TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy.

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt.

Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase (ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1-stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli.

Role of miR-181c in Diet-induced obesity through regulation of lipid synthesis in liver.

We recently identified a nuclear-encoded miRNA (miR-181c) in cardiomyocytes that can translocate into mitochondria to regulate mitochondrial gene mt-COX1 and influence obesity-induced cardiac dysfunction through the mitochondrial pathway. Because liver plays a pivotal role during obesity, we hypothesized that miR-181c might contribute to the pathophysiological complications associated with obesity. Therefore, we used miR-181c/d-/- mice to study the role of miR-181c in hepatocyte lipogenesis during diet-induced obesity. The mice were fed a high-fat (HF) diet for 26 weeks, during which indirect calorimetric measurements were made. Quantitative PCR (qPCR) was used to examine the expression of genes involved in lipid synthesis. We found that miR-181c/d-/- mice were not protected against all metabolic consequences of HF exposure. After 26 weeks, the miR-181c/d-/- mice had a significantly higher body fat percentage than did wild-type (WT) mice. Glucose tolerance tests showed hyperinsulinemia and hyperglycemia, indicative of insulin insensitivity in the miR-181c/d-/- mice. miR-181c/d-/- mice fed the HF diet had higher serum and liver triglyceride levels than did WT mice fed the same diet. qPCR data showed that several genes regulated by isocitrate dehydrogenase 1 (IDH1) were more upregulated in miR-181c/d-/- liver than in WT liver. Furthermore, miR-181c delivered in vivo via adeno-associated virus attenuated the lipogenesis by downregulating these same lipid synthesis genes in the liver. In hepatocytes, miR-181c regulates lipid biosynthesis by targeting IDH1. Taken together, the data indicate that overexpression of miR-181c can be beneficial for various lipid metabolism disorders.

Inhibition of phosphodiesterase type 9 reduces obesity and cardiometabolic syndrome in mice.

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP-selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I-induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism-regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.

MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury.

RATIONALE: The mTORC1 (mechanistic target of rapamycin complex-1) controls metabolism and protein homeostasis and is activated following ischemia reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little reported. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied before ischemic stress. This can be circumvented by regulating one serine (S1365) on TSC2 (tuberous sclerosis complex) to achieve bidirectional mTORC1 modulation but only with TCS2-regulated costimulation. OBJECTIVE: We tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and is required for IPC by amplifying mTORC1 activity to favor glycolytic metabolism. METHODS AND RESULTS: Mice with either S1365A (TSC2SA; phospho-null) or S1365E (TSC2SE; phosphomimetic) knockin mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2SA mice had amplified mTORC1 activation and improved heart function compared with wild-type and TSC2SE hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2SE hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2SA, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acylcarnitine levels declined during ischemia. The relative IR protection in TSC2SA was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in wild type and TSC2SE but not TSC2SA which had the worst post-IR function under these conditions. CONCLUSIONS: TSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC. Graphic Abstract: A graphic abstract is available for this article.

Phosphorylation Modifications Regulating Cardiac Protein Quality Control Mechanisms.

Many forms of cardiac disease, including heart failure, present with inadequate protein quality control (PQC). Pathological conditions often involve impaired removal of terminally misfolded proteins. This results in the formation of large protein aggregates, which further reduce cellular viability and cardiac function. Cardiomyocytes have an intricately collaborative PQC system to minimize cellular proteotoxicity. Increased expression of chaperones or enhanced clearance of misfolded proteins either by the proteasome or lysosome has been demonstrated to attenuate disease pathogenesis, whereas reduced PQC exacerbates pathogenesis. Recent studies have revealed that phosphorylation of key proteins has a potent regulatory role, both promoting and hindering the PQC machinery. This review highlights the recent advances in phosphorylations regulating PQC, the impact in cardiac pathology, and the therapeutic opportunities presented by harnessing these modifications.

CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

Targeting Protein Kinase G to Treat Cardiac Proteotoxicity.

