The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho

Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.     

Tumor Suppressors TSC1 and TSC2 Differentially Modulate Actin Cytoskeleton and Motility of Mouse Embryonic Fibroblasts

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.     

Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction, resulting from proliferation of smooth-muscle-like cells, which have mutations in the tumor suppressor genes TSC1 or TSC2. Among 277 LAM patients, severe disease was associated with hypoxia and elevated red blood cell indexes that accompanied reduced pulmonary function. Because high red cell indexes could result from hypoxemia-induced erythropoietin (EPO) production, and EPO is a smooth muscle cell mitogen, we investigated effects of EPO in human cells with genetic loss of tuberin function, and we found that EPO increased proliferation of human TSC2-/-, but not of TSC2+/-, cells. A discrete population of cells grown from explanted lungs was characterized by the presence of EPO receptor and loss of heterozygosity for TSC2, consistent with EPO involvement. In LAM cells from lung nodules, EPO was localized to the extracellular matrix, supporting evidence for activation of an EPO-driven signaling pathway. Although the high red cell mass of LAM patients could be related to advanced disease, we propose that EPO, synthesized in response to episodic hypoxia, may increase disease progression by enhancing the proliferation of LAM cells.     

TSC2 epigenetic defect in primary LAM cells. Evidence of an anchorage‐independent survival

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α‐smooth muscle (ASM)‐like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)‐dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non‐adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem‐cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)‐6 and IL‐8. Anti‐EGF receptor antibodies and rapamycin affect proliferation and viability of non‐adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.     

TSC1 Controls Distribution of Actin Fibers through Its Effect on Function of Rho Family of Small GTPases and Regulates Cell Migration and Polarity

The tumor-suppressor genes TSC1 and TSC2 are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of TSC1 and TSC2 form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a Tsc1-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.     

Autophagy: an 'Achilles' heel of tumorigenesis in TSC and LAM

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.     

The TSC1 and TSC2 tumor suppressors are required for proper ER stress response and protect cells from ER stress-induced apoptosis

Tuberous sclerosis complex (TSC)1 and TSC2 are tumor suppressors that inhibit cell growth and mutation of either gene causes benign tumors in multiple tissues. The TSC1 and TSC2 gene products form a functional complex that has GTPase-activating protein (GAP) activity toward Ras homolog enriched in brain (Rheb) to inhibit mammalian target of rapamycin complex 1 (mTORC1), which is constitutively activated in TSC mutant tumors. We found that cells with mutation in either TSC1 or TSC2 are hypersensitive to endoplasmic reticulum (ER) stress and undergo apoptosis. Although the TSC mutant cells show elevated eIF2α phosphorylation, an early ER stress response marker, at both basal and induced conditions, induction of other ER stress response markers, including ATF4, ATF6 and C/EBP homologous protein (CHOP), is severely compromised. The defects in ER stress response are restored by raptor knockdown but not by rapamycin treatment in the TSC mutant cells, indicating that a rapamycin-insensitive mTORC function is responsible for the defects in ER stress response. Consistently, activation of Rheb sensitizes cells to ER stress. Our data show an important role of TSC1/TSC2 and Rheb in unfolded protein response and cell survival. We speculate that an important physiological function of the TSC1/2 tumor suppressors is to protect cells from harmful conditions. These observations indicate a potential therapeutic application of using ER stress agents to selectively kill TSC1 or TSC2 mutant cells for TSC treatment.     

