Mutations of the tumor suppressor genes tuberous sclerosis complex (TSC)1 and TSC2 cause pulmonary lymphangioleiomyomatosis (LAM) and tuberous sclerosis (TS). Current rapamycin-based therapies for TS and LAM have a predominantly cytostatic effect, and disease progression resumes with therapy cessation. Evidence of RhoA GTPase activation in LAM-derived and human TSC2-null cells suggests that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor statins can be used as potential adjuvant agents. The goal of this study was to determine which statin (simvastatin or atorvastatin) is more effective in suppressing TSC2-null cell growth and signaling. Simvastatin, but not atorvastatin, showed a concentration-dependent (0.5–10 μM) inhibitory effect on mouse TSC2-null and human LAM–derived cell growth. Treatment with 10 μM simvastatin induced dramatic disruption of TSC2-null cell monolayer and cell rounding; in contrast, few changes were observed in cells treated with the same concentration of atorvastatin. Combined treatment of rapamycin with simvastatin but not with atorvastatin showed a synergistic growth-inhibitory effect on TSC2-null cells. Simvastatin, but not atorvastatin, inhibited the activity of prosurvival serine-threonine kinase Akt and induced marked up-regulation of cleaved caspase-3, a marker of cell apoptosis. Simvastatin, but not atorvastatin, also induced concentration-dependent inhibition of p42/p44 Erk and mTORC1. Thus, our data show growth-inhibitory and proapoptotic effects of simvastatin on TSC2-null cells compared with atorvastatin. These findings have translational significance for combinatorial therapeutic strategies of simvastatin to inhibit TSC2-null cell survival in TS and LAM.
TSC; LAM; apoptosis; TSC2; mTOR
Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome caused by mutations of the tumor suppressor gene folliculin (FLCN) by unknown mechanism. BHD lungs exhibit increased alveolar epithelial cell apoptosis. We show that Flcn deletion in lung epithelium leads to cell apoptosis, alveolar enlargement and impaired lung function. FLCN loss also impairs alveolar epithelial barrier function. 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. Flcn-null cell survival is rescued by AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data show that Flcn regulates lung epithelial cell survival and alveolar size and 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.
Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease found almost exclusively in women that is characterized by neoplastic growth of atypical smooth muscle-like cells in the lung, destruction of lung parenchyma, and obstruction of lymphatics. These processes lead to the formation of lung cysts, rupture of which results in spontaneous pneumothorax. Progression of LAM often results in loss of pulmonary function and death. LAM affects predominantly women of childbearing age and is exacerbated by pregnancy. The only proven treatment for LAM is lung transplantation, and even then LAM cells will often return to the transplanted lung. However, methodical and targeted approaches toward understanding LAM pathophysiology have led to the discovery of new potential therapeutic avenues. For example, the mutational inactivation of tumor suppressor complex genes tuberous sclerosis complex 1 (TSC1) or TSC2 has been shown to be present in lung LAM cells. These mutations occur sporadically or in association with inherited hamartoma syndrome tuberous sclerosis (TSC). Since TSC genes function as negative regulators of the mammalian target of rapamycin (mTOR), a major controller of cell growth, metabolism and survival, rapamycin analogs have recently been used to treat LAM patients with promising results. Similarly, studies focusing on the importance of estrogen in LAM progression have suggested that anti-estrogen therapy might prove to be an alternative means of treating LAM. This mini-review summarizes recent progress in understanding LAM pathophysiology, including the latest preclinical and clinical studies, and insights regarding the role of hormones in LAM.
interstitial lung disease; cancer; rare disease; TSC
Dysregulated activity of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) is characteristic feature of hamartoma syndromes. Hamartoma syndromes, dominantly inherited cancer predisposition disorders, affect multiple organs and are manifested by benign tumors consisting of various cell types native to the tissues in which they arise. In the past few years, three inherited hamartoma syndromes, Cowden syndrome (CS), tuberous sclerosis complex (TSC) syndrome, and Peutz-Jeghens syndrome (PJS), have all been linked to a common biochemical pathway: the hyperactivation of PI3K/mTORC1 intracellular signaling. Three tumor suppressors, PTEN (phosphatases and tensin homolog), tuberous sclerosis complex TSC1/TSC2, and LKB1, are negative regulators of PI3K/mTORC1 signaling; disease-related inactivation of these tumor suppressors results in the development of PTEN-associated hamartoma syndromes, TSC and PJS, respectively. The goal of this review is to provide a roadmap for navigating the inherently complex regulation of PI3K/mTORC1 signaling while highlighting the progress that has been made in elucidating the cellular and molecular mechanisms of hamartoma syndromes and identificating potential therapeutic targets for their treatment. Importantly, because the PI3K/mTORC1 pathway is activated in the majority of common human cancers, the identification of novel molecular target(s) for the treatment of hamartoma syndromes may have a broader translational potential, and is critically important not only for therapeutic intervention in hamartoma disorders, but also for the treatment of cancers.
PI3K; mTORC1; PTEN; cancer; lung; kidney; TSC; LAM; biomarker; therapy
TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1−/− MEFs have decreased migration compared to littermate-derived Tsc1+/+ MEFs. Migration of Tsc1−/− MEFs with re-expressed TSC1 was comparable to Tsc1+/+ MEF migration. In contrast, Tsc2−/− MEFs showed an increased migration compared to Tsc2+/+ MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1−/− MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2−/− MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1−/− or Tsc2−/− MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s) by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2−/− MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2-null cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes mTOR- and mTORC2-dependent pro-migratory cell phenotype.
Proliferation, migration, and differentiation of smooth muscle (SM)–like lymphangioleiomyomatosis (LAM) cells in the lungs are pathologic manifestations of pulmonary LAM, a rare lung disease predominantly afflicting women and exacerbated by pregnancy. LAM cells form nodules throughout the lung without any predominant localization, but can also form small cell clusters dispersed within lung parenchyma. LAM cells have the appearance of “immature” SM-like cells, irregularly distributed within the nodule in contrast to organized SM cell layers in airways and vasculature. Progressive growth of LAM cells leads to the cystic destruction of the lung parenchyma, obstruction of airways and lymphatics, and loss of pulmonary function. Pathogenetically, LAM occurs from somatic or genetic mutations of tumor suppressor genes tuberous sclerosis complex 1 (TSC1) or TSC2. The TSC1/TSC2 protein complex is an integrator of signaling networks regulated by growth factors, insulin, nutrients, and energy. The observation that the TSC1/TSC2 functions as a negative regulator of the mammalian target of rapamycin (mTOR)/p70 S6 kinase (S6K1) signaling pathway yielded the first rapamycin clinical trial for LAM. Although LAM is a rare lung disease, the elucidation of disease-relevant mechanisms of LAM will provide a better understanding of not only SM-like cell growth, migration, and differentiation in LAM but may also offer insights into other metabolic diseases such as cardiovascular diseases, diabetes, and cancer. In this article, we will summarize the progress made in our understanding of LAM, and we will focus on how dysregulation of TSC1/TSC2 signaling results in abnormal proliferation and migration of SM-like LAM cells.
lung cancer; TSC1; TSC2; signal transduction; mesenchymal cells
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.
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.
Airways diseases such as asthma, emphysema, and chronic bronchitis are, in part, characterized by reversible airflow obstruction and inflammation. In severe disease, marked decreases in lung function are associated with airway smooth muscle proliferation and airway neutrophilia. Inhaled glucocorticoids attenuate increased airflow obstruction and airway inflammation that occur, in part, due to increased smooth muscle migration and proliferation, as well as the airway neutrophilia. Glucocorticoids, however, have adverse side effects and, in some patients, are ineffective despite high doses. Recent research has explored the effects of non-traditional steroids on attenuation of inflammation associated with airways diseases. These non-traditional steroids have improved side effect profiles in comparison to glucocorticoid therapy. Our studies assessed effects of dehydroepiandrosterone-3-sulfate (DHEA-S) on migration of both human peripheral blood neutrophils (PMN) and human airway smooth muscle cells (HASM). DHEA-S dose-dependently inhibited chemotaxis of PMN and HASM while having no effect on the phosphorylation levels of Akt, ERK1/2, p38 MAPK or PKC, canonical positive regulators of cell migration. These studies demonstrate direct effects of DHEA-S on cell migration, thereby suggesting that DHEA-S may attenuate airway inflammation and cell migration.
airway inflammation; DHEA-S; airway remodeling
Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle–like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor–D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.
Severe asthma manifests as airway remodeling and irreversible airway obstruction, in part because of the proliferation and migration of human airway smooth muscle (HASM) cells. We previously reported that cyclic adenosine monophosphate–mobilizing agents, including β2-adrenergic receptor (β2AR) agonists, which are mainstay of asthma therapy, and prostaglandin E2 (PGE2), inhibit the migration of HASM cells, although the mechanism for this migration remains unknown. Vasodilator-stimulated phosphoprotein (VASP), an anticapping protein, modulates the formation of actin stress fibers during cell motility, and is negatively regulated by protein kinase A (PKA)–specific inhibitory phosphorylation at serine 157 (Ser157). Here, we show that treatment with β2AR agonists and PGE2 induces the PKA-dependent phosphorylation of VASP and inhibits the migration of HASM cells. The stable expression of PKA inhibitory peptide and the small interfering (si) RNA-induced depletion of VASP abolish the inhibitory effects of albuterol and PGE2 on the migration of HASM cells. Importantly, prolonged treatment with albuterol prevents the agonist-induced phosphorylation of VASP at Ser157, and reverses the inhibitory effects of albuterol and formoterol, but not PGE2, on the basal and PDGF-induced migration of HASM cells. Collectively, our data demonstrate that β2AR agonists selectively inhibit the migration of HASM cells via a β2AR/PKA/VASP signaling pathway, and that prolonged treatment with albuterol abolishes the inhibitory effect of β-agonists on the phosphorylation of VASP and migration of HASM cells because of β2AR desensitization.
airway hyperresponsiveness; β2-adrenergic receptor desensitization; protein kinase A; albuterol; formoterol
Increased pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysiological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PH). The long-acting β2-adrenergic receptor (β2AR) agonist formoterol, a racemate comprised of (R,R)- and (S,S)-enantiomers, is commonly used as a vasodilator in chronic obstructive pulmonary disease (COPD). PH, a common complication of COPD, increases patients’ morbidity and reduces survival. Recent studies demonstrate that formoterol has anti-proliferative effects on airway smooth muscle cells and bronchial fibroblasts. The effects of formoterol and its enantiomers on PAVSM cell proliferation are not determined. The goals of this study were to examine effects of racemic formoterol and its enantiomers on PAVSM cell proliferation as it relates to COPD-associated PH.
Basal, thrombin-, PDGF- and chronic hypoxia-induced proliferation of primary human PAVSM cells was examined by DNA synthesis analysis using BrdU incorporation assay. ERK1/2, mTORC1 and mTORC2 activation were determined by phosphorylation levels of ERK1/2, ribosomal protein S6 and S473-Akt using immunoblot analysis.
We found that (R,R) and racemic formoterol inhibited basal, thrombin- and chronic hypoxia-induced proliferation of human PAVSM cells while (S,S) formoterol had lesser inhibitory effect. The β2AR blocker propranolol abrogated the growth inhibitory effect of formoterol. (R,R), but not (S,S) formoterol attenuated basal, thrombin- and chronic hypoxia-induced ERK1/2 phosphorylation, but had little effect on Akt and S6 phosphorylation levels. Formoterol and its enantiomers did not significantly affect PDGF-induced DNA synthesis and PDGF-dependent ERK1/2, S473-Akt and S6 phosphorylation in human PAVSM cells.
Formoterol inhibits basal, thrombin-, and chronic hypoxia-, but not PDGF-induced human PAVSM cell proliferation and ERK1/2, but has little effect on mTORC1 and mTORC2 signaling. Anti-proliferative effects of formoterol depend predominantly on its (R,R) enantiomer and require the binding with β2AR. These data suggest that (R,R) formoterol may be considered as potential adjuvant therapy to inhibit PAVSM cell proliferation in COPD-associated PH.
(R; R) formoterol; ERK1/2; Pulmonary hypertension
Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
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.
The TSC1 and TSC2 proteins, which function as a TSC1/TSC2 tumor suppressor complex, are associated with lymphangioleiomyomatosis (LAM), a genetic disorder characterized by the abnormal growth of smooth muscle–like cells in the lungs. The precise molecular mechanisms that modulate LAM cell growth remain unknown. We demonstrate that TSC2 regulates LAM cell growth. Cells dissociated from LAM nodules from the lungs of five different patients with LAM have constitutively activated S6K1, hyperphosphorylated ribosomal protein S6, activated Erk, and increased DNA synthesis compared with normal cells from the same patients. These effects were augmented by PDGF stimulation. Akt activity was unchanged in LAM cells. Rapamycin, a specific S6K1 inhibitor, abolished increased LAM cell growth. The full-length TSC2 was necessary for inhibition of S6 hyperphosphorylation and DNA synthesis in LAM cells, as demonstrated by co-microinjection of the C-terminus, which contains the GTPase activating protein homology domain, and the N-terminus, which binds TSC1. Our data demonstrate that increased LAM cell growth is associated with constitutive S6K1 activation, which is extinguishable by TSC2 expression. Loss of TSC2 GAP activity or disruption of the TSC1/TSC2 complex dysregulates S6K1 activation, which leads to abnormal cell proliferation associated with LAM disease.
interstitial lung disease; smooth muscle; TSC
The loss of TSC2 function is associated with the pathobiology of lymphangioleiomyomatosis (LAM), which is characterized by the abnormal proliferation, migration, and differentiation of smooth muscle–like cells within the lungs. Although the etiology of LAM remains unknown, clinical and genetic evidence provides support for the neoplastic nature of LAM. The goal of this study was to determine the role of tumor suppressor TSC2 in the neoplastic potential of LAM cells. We show that primary cultures of human LAM cells exhibit increased migratory activity and invasiveness, which is abolished by TSC2 re-expression. We found that TSC2 also inhibits cell migration through its N-terminus, independent of its GTPase-activating protein activity. LAM cells show increased stress fiber and focal adhesion formation, which is attenuated by TSC2 re-expression. The small GTPase RhoA is activated in LAM cells compared with normal human mesenchymal cells. Pharmacologic inhibition of Rho activity abrogates LAM cell migration; RhoA activity was also abolished by TSC2 re-expression or TSC1 knockdown with specific siRNA. These data demonstrate that TSC2 controls cell migration through its N-terminus by associating with TSC1 and regulating RhoA activity, suggesting that TSC2 may play a critical role in modulating cell migration and invasiveness, which contributes to the pathobiology of LAM.
lung; RhoA GTPase; smooth muscle cells; TSC1