Impaired or insufficient protein kinase G (PKG) signaling and protein quality control (PQC) are hallmarks of most forms of cardiac disease, including heart failure. Their dysregulation has been shown to contribute to and exacerbate cardiac hypertrophy and remodeling, reduced cell survival and disease pathogenesis. Enhancement of PKG signaling and PQC are associated with improved cardiac function and survival in many pre-clinical models of heart disease. While many clinically used pharmacological approaches exist to stimulate PKG, there are no FDA-approved therapies to safely enhance cardiomyocyte PQC. The latter is predominantly due to our lack of knowledge and identification of proteins regulating cardiomyocyte PQC. Recently, multiple studies have demonstrated that PKG regulates PQC in the heart, both during physiological and pathological states. These studies tested already FDA-approved pharmacological therapies to activate PKG, which enhanced cardiomyocyte PQC and alleviated cardiac disease. This review examines the roles of PKG and PQC during disease pathogenesis and summarizes the experimental and clinical data supporting the utility of stimulating PKG to target cardiac proteotoxicity.

PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Myocardial Phosphodiesterases and Their Role in cGMP Regulation.

Cyclic nucleotide phosphodiesterases comprise an 11-member superfamily yielding near 100 isoform variants that hydrolyze cAMP or cGMP to their respective 5'-monophosphate form. Each plays a role in compartmentalized cyclic nucleotide signaling, with varying selectivity for each substrate, and conveying cell and intracellular-specific localized control. This review focuses on the 5 phosphodiesterases (PDEs) expressed in the cardiac myocyte capable of hydrolyzing cGMP and that have been shown to play a role in cardiac physiological and pathological processes. PDE1, PDE2, and PDE3 catabolize cAMP as well, whereas PDE5 and PDE9 are cGMP selective. PDE3 and PDE5 are already in clinical use, the former for heart failure, and PDE1, PDE9, and PDE5 are all being actively studied for this indication in patients. Research in just the past few years has revealed many novel cardiac influences of each isoform, expanding the therapeutic potential from their selective pharmacological blockade or in some instances, activation. PDE1C inhibition was found to confer cell survival protection and enhance cardiac contractility, whereas PDE2 inhibition or activation induces beneficial effects in hypertrophied or failing hearts, respectively. PDE3 inhibition is already clinically used to treat acute decompensated heart failure, although toxicity has precluded its long-term use. However, newer approaches including isoform-specific allosteric modulation may change this. Finally, inhibition of PDE5A and PDE9A counter pathological remodeling of the heart and are both being pursued in clinical trials. Here, we discuss recent research advances in each of these PDEs, their impact on the myocardium, and cardiac therapeutic potential.

PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress.

The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3ß) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.

Divergent Effects of miR-181 Family Members on Myocardial Function Through Protective Cytosolic and Detrimental Mitochondrial microRNA Targets.

BACKGROUND: MicroRNA (miRNA) is a type of noncoding RNA that can repress the expression of target genes through posttranscriptional regulation. In addition to numerous physiologic roles for miRNAs, they play an important role in pathophysiologic processes affecting cardiovascular health. Previously, we reported that nuclear encoded microRNA (miR-181c) is present in heart mitochondria, and importantly, its overexpression affects mitochondrial function by regulating mitochondrial gene expression. METHODS AND RESULTS: To investigate further how the miR-181 family affects the heart, we suppressed miR-181 using a miR-181-sponge containing 10 repeated complementary miR-181 "seed" sequences and generated a set of H9c2 cells, a cell line derived from rat myoblast, by stably expressing either a scrambled or miR-181-sponge sequence. Sponge-H9c2 cells showed a decrease in reactive oxygen species production and reduced basal mitochondrial respiration and protection against doxorubicin-induced oxidative stress. We also found that miR-181a/b targets phosphatase and tensin homolog (PTEN), and the sponge-expressing stable cells had increased PTEN activity and decreased PI3K signaling. In addition, we have used miR-181a/b-/- and miR-181c/d-/- knockout mice and subjected them to ischemia-reperfusion injury. Our results suggest divergent effects of different miR-181 family members: miR-181a/b targets PTEN in the cytosol, resulting in an increase in infarct size in miR-181a/b-/- mice due to increased PTEN signaling, whereas miR-181c targets mt-COX1 in the mitochondria, resulting in decreased infarct size in miR-181c/d-/- mice. CONCLUSIONS: The miR-181 family alters the myocardial response to oxidative stress, notably with detrimental effects by targeting mt-COX1 (miR-181c) or with protection by targeting PTEN (miR-181a/b).

miR-181b regulates vascular stiffness age dependently in part by regulating TGF-ß signaling.

BACKGROUND: Endothelial dysfunction and arterial stiffening play major roles in cardiovascular diseases. The critical role for the miR-181 family in vascular inflammation has been documented. Here we tested whether the miR-181 family can influence the pathogenesis of hypertension and vascular stiffening. METHODS AND RESULTS: qPCR data showed a significant decrease in miR-181b expression in the aorta of the older mice. Eight miR-181a1/b1-/- mice and wild types (C57BL6J:WT) were followed weekly for pulse wave velocity (PWV) and blood pressure measurements. After 20 weeks, the mice were tested for endothelial function and aortic modulus. There was a progressive increase in PWV and higher systolic blood pressure in miR-181a1/b1-/- mice compared with WTs. At 21 weeks, aortic modulus was significantly greater in the miR-181a1/b1-/- group, and serum TGF-ß was found to be elevated at this time. A luciferase reporter assay confirmed miR-181b targets TGF-ßi (TGF-ß induced) in the aortic VSMCs. In contrast, wire myography revealed unaltered endothelial function along with higher nitric oxide production in the miR-181a1/b1-/- group. Cultured VECs and VSMCs from the mouse aorta showed more secreted TGF-ß in VSMCs of the miR-181a1/b1-/- group; whereas, no change was observed from VECs. Circulating levels of angiotensin II were similar in both groups. Treatment with losartan (0.6 g/L) prevented the increase in PWV, blood pressure, and vascular stiffness in miR-181a1/b1-/- mice. Immunohistochemistry and western blot for p-SMAD2/3 validated the inhibitory effect of losartan on TGF-ß signaling in miR-181a1/b1-/- mice. CONCLUSIONS: Decreased miR-181b with aging plays a critical role in ECM remodeling by removing the brake on the TGF-ß, pSMAD2/3 pathway.

Exosomal MicroRNA-15a Transfer from the Pancreas Augments Diabetic Complications by Inducing Oxidative Stress.

AIMS: MicroRNAs (miRNAs), one type of noncoding RNA, modulate post-transcriptional gene expression in various pathogenic pathways in type 2 diabetes (T2D). Currently, little is known about how miRNAs influence disease pathogenesis by targeting cells at a distance. The purpose of this study was to investigate the role of exosomal miRNAs during T2D. RESULTS: We show that miR-15a is increased in the plasma of diabetic patients, correlating with disease severity. miR-15 plays an important role in insulin production in pancreatic ß-cells. By culturing rat pancreatic ß-cells (INS-1) cells in high-glucose media, we identified a source of increased miR-15a in the blood as exosomes secreted by pancreatic ß-cells. We postulate that miR-15a, produced in pancreatic ß-cells, can enter the bloodstream and contribute to retinal injury. miR-15a overexpression in Müller cells can be induced by exposing Müller cells to exosomes derived from INS-1 cells under high-glucose conditions and results in oxidative stress by targeting Akt3, which leads to apoptotic cell death. The in vivo relevance of these findings is supported by results from high-fat diet and pancreatic ß-cell-specific miR-15a-/- mice. INNOVATION: This study highlights an important and underappreciated mechanism of remote cell-cell communication (exosomal transfer of miRNA) and its influence on the development of T2D complications. CONCLUSION: Our findings suggest that circulating miR-15a contributes to the pathogenesis of diabetes and supports the concept that miRNAs released by one cell type can travel through the circulation and play a role in disease progression via their transfer to different cell types, inducing oxidative stress and cell injury. Antioxid. Redox Signal. 27, 913-930.