Resveratrol prevents rapamycin-induced upregulation of autophagy and selectively induces apoptosis in TSC2-deficient cells

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is hyperactivated in a variety of cancers and disorders, including lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC), which are characterized by mutations in tumor suppressors TSC1 or TSC2. The concern with the use of mTORC1 inhibitors, such as rapamycin or its analogs (rapalogs), is that they cause upregulation of autophagy and suppress the negative feedback loop to Akt, which promotes cell survival, causing the therapy to be only partially effective, and relapse occurs upon cessation of treatment. In this study, we investigate the use of rapamycin in combination with resveratrol, a naturally occurring polyphenol, in TSC2-deficient cells. We tested whether such combination would prevent rapamycin-induced upregulation of autophagy and shift the cell fate toward apoptosis. We found that this combination treatment blocked rapamycin-induced upregulation of autophagy and restored inhibition of Akt. Interestingly, the combination of rapamycin and resveratrol selectively promoted apoptosis of TSC2-deficient cells. Thus, the addition of resveratrol to rapamycin treatment may be a promising option for selective and targeted therapy for diseases with TSC loss and mTORC1 hyperactivation.     

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2^Y1571H cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2^R611Q, which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.     

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

Activated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1-oestrogen receptor fusion protein (c-Raf-1-BxB-ER, N-BxB-ER cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ER protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ER-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21(Cip1). These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control.     

The Small GTPases Rho and Rac Are Required for the Establishment of Cadherin-dependent Cell–Cell Contacts

Cadherins are calcium-dependent cell–cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell–cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell–cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell–cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell– cell contact formation and the remodelling of actin filaments in epithelial cells.     

Rapid suppression of activated Rac1 by cadherins and nectins during de novo cell-cell adhesion

Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ～66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions.     

Pulmonary lymphangioleiomyomatosis (LAM): progress and current challenges

Lymphangioleiomyomatosis (LAM), a rare lung disease, is characterized by the progressive proliferation, migration, and differentiation of smooth muscle (SM)-like LAM cells, which lead to the cystic destruction of the lung parenchyma, obstruction of airways and lymphatics, and loss of pulmonary function. LAM is a disease predominantly affecting women and is exacerbated by pregnancy; only a lung transplant can save the life of a patient. It has been discovered that in LAM, somatic or genetic mutations of tumor suppressor genes tuberous sclerosis complex 1 (TSC1) or TSC2 occur and the TSC1/TSC2 protein complex functions as a negative regulator of the mTOR/S6K1 signaling pathway. These two pivotal observations paved the way for the first rapamycin clinical trial for LAM. The recent discoveries that TSC1/TSC2 complex functions as an integrator of signaling networks regulated by growth factors, insulin, nutrients, and energy heightened the interest regarding this rare disease because the elucidation of disease-relevant mechanisms of LAM will promote a better understanding of other metabolic diseases such as diabetes, cancer, and cardiovascular diseases. In this review, we will summarize the progress made in our understanding of TSC1/TSC2 cellular signaling and the molecular mechanisms of LAM; we will also highlight some of the lesser explored directions and challenges in LAM research.     

TSC1/TSC2 signaling in the CNS

Over the past several years, the study of a hereditary tumor syndrome, tuberous sclerosis complex (TSC), has shed light on the regulation of cellular proliferation and growth. TSC is an autosomal dominant disorder that is due to inactivating mutations in TSC1 or TSC2 and characterized by benign tumors (hamartomas) involving multiple organ systems. The TSC1/2 complex has been found to play a crucial role in an evolutionarily-conserved signaling pathway that regulates cell growth: the mTORC1 pathway. This pathway promotes anabolic processes and inhibits catabolic processes in response to extracellular and intracellular factors. Findings in cancer biology have reinforced the critical role for TSC1/2 in cell growth and proliferation. In contrast to cancer cells, in the CNS, the TSC1/2 complex not only regulates cell growth/proliferation, but also orchestrates an intricate and finely tuned system that has distinctive roles under different conditions, depending on cell type, stage of development, and subcellular localization. Overall, TSC1/2 signaling in the CNS, via its multi-faceted roles, contributes to proper neural connectivity. Here, we will review the TSC signaling in the CNS.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.     

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.     

Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM)

Although the cellular functions of TSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2-/- smooth muscle cells (ELT3) and TSC2-/- epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM.