mice, we found significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression and 4) inflammation, which are collectively referred to as the “pro-thrombotic phenotype” in vivo . These data suggest that pathological flow-induced LATS1/2 degradation promotes EC apoptosis and proliferation simultaneously. This cycle of EC damage-thrombus formation-re-endothelialization results in rapid thrombus enlargement and distal pulmonary thrombosis.

Endothelial TAZ inhibits capillarization of liver sinusoidal endothelium and damage-induced liver fibrosis via nitric oxide production.

Background: Endothelial dysfunction is a systemic disorder and is involved in the pathogenesis of several human diseases. Hemodynamic shear stress plays an important role in vascular homeostasis including nitric oxide (NO) production. Impairment of NO production in endothelial cells stimulates the capillarization of liver sinusoidal endothelial cells, followed by hepatic stellate cell activation, inducing liver fibrosis. However, the detailed mechanism underlying NO production is not well understood. In hepatocytes, transcriptional co-activator with PDZ-binding motif (TAZ) has been reported to be involved in liver fibrosis. However, the role of endothelial TAZ in liver fibrosis has not been investigated. In this study, we uncovered the role TAZ in endothelial cell NO production, and its subsequent effects on liver fibrosis. Methods: TAZ-floxed mice were crossed with Tie2-cre transgenic mice, to generate endothelium-specific TAZ-knockout (eKO) mice. To induce liver damage, a 3,5-diethoxycarboncyl-1,4-dihydrocollidine, methionine-choline-deficient diet, or partial hepatectomy was applied. Liver fibrosis and endothelial dysfunction were analyzed in wild-type and eKO mice after liver damage. In addition, liver sinusoidal endothelial cell (LSEC) was used for in vitro assays of protein and mRNA levels. To study transcriptional regulation, chromatin immunoprecipitation and luciferase reporter assays were performed. Results: In liver of eKO mice, LSEC capillarization was observed, evidenced by loss of fenestrae and decreased LSEC-specific marker gene expression. LSEC capillarization of eKO mouse is caused by downregulation of endothelial nitric oxide synthase expression and subsequent decrease in NO concentration, which is transcriptionally regulated by TAZ-KLF2 binding to Nos3 promoter. Diminished NO concentration by TAZ knockout in endothelium accelerates liver fibrosis induced by liver damages. Conclusions: Endothelial TAZ inhibits damage-induced liver fibrosis via NO production. This highlights an unappreciated role of TAZ in vascular health and liver diseases.

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Endothelial ATP-Sensitive Potassium Channel Protects Against the Development of Hypertension and Atherosclerosis.

In the endothelium, ATP-sensitive potassium (KATP) channels are thought to couple cellular metabolism with membrane excitability, calcium entry, and endothelial mediator release. We hypothesized that endothelial KATP channels have a broad role protecting against high blood pressure and atherosclerosis. Endothelial-specific Kir6.1 KO mice (eKO) and eKO mice on an apolipoprotein E KO background were generated (A-eKO) to investigate the role of KATP channels in the endothelium. Basal blood pressure was not elevated in eKO mice. However, when challenged with a high-salt diet and the eNOS inhibitor L-NAME, eKO mice became more hypertensive than their littermate controls. In aorta, NO release at least partly contributes to the endothelium-dependent vasorelaxation induced by pinacidil. In A-eKO mice atherosclerotic plaque density was significantly greater than in their littermate controls when challenged with a high-fat diet, particularly in the aortic arch region. Levels of endothelial dysfunction markers were higher in eKO compared with WT mice; however, these were not significant for A-eKO mice compared with their littermate controls. Furthermore, decreased vascular reactivity was observed in the mesenteric arteries of A-eKO mice, but not in aorta when on a high-fat diet. Our data support a role for endothelial Kir6.1-containing KATP channels in the endothelial protection against environmental stressors: the maintenance of blood pressure homeostasis in response to high salt and endothelial integrity when challenged with a high-fat diet.

Vascular KATP channels protect from cardiac dysfunction and preserve cardiac metabolism during endotoxemia

KATP channels in the vasculature composed of Kir6.1 regulate vascular tone and may contribute to the pathogenesis of endotoxemia. We used mice with cell-specific deletion of Kir6.1 in smooth muscle (smKO) and endothelium (eKO) to investigate this question. We found that smKO mice had a significant survival disadvantage compared with their littermate controls when treated with a sub-lethal dose of lipopolysaccharide (LPS). All cohorts of mice became hypotensive following bacterial LPS administration; however, mean arterial pressure in WT mice recovered to normal levels, whereas smKO struggled to overcome LPS-induced hypotension. In vivo and ex vivo investigations revealed pronounced cardiac dysfunction in LPS-treated smKO, but not in eKO mice. Similar results were observed in a cecal slurry injection model. Metabolomic profiling of hearts revealed significantly reduced levels of metabolites involved in redox/energetics, TCA cycle, lipid/fatty acid and amino acid metabolism. Vascular smooth muscle-localised KATP channels have a critical role in the response to systemic infection by normalising cardiac function and haemodynamics through metabolic homeostasis.Key messages• Mice lacking vascular KATP channels are more susceptible to death from infection.• Absence of smooth muscle KATP channels depresses cardiac function during infection.• Cardiac dysfunction is accompanied by profound changes in cellular metabolites.• Findings from this study suggest a protective role for vascular KATP channels in response to systemic infection.

