Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

Down-regulation of AKT proteins slows the growth of mutant-KRAS pancreatic tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinoma (PDAC) harbors activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilizes proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. Concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions and the IGF-1 growth stimulation effect was AKT dependent. RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth and pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

Propionyl-CoA carboxylase subunit B regulates anti-tumor T cells in a pancreatic cancer mouse model.

Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca-/- KPC (named αKO) cancer cells induces clonal expansion of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally expanded CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally expanded T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

Trends in myocardial infarction and coronary revascularisation procedures in Australia, 1993-2017.

OBJECTIVE: Prior data have shown rising acute myocardial infarction (MI) trends in Australia; whether these increases have continued in recent years is not known. This study thus sought to characterise contemporary nationwide trends in MI hospitalisations and coronary procedures in Australia and their associated economic burden. METHODS: The primary outcome measure was the incidence and time trends of total MI, ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI) hospitalisations from 1993 to 2017. The incidence and time trends of coronary procedures were additionally collected, alongside MI hospitalisation costs. RESULTS: Adjusted for population changes, annual MI incidence increased from 216.2 cases per 100 000 to a peak of 270.4 in 2007 with subsequent decline to 218.7 in 2017. Similarly, NSTEMI incidence increased from 68.0 cases per 100 000 in 1993 to a peak of 192.6 in 2007 with subsequent decline to 162.6 in 2017. STEMI incidence decreased from 148.3 cases per 100 000 in 1993 to 56.2 in 2017. Across the study period, there were annual increases in MI hospitalisations of 0.7% and NSTEMI hospitalisations of 5.6%, and an annual decrease in STEMI hospitalisations of 4.8%. Angiography and percutaneous coronary intervention increased by 3.4% and 3.3% annually, respectively, while coronary artery bypass graft surgery declined by 2.2% annually. MI hospitalisation costs increased by 100% over the study period, despite a decreased average length of stay by 45%. CONCLUSIONS: The rising incidence of MI hospitalisations appear to have stabilised in Australia. Despite this, associated healthcare expenditure remains significant, suggesting a need for continual implementation of public health policies and preventative strategies.

Intercellular delivery of therapeutic oligonucleotides.

One promising approach to cancer therapeutics is to induce changes in gene expression that either reduce cancer cell proliferation or induce cancer cell death. Therefore, delivering oligonucleotides (siRNA/miRNA) that target specific genes or gene programs might have a potential therapeutic benefit. The aim of this study was to examine the potential of cell-based delivery of oligonucleotides to cancer cells via two naturally occurring intercellular pathways: gap junctions and vesicular/exosomal traffic. We utilized human mesenchymal stem cells (hMSCs) as delivery cells and chose to deliver in vitro two synthetic oligonucleotides, AllStars HS Cell Death siRNA and miR-16 mimic, as toxic (therapeutic) oligonucleotides targeting three cancer cell lines: prostate (PC3), pancreatic (PANC1) and cervical (HeLa). Both oligonucleotides dramatically reduced cell proliferation and/or induced cell death when transfected directly into target cells and delivery hMSCs. The delivery and target cells we chose express gap junction connexin 43 (Cx43) endogenously (PC3, PANC1, hMSC) or via stable transfection (HeLaCx43). Co-culture of hMSCs (transfected with either toxic oligonucleotide) with any of Cx43 expressing cancer cells induced target cell death (~20% surviving) or senescence (~85% proliferation reduction) over 96 hours. We eliminated gap junction-mediated delivery by using connexin deficient HeLaWT cells or knocking out endogenous Cx43 in PANC1 and PC3 cells via CRISPR/Cas9. Subsequently, all Cx43 deficient target cells co-cultured with the same toxic oligonucleotide loaded hMSCs proliferated, albeit at significantly slower rates, with cell number increasing on average ~2.2-fold (30% of control cells) over 96 hours. Our results show that both gap junction and vesicular/exosomal intercellular delivery pathways from hMSCs to target cancer cells deliver oligonucleotides and function to either induce cell death or significantly reduce their proliferation. Thus, hMSC-based cellular delivery is an effective method of delivering synthetic oligonucleotides that can significantly reduce tumor cell growth and should be further investigated as a possible approach to cancer therapy.

