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

Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

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

Normalization of Enzyme Expression and Activity Regulating Vitamin A Metabolism Increases RAR-Beta Expression and Reduces Cellular Migration and Proliferation in Diseases Caused by Tuberous Sclerosis Gene Mutations

BackgroundMutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARβ) expression. The goal of the present study was to investigate the molecular connection between vitamin A metabolism and TSC mutation. We also aimed to investigate the effect of the FDA approved drug rapamycin and the vitamin A metabolite retinoic acid (RA) in cell lines with TSC mutation.MethodsExpression and activity of vitamin A associated metabolic enzymes and RARβ were assessed in human kidney angiomyolipoma derived cell lines, primary lymphangioleiomyomatosis (LAM) tissue derived LAM cell lines. RARβ protein levels were also tested in primary LAM lung tissue sections. TaqMan arrays, enzyme activities, qRT-PCRs, immunohistochemistry, immunofluorescent staining, and western blotting were performed and analysed. The functional effects of retinoic acid (RA) and rapamycin were tested in a scratch and a BrDU assay to assess cell migration and proliferation.ResultsMetabolic enzyme arrays revealed a general deregulation of many enzymes involved in vitamin A metabolism including aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases (ADHs) and Cytochrome P450 2E1 (CYP2E1). Furthermore, RARβ downregulation was a characteristic feature of all TSC-deficient cell lines and primary tissues. Combination of the two FDA approved drugs -RA for acute myeloid leukaemia and rapamycin for TSC mutation- normalised ALDH and ADH expression and activity, restored RARβ expression and reduced cellular proliferation and migration.ConclusionDeregulation of vitamin A metabolizing enzymes is a feature of TSC mutation. RA can normalize RARβ levels and limit cell migration but does not have a significant effect on proliferation. Based on our data, translational studies could confirm whether combination of RA with reduced dosage of rapamycin would have more beneficial effects to higher dosage of rapamycin monotherapy meanwhile reducing adverse effects of rapamycin for patients with TSC mutation

Smooth Muscle–like Cells in Pulmonary Lymphangioleiomyomatosis

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

Wild type mesenchymal cells contribute to the lung pathology of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease

CrossTORC and WNTegration in Disease: Focus on Lymphangioleiomyomatosis

The mechanistic target of rapamycin (mTOR) and wingless-related integration site (Wnt) signal transduction networks are evolutionarily conserved mammalian growth and cellular development networks. Most cells express many of the proteins in both pathways, and this review will briefly describe only the key proteins and their intra- and extracellular crosstalk. These complex interactions will be discussed in relation to cancer development, drug resistance, and stem cell exhaustion. This review will also highlight the tumor-suppressive tuberous sclerosis complex (TSC) mutated, mTOR-hyperactive lung disease of women, lymphangioleiomyomatosis (LAM). We will summarize recent advances in the targeting of these pathways by monotherapy or combination therapy, as well as future potential treatments

Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p