Loss of dynamin-related protein 1 (Drp1) does not affect epidermal development or UVB-induced apoptosis but does accelerate UVB-induced carcinogenesis

BackgroundMitochondrial morphology is controlled by fission and fusion. Dynamin-related protein 1 (Drp1, dynamin-1-like protein (Dnml1)) regulates mitochondrial fission, which is associated with cell division and apoptosis. We previously reported that DRP1 is indispensable for cell growth in cutaneous squamous cell carcinoma. However, little is known about Drp1 in normal epidermis/keratinocytes. ObjectivesWe investigated the function of Drp1 in normal epidermis/keratinocytes. MethodsEpidermis-specific Drp1 knockout (EKO) mice were analyzed. ResultsEpidermal development in the EKO mice were indistinguishable from those in the wild-type (WT) mice. Ultrastructural analysis and immunohistochemistry revealed that the mitochondria of keratinocytes in the EKO mice were neither elongated nor constricted. Drp1 knockdown did not diminish the cell growth of normal human keratinocytes. Both in vivo and in vitro, UVB-induced apoptosis in the EKO epidermis and keratinocytes did not differ from that in the WT mice. In chronic UVB-irradiation, the loss of Drp1 sensitized the epidermis to the development of skin tumors. Clinically, DRP1 is expressed more highly in sun-exposed skin than in non-exposed skin in individuals under age 40, but not in those over age 60. ConclusionEKO mice demonstrate that Drp1 is dispensable for the development and apoptosis of the epidermis. Drp1 plays critical roles in malignant tumors; thus, the molecular machinery of mitochondrial dynamics involving Drp1 could be a novel therapeutic target for malignant keratinocytic lesions. On the other hand, the anti-tumorigenic role of Drp1 in chronic UVB-induced carcinogenesis need to be further investigated.

Immunoproteasome subunit β5i regulates diet-induced atherosclerosis through altering MERTK-mediated efferocytosis in Apoe knockout mice.

The immunoproteasome contains three catalytic subunits (β1i, β2i and β5i) that are important modulators of immune cell homeostasis. A previous study showed a correlation between β5i and human atherosclerotic plaque instability; however, the causative role of β5i in atherosclerosis and the underlying mechanisms remain unknown. Here we explored this issue in apolipoprotein E (Apoe) knockout (eKO) mice with genetic deletion or pharmacological inhibition of β5i. We found that β5i expression was upregulated in lesional macrophages after an atherogenic diet (ATD). β5i/Apoe double KO (dKO) mice fed on the ATD had a significant decrease in both lesion area and necrotic core area, compared with eKO controls. Moreover, dKO mice had less caspase-3+ apoptotic cell accumulation but enhanced efferocytosis of apoptotic cells and increased expression of Mer receptor tyrosine kinase (MERTK). Consistently, similar phenotypes were observed in eKO mice transplanted with dKO bone marrow or treated with β5i-specific inhibitor PR-957. Mechanistic studies in vitro revealed that β5i deletion reduced IκBα degradation and inhibited NF-κB activation, promoting Mertk transcription and efferocytosis, thereby attenuating apoptotic cell accumulation. In conclusion, we demonstrate that β5i plays an important role in diet-induced atherosclerosis by altering MERTK-mediated efferocytosis. β5i might be a potential pharmaceutical target against atherosclerosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fenretinide treatment accelerates atherosclerosis development in apoE-deficient mice in spite of beneficial metabolic effects.

Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action. To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelveweeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses. Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice. We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis.

Molecular and functional characterization of the endothelial ATP-sensitive potassium channel

ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system, where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells, but their presence and function in this context are less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch clamp, ex vivo coronary perfusion Langendorff heart experiments, and endothelial cell Ca2+ imaging comparing eKO and wild-type mice show that Kir6.1-containing KATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca2+], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the KATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation, and this resulted in greater cardiac injury during ischemia-reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial KATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation, and has a protective role in ischemia reperfusion.