Loss of TRIM21 alleviates cardiotoxicity by suppressing ferroptosis induced by the chemotherapeutic agent doxorubicin.

BACKGROUND: Doxorubicin, an anthracycline chemotherapeutic agent, is widely used in the treatment of many cancers. However, doxorubicin posts a great risk of adverse cardiovascular events, which are thought to be caused by oxidative stress. We recently reported that the ubiquitin E3 ligase TRIM21 interacts and ubiquitylates p62 and negatively regulates the p62-Keap1-Nrf2 antioxidant pathway. Therefore, we sought to determine the role TRIM21 in cardiotoxicity induced by oxidative damage. METHODS: Using TRIM21 knockout mice, we examined the effects of TRIM21 on cardiotoxicity induced by two oxidative damage models: the doxorubicin treatment model and the Left Anterior Descending (LAD) model. We also explored the underlying mechanism by RNA-sequencing of the heart tissues, and by treating the mouse embryonic fibroblasts (MEFs), immortalized rat cardiomyocyte line H9c2, and immortalized human cardiomyocyte line AC16 with doxorubicin. FINDINGS: TRIM21 knockout mice are protected from heart failure and fatality in both the doxorubicin and LAD models. Hearts of doxorubicin-treated wild-type mice exhibit deformed mitochondria and elevated level of lipid peroxidation reminiscent of ferroptosis, which is alleviated in TRIM21 knockout hearts. Mechanistically, TRIM21-deficient heart tissues and cultured MEFs and H9c2 cells display enhanced p62 sequestration of Keap1 and are protected from doxorubicin-induced ferroptosis. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient TRIM21 mutants impedes the protection from doxorubicin-induced cell death. INTERPRETATION: Our study demonstrates that TRIM21 ablation protects doxorubicin-induced cardiotoxicity and illustrates a new function of TRIM21 in ferroptosis, and suggests TRIM21 as a therapeutic target for reducing chemotherapy-related cardiotoxicity. FUNDING: NIH (CA129536; DK108989): data collection, analysis. Shanghai Pujiang Program (19PJ1401900): data collection. National Natural Science Foundation (31971161): data collection. Department of Veteran Affairs (BX004083): data collection. Tianjin Science and Technology Plan Project (17ZXMFSY00020): data collection.

Endothelial JAK2V617F mutation leads to thrombosis, vasculopathy, and cardiomyopathy in a murine model of myeloproliferative neoplasm.

OBJECTIVE: Cardiovascular complications are the leading cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The acquired kinase mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for cardiovascular dysfunction in MPNs are not fully understood, limiting the effectiveness of current treatment. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs. The goal of this study was to test the hypothesis that the JAK2V617F mutation alters endothelial function to promote cardiovascular complications in patients with MPNs. APPROACH AND RESULTS: We employed murine models of MPN in which the JAK2V617F mutation is expressed in specific cell lineages. When JAK2V617F is expressed in both blood cells and vascular ECs, the mice developed MPN and spontaneous, age-related dilated cardiomyopathy with an increased risk of sudden death as well as a prothrombotic and vasculopathy phenotype on histology evaluation. In contrast, despite having significantly higher leukocyte and platelet counts than controls, mice with JAK2V617F-mutant blood cells alone did not demonstrate any cardiac dysfunction, suggesting that JAK2V617F-mutant ECs are required for this cardiovascular disease phenotype. Furthermore, we demonstrated that the JAK2V617F mutation promotes a pro-adhesive, pro-inflammatory, and vasculopathy EC phenotype, and mutant ECs respond to flow shear differently than wild-type ECs. CONCLUSIONS: These findings suggest that the JAK2V617F mutation can alter vascular endothelial function to promote cardiovascular complications in MPNs. Therefore, targeting the MPN vasculature represents a promising new therapeutic strategy for patients with MPNs.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

CEMIP upregulates BiP to promote breast cancer cell survival in hypoxia.

Cell migration-inducing protein (CEMIP) and binding immunoglobulin protein (BiP) are upregulated in human cancers, where they drive cancer progression and metastasis. It has been shown that CEMIP resides in the endoplasmic reticulum (ER) where it interacts with BiP to induce cell migration, but the relationship between the two proteins was previously unknown. Here we show that CEMIP mediates activation of the BiP promoter and upregulates BiP transcript and protein levels in breast cancer cell lines. Moreover, CEMIP overexpression confers protective adaptations to cancer cells under hypoxic conditions, by decreasing apoptosis, activating autophagy, and increasing glucose uptake, to facilitate tumor growth. We demonstrate that BiP signals downstream of CEMIP, modulating cellular resistance to hypoxia. Reducing BiP in CEMIP-expressing cells sensitized cells to hypoxia treatment, decreased glucose uptake, and resulted in tumor regression in vivo. Our study provides insights into the link between CEMIP and BiP expression and the pro-survival role they play in hypoxia. Better understanding of the mechanisms behind cancer cell adaptations to harsh tumor environments could lead to development of improved cancer treatments.

Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling.

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, I F, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of I F are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

Tumor-intrinsic PIK3CA represses tumor immunogenecity in a model of pancreatic cancer.

The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapies. Here we show using a syngeneic orthotopic implantation model of pancreatic cancer that Pik3ca regulates tumor immunogenicity. Genetic silencing of Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors resulted in infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progressed and killed all of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminated Pik3ca-null tumors in immunodeficient mice. Loss of PIK3CA or inhibition of its effector, AKT, increased the expression of MHC Class I and CD80 on tumor cells. These changes contributed to the increased susceptibility of Pik3ca-null tumors to T cell surveillance. Our results indicate that tumor cell PIK3CA-AKT signaling limits T cell recognition and clearance of pancreatic cancer cells. Strategies that target this pathway may yield an effective immunotherapy for this cancer.

Phospholipase D2 loss results in increased blood pressure via inhibition of the endothelial nitric oxide synthase pathway.

The Phospholipase D (PLD) superfamily is linked to neurological disease, cancer, and fertility, and a recent report correlated a potential loss-of-function PLD2 polymorphism with hypotension. Surprisingly, PLD2 -/- mice exhibit elevated blood pressure accompanied by associated changes in cardiac performance and molecular markers, but do not have findings consistent with the metabolic syndrome. Instead, expression of endothelial nitric oxide synthase (eNOS), which generates the potent vasodilator nitric oxide (NO), is decreased. An eNOS inhibitor phenocopied PLD2 loss and had no further effect on PLD2 -/- mice, confirming the functional relationship. Using a human endothelial cell line, PLD2 loss of function was shown to lower intracellular free cholesterol, causing upregulation of HMG Co-A reductase, the rate-limiting enzyme in cholesterol synthesis. HMG Co-A reductase negatively regulates eNOS, and the PLD2-deficiency phenotype of decreased eNOS expression and activity could be rescued by cholesterol supplementation and HMG Co-A reductase inhibition. Together, these findings identify a novel pathway through which the lipid signaling enzyme PLD2 regulates blood pressure, creating implications for on-going therapeutic development of PLD small molecule inhibitors. Finally, we show that the human PLD2 polymorphism does not trigger eNOS loss, but rather creates another effect, suggesting altered functioning for the allele.

The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth.

PURPOSE: Signal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110ß catalytic subunits of PI3K were conditionally deleted in the mouse lens. METHODS: Floxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice. Lenses of age-matched animals were dissected and photographed. Postnatal lenses were fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin. Cell proliferation was quantified by labeling S-phase cells in intact lenses with 5-ethynyl-2'-deoxyuridine. Protein kinase B (AKT) activation was examined by Western blotting. RESULTS: Lens-specific deletion of p110α resulted in a significant reduction of eye and lens size, without compromising lens clarity. Conditional knockout of p110ß had no effect on lens size or clarity, and deletion of both the p110α and p110ß subunits resulted in a phenotype that resembled the p110α single-knockout phenotype. Levels of activated AKT were decreased more in p110α- than in p110ß-deficient lenses. A significant reduction in proliferating cells in the germinative zone was observed on postnatal day 0 in p110α knockout mice, which was temporally correlated with decreased lens volume. CONCLUSIONS: These data suggest that the class 1A PI3K signaling pathway plays an important role in the regulation of lens size by influencing the extent and spatial location of cell proliferation in the perinatal period.

