Dmitry A. Goncharov

TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase

Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2−/− cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2−/− cells activated Rac1 and induced loss of stress fibers. Our data indicate that TSC1 inhibits Rac1 and TSC2 blocks this activity of TSC1. Because TSC1 and TSC2 regulate Rho and Rac1, whose activities are interconnected in a reciprocal fashion, loss of either TSC1 or TSC2 function may result in the deregulation of cell motility and adhesion, which are associated with the pathobiology of TSC and LAM.

Src is necessary and sufficient for human airway smooth muscle cell proliferation and migration.

Airway smooth muscle (ASM) hypertrophy and hyperplasia, important pathological features in chronic severe asthma, likely contribute to irreversible airflow obstruction. Despite considerable research effort, the precise cellular mechanisms that modulate ASM growth remain unknown. Src, a nonreceptor tyrosine kinase proto‐oncogene, reportedly modulates cell proliferative responses to growth factors, contractile agonists, and inflammatory mediators. Here, we show that Src activation is required for human ASM mitogenesis and motility. Platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), and thrombin induce rapid activation of Src, and inhibition of Src induces a concentration‐dependent abrogation of PDGF‐, EGF‐, and thrombin‐induced ASM cell proliferation. Src immunoprecipitates had associated phosphatidylinositol 3‐kinase, or PI3K, activation in response to PDGF and thrombin but not EGF. Further, Src activation is both necessary and sufficient for the stimulation of DNA synthesis as demonstrated by dominant negative Src inhibition of PDGF‐, EGF‐, and thrombin‐induced DNA synthesis. Human ASM cell migration was also attenuated by transfection of cells with dominant negative Src. Further, expression of constitutively active Src promoted cell migration. Collectively, these data demonstrate that Src modulates human ASM cell proliferation and migration, suggesting that Src may play an important role in promoting ASM cell growth and migration that occur in airway remodeling found in asthma and chronic obstructive pulmonary disease, or COPD.

mTORC2 is required for proliferation and survival of TSC2-null cells.

ABSTRACT Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

SIRT3–AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Background— Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension. Methods and Results— To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase. Conclusions— These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Mammalian Target of Rapamycin Complex 2 (mTORC2) Coordinates Pulmonary Artery Smooth Muscle Cell Metabolism, Proliferation, and Survival in Pulmonary Arterial Hypertension

Background— Enhanced proliferation, resistance to apoptosis, and metabolic shift to glycolysis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiological components of pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (PAH). The role of the distinct mammalian target of rapamycin (mTOR) complexes mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-Rictor) in PAVSMC proliferation and survival in PAH and their therapeutic relevance are unknown. Methods and Results— Immunohistochemical and immunoblot analyses revealed that mTORC1 and mTORC2 pathways are markedly upregulated in small remodeled pulmonary arteries and isolated distal PAVSMCs from subjects with idiopathic PAH that have increased ATP levels, proliferation, and survival that depend on glycolytic metabolism. Small interfering RNA– and pharmacology-based analysis showed that although both mTORC1 and mTORC2 contribute to proliferation, only mTORC2 is required for ATP generation and survival of idiopathic PAH PAVSMCs. mTORC2 downregulated the energy sensor AMP-activated protein kinase, which led to activation of mTORC1-S6 and increased proliferation, as well as a deficiency of the proapoptotic protein Bim and idiopathic PAH PAVSMC survival. NADPH oxidase 4 (Nox4) protein levels were increased in idiopathic PAH PAVSMCs, which was necessary for mTORC2 activation, proliferation, and survival. Nox4 levels and mTORC2 signaling were significantly upregulated in small pulmonary arteries from hypoxia-exposed rats at days 2 to 28 of hypoxia. Treatment with the mTOR kinase inhibitor PP242 at days 15 to 28 suppressed mTORC2 but not Nox4, induced smooth muscle–specific apoptosis in small pulmonary arteries, and reversed hypoxia-induced pulmonary vascular remodeling in rats. Conclusions— These data provide a novel mechanistic link of Nox4-dependent activation of mTORC2 via the energy sensor AMP-activated protein kinase to increased proliferation and survival of PAVSMCs in PAH, which suggests a new potential pathway for therapeutic interventions.

mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia

Pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysi‐ological component of vascular remodeling in pulmonary arterial hypertension (PAH) for which cellular and molecular mechanisms are poorly understood. The goal of our study was to determine the role of mammalian target of rapamycin (mTOR) in PAVSM cell proliferation, a major pathological manifestation of vascular remodeling in PAH. Our data demonstrate that chronic hypoxia promoted mTOR(Ser‐2481) phosphorylation, an indicator of mTOR intrinsic catalytic activity, mTORC1‐specific S6 and mTORC2‐specific Akt (Ser‐473) phosphorylation, and proliferation of human and rat PAVSM cells that was inhibited by siRNA mTOR PAVSM cells derived from rats exposed to chronic hypoxia (VSM‐H cells) retained increased mTOR(Ser‐2481), S6, Akt (Ser‐473) phosphorylation, and DNA synthesis compared to cells from nor‐moxia‐exposed rats. Suppression of mTORC2 signaling with siRNA rictor, or inhibition of mTORC1 signaling with rapamycin and metformin, while having little effect on other complex activities, inhibited VSM‐H and chronic hypoxia‐induced human and rat PAVSM cell proliferation. Collectively, our data demonstrate that up‐regulation of mTOR activity and activation of both mTORC1 and mTORC2 are required for PAVSM cell proliferation induced by in vitro and in vivo chronic hypoxia and suggest that mTOR may serve as a potential therapeutic target to inhibit vascular remodeling in PAH.—Krymskaya, V. P., Snow, J., Cesarone, G., Khavin, I., Goncharov, D. A., Lim, P. N., Veasey, S. C, Ihida‐Stansbury, K., Jones, P. L., Goncharova, E. A. mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J. 25, 1922‐1933 (2011). www.fasebj.org

Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder

Blocking mTORC1 rescues the neural progenitor cell migratory defect caused by depletion of the STRADA pseudokinase and reduces seizures in patients with a rare neurodevelopmental disorder. Preventing Seizures with Rapamycin The discovery of new treatments for rare neurodevelopmental disorders associated with epilepsy and intellectual disability is often limited by small patient sample sizes that delay initiation of clinical trials. Mutations in the gene STRADA cause brain malformations, seizures, and failure to develop social language in children, with no known successful treatment. In a new study, Parker and colleagues now show that the protein STRADA modulates the mammalian target of rapamycin (mTOR) signaling pathway and that loss of STRADA results in unchecked mTOR activity. Depletion of STRADA in mouse neural progenitor cells resulted in loss of polarity, impaired migration, and inability to form layers in the cerebral cortex. Impaired migration was also identified in fibroblasts from patients lacking STRADA, and all of these effects were prevented with the mTOR inhibitor rapamycin, an immunosuppressant drug in clinical use. The authors then treated five children with rapamycin (sirolimus) beginning at 3 to 8 months of age, and abatement of seizures was observed in all of the children. Early treatment with mTOR pathway inhibitors may be beneficial for children with this neurodevelopmental disorder or with other conditions associated with enhanced mTOR signaling such as tuberous sclerosis complex and fragile X syndrome. A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders.

Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact

Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein‐coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT‐PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle‐positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a‐induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c‐kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c‐kit failed to inhibit ASM cell‐mediated enhancement of mast cell degranulation. However, dexamethasone‐treated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a‐induced mast cell degranulation when compared with untreated cells. These findings suggest that cell‐cell interaction between ASM cells and mast cells, via a SCF‐c‐kit‐independent but dexamethasone‐sensitive mechanism, enhances C3a‐induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.

Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration

Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.

Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-cadherin, LKB1 and AMPK

Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome, which is caused by mutations of the tumor suppressor gene folliculin (FLCN). The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcn deletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcn(f/f):SP-C-Cre mice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.