Abstract A27: Redefining the role of Notch in acinar to ductal metaplasia

Abstract The EGFR and Notch signaling pathways have been widely implicated in relation to both chronic pancreatitis and pancreatic ductal adenocarcinoma in humans, however the link between the two pathways has remained obscure. Acinar to ductal metaplasia (ADM,) a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells, has been linked to both the Notch and EGFR signaling pathways. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasia, a precursor lesion to pancreatic cancer. Recently we and others have shown that genetic ablation of EGFR in the pancreas effectively prevents acinar to ductal metaplasia in cerulein model of pancreatitis in mice, and prevents tumorigenesis driven by oncogenic Kras. Additionally, we have found that acinar cells harvested from EGFRf/f;Ptf1aCre/+ mice (EKO) mice are resistant to ADM in vitro upon cerulein treatment, or by the expression of oncogenic Kras. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has previously been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has also been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EKO mice. Interestingly, these acinar cells fail to transdifferentiate in vitro upon transduction of N2ICD in 3D culture; however the transdifferentiation event can be rescued by co-expression of oncogenic Kras with N2ICD in acinar cells lacking EGFR. These results indicate that EGFR is important in Notch induced ADM, however there are other pathways that possibly can be utilized by acinar cells expressing oncogenic Kras to drive ADM. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Redefining the role of Notch in acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A27.

Abstract A09: Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia

Abstract Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase ϵ Tunes Neuronal Excitability

Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.

Hyperglycemia and advanced glycosylation end products suppress adipocyte apoE expression: implications for adipocyte triglyceride metabolism

Endogenous adipocyte apolipoprotein E (apoE) plays an important role in adipocyte lipoprotein metabolism and lipid flux. A potential role for hyperglycemia in regulating adipocyte apoE expression and triglyceride metabolism was examined. Exposure of adipocytes to high glucose or advanced glycosylation end product-BSA significantly suppressed apoE mRNA and protein levels. This suppression was significantly attenuated by antioxidants or inhibitors of the NF-κB transcription pathway. Hyperglycemia in vivo led to adipose tissue oxidant stress and significant reduction in adipose tissue and adipocyte apoE mRNA level. Incubation with antioxidant in organ culture completely reversed this suppression. Hyperglycemia also reduced adipocyte triglyceride synthesis, and this could be completely reversed by adenoviral-mediated increases in apoE. To more specifically evaluate an in vivo role for adipocyte apoE expression on organismal triglyceride distribution in vivo, WT or apoE knockout (EKO) adipose tissue was transplanted in EKO recipient mice. After 12 wk, WT adipocytes transplanted in EKO mice accumulated more triglyceride compared with transplanted EKO adipocytes. In addition, EKO recipients of WT adipose tissue had reduced hepatic triglyceride content compared with EKO recipients transplanted with EKO adipose tissue. Our results demonstrate that hyperglycemia and advanced glycosylation end products suppress the expression of adipocyte apoE in vitro and in vivo and thereby reduce adipocyte triglyceride synthesis. In vivo results using adipose tissue transplantation suggest that reduction of adipocyte apoE, and subsequent reduction of adipocyte triglyceride accumulation, could influence lipid accumulation in nonadipose tissue.

Protein Tyrosine Phosphatase Epsilon Regulates Integrin-mediated Podosome Stability in Osteoclasts by Activating Src

The nonreceptor isoform of tyrosine phosphatase epsilon (cyt-PTPe) supports osteoclast adhesion and activity in vivo, leading to increased bone mass in female mice lacking PTPe (EKO mice). The structure and organization of the podosomal adhesion structures of EKO osteoclasts are abnormal; the molecular mechanism behind this is unknown. We show here that EKO podosomes are disorganized, unusually stable, and reorganize poorly in response to physical contact. Phosphorylation and activities of Src, Pyk2, and Rac are decreased and Rho activity is increased in EKO osteoclasts, suggesting that integrin signaling is defective in these cells. Integrin activation regulates cyt-PTPe by inducing Src-dependent phosphorylation of cyt-PTPe at Y638. This phosphorylation event is crucial because wild-type-but not Y638F-cyt-PTPe binds and further activates Src and restores normal stability to podosomes in EKO osteoclasts. Increasing Src activity or inhibiting Rho or its downstream effector Rho kinase in EKO osteoclasts rescues their podosomal stability phenotype, indicating that cyt-PTPe affects podosome stability by functioning upstream of these molecules. We conclude that cyt-PTPe participates in a feedback loop that ensures proper Src activation downstream of integrins, thus linking integrin signaling with Src activation and accurate organization and stability of podosomes in osteoclasts.