Control of cardiac repolarization by phosphoinositide 3-kinase signaling to ion channels.

Upregulation of phosphoinositide 3-kinase (PI3K) signaling is a common alteration in human cancer, and numerous drugs that target this pathway have been developed for cancer treatment. However, recent studies have implicated inhibition of the PI3K signaling pathway as the cause of a drug-induced long-QT syndrome in which alterations in several ion currents contribute to arrhythmogenic drug activity. Surprisingly, some drugs that were thought to induce long-QT syndrome by direct block of the rapid delayed rectifier (IKr) also seem to inhibit PI3K signaling, an effect that may contribute to their arrhythmogenicity. The importance of PI3K in regulating cardiac repolarization is underscored by evidence that QT interval prolongation in diabetes mellitus also may result from changes in multiple currents because of decreased insulin activation of PI3K in the heart. How PI3K signaling regulates ion channels to control the cardiac action potential is poorly understood. Hence, this review summarizes what is known about the effect of PI3K and its downstream effectors, including Akt, on sodium, potassium, and calcium currents in cardiac myocytes. We also refer to some studies in noncardiac cells that provide insight into potential mechanisms of ion channel regulation by this signaling pathway in the heart. Drug development and safety could be improved with a better understanding of the mechanisms by which PI3K regulates cardiac ion channels and the extent to which PI3K inhibition contributes to arrhythmogenic susceptibility.

PI3K regulation of RAC1 is required for KRAS-induced pancreatic tumorigenesis in mice.

BACKGROUND & AIMS: New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS: Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110ß (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS: Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS: KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Kisspeptin cell-specific PI3K signaling regulates hypothalamic kisspeptin expression and participates in the regulation of female fertility.

Hypothalamic kisspeptin neurons integrate and translate cues from the internal and external environments that regulate gonadotropin-releasing hormone (GnRH) secretion and maintain fertility in mammals. However, the intracellular signaling pathways utilized to translate such information into changes in kisspeptin expression, release, and ultimately activation of the kisspeptin-receptive GnRH network have not yet been identified. PI3K is an important signaling node common to many peripheral factors known to regulate kisspeptin expression and GnRH release. We investigated whether PI3K signaling regulates hypothalamic kisspeptin expression, pubertal development, and adult fertility in mice. We generated mice with a kisspeptin cell-specific deletion of the PI3K catalytic subunits p110α and p110ß (kiss-p110α/ß-KO). Using in situ hybridization, we examined Kiss1 mRNA expression in gonad-intact, gonadectomized (Gdx), and Gdx + steroid-replaced mice. Kiss1 cell number in the anteroventral periventricular hypothalamus (AVPV) was significantly reduced in intact females but not in males. In contrast, compared with WT and regardless of steroid hormone status, Kiss1 cell number was lower in the arcuate (ARC) of kiss-p110α/ß-KO males, but it was unaffected in females. Both intact Kiss-p110α/ß-KO males and females had reduced ARC kisspeptin-immunoreactive (IR) fibers compared with WT animals. Adult kiss-p110α/ß-KO males had significantly lower circulating luteinizing hormone (LH) levels, whereas pubertal development and fertility were unaffected in males. Kiss-p110α/ß-KO females exhibited a reduction in fertility despite normal pubertal development, LH levels, and estrous cyclicity. Our data show that PI3K signaling is important for the regulation of hypothalamic kisspeptin expression and contributes to normal fertility in females.

AKT activation promotes PTEN hamartoma tumor syndrome-associated cataract development.

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.

Suppression of phosphoinositide 3-kinase signaling and alteration of multiple ion currents in drug-induced long QT syndrome.

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K(+)) current I(Kr). Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K(+) currents I(Kr) and I(Ks), the L-type calcium ion (Ca(2+)) current I(Ca,L), and the peak sodium ion (Na(+)) current I(Na) and increased the persistent Na(+) current I(NaP). Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in I(NaP). These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function.

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

VIP and endothelin receptor antagonist: an effective combination against experimental pulmonary arterial hypertension.

BACKGROUND: Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy? METHODS: Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival. RESULTS: Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days. CONCLUSIONS: 1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.