Enhanced atherothrombotic formation after oxidative injury by FeCl 3 to the common carotid artery in severe combined hyperlipidemic mice

Enhanced susceptibility to atherosclerosis from severe hypertriglyceridemia (HTG) resulting from lipoprotein lipase (LPL) deficiency has been demonstrated in our recent findings which employed a unique mouse model. In the present study we provide further evidence that severe HTG due to LPL deficiency also promotes an atherothrombotic response to arterial injury induced by ferric chloride in a severe combined hyperlipidemic mouse model. Methods and results: A mouse model (LPL −/−XApoE −/− double knockout, DKO) with severe combined hyperlipidemia was established by crossing ApoE and LPL-deficient mice. The common carotid arteries of ApoE knockout (EKO) and DKO mice were subjected to injury by ferric chloride, and the formation of arterial thrombosis together with various markers were compared in these lesions. DKO mice demonstrated significantly enhanced thrombus formation overlying atherosclerotic plaque after injury, which contained smooth muscle cells, macrophages, and neutral lipid. The area of neointima, mean intima/media ratios, and the percentage of luminal stenosis were significantly greater ( P < 0.01) in DKO mice. Compared with EKO mice, the expression of von Willebrand factor (vWF) and plasminogen activator inhibitor type 1 (PAI-1) were increased in DKO mice. Conclusions: Severe combined hyperlipidemia promotes thrombosis after ferric chloride injury to atherosclerotic vessels and HTG plays a major role in the process.

Peroxisome Proliferator-activated Receptor γ Stimulation of Adipocyte ApoE Gene Transcription Mediated by the Liver Receptor X Pathway

Peroxisome proliferator-activated receptor (PPARgamma) agonists increase insulin sensitivity in humans and are useful for treating human diabetes. Treatment with these agonists leads to increased apoE expression and triglyceride accumulation in adipocytes. The importance of apoE for adipocyte triglyceride accumulation is demonstrated by observations that triglyceride accumulation is impaired in apoE knockout adipocytes treated with PPARgamma agonists. The current studies investigate the molecular mechanism for PPARgamma stimulation of the adipocyte apoE gene and demonstrate that the liver receptor X (LXR) response element within an apoE gene downstream enhancer is required for the apoE response to PPARgamma agonists. The response of the apoE gene to treatment with PPARgamma agonists was delayed beyond 12 h suggesting the involvement of an intermediary pathway. The combined addition of PPARgamma and LXR agonists did not increase apoE response beyond that observed with addition of either alone. Deletion or mutation of the LXR response element completely eliminated the adipocyte apoE gene response to a PPARgamma agonist. Chromatin immunoprecipitation analyses performed using isolated adipocytes, or adipose tissue from mice treated with PPARgamma agonists, showed increased LXR binding to the apoE gene after PPARgamma agonist treatment. Knockdown of LXR expression completely eliminated the increase in apoE message, protein, and triglyceride in response to PPARgamma stimulation. The LXR response element has been previously shown to mediate sterol responsiveness of the apoE gene, and apoE expression plays an important role in adipocyte triglyceride balance. The current observations suggest that the PPARgamma-LXR-apoE regulatory cascade could be an important molecular link for cross-talk between adipocyte triglyceride and cholesterol homeostasis.

Role of adipocyte-derived apoE in modulating adipocyte size, lipid metabolism, and gene expression in vivo

Adipocytes isolated from apolipoprotein E (apoE)-knockout (EKO) mice display alterations in triglyceride (TG) metabolism and gene expression. The present studies were undertaken to evaluate the impact of endogenously produced adipocyte apoE on these adipocyte parameters in vivo, independent of the profoundly disturbed metabolic milieu of EKO mice. Adipose tissue from wild-type (WT) or EKO mice was transplanted into WT recipients, which were then fed chow or high-fat diet for 8-10 wk. After a chow diet, freshly isolated transplanted EKO adipocytes were significantly (P < 0.05) smaller (70%) than transplanted WT adipocytes and displayed significantly lower rates of TG synthesis and higher rates of TG hydrolysis. Transplanted EKO adipocytes also had higher mRNA levels for adiponectin, perilipin, and genes coding for enzymes in the fatty acid oxidation pathway and lower levels of caveolin. After a high-fat diet and consequent increase in circulating lipid and apoE levels, transplanted WT adipocyte size increased by 106 x 10(3) microm(3), whereas EKO adipocyte size increased only by 19 x 10(3) microm(3). Endogenous host adipose tissue harvested from WT recipients of transplanted WT or EKO adipose tissue did not demonstrate any difference in adipocyte size. Consistent with the in vivo observations, EKO adipocytes synthesized less TG when incubated with apoE-containing TG-rich lipoproteins than WT adipocytes. Our results establish a novel in vivo role for endogenously produced apoE, distinct from circulating apoE, in modulation of adipocyte TG metabolism and gene expression. They support a model in which endogenously produced adipocyte apoE facilitates adipocyte lipid acquisition from circulating TG-rich lipoproteins.