Since LAM occurs primarily in women, we questioned whether reproductive hormones such as prolactin might affect signaling and proliferation in TSC2-deficient cells and, if that is the case, perhaps disease progression. Prolactin (Prl) is an important hormone for women of childbearing age, with key roles in proliferation, differentiation, and survival of wide variety of cells (e.g., lymphocytes, breast cancer cells). Although PRL is produced mainly by the anterior pituitary gland and acts systemically as a classical endocrine factor, it is also produced locally at multiple extra-pituitary sites, where it acts in an autocrine/paracrine manner. It is also produced by human tumors, including breast and lung. Prolactin works through the PRL receptor (PRLr), a type I transmembrane protein, which consists of an extracellular domain, a short transmembrane domain, and a variable intracellular domain (ICD) that mediates signaling. Alternative splicing generates PRLr isoforms classified by the length of their ICDs as short, intermediate, or long. The signaling properties of the intermediate and short isoforms differ from those of the long form due to different interactions of the shorter ICDs with scaffolding and signaling proteins. Major signaling networks downstream of PRL/PRLr include Janus kinases (JAKs) and the signal transducers and activators of transcription (STAT) signaling pathways as well as the ShcRasRaf mitogen-activated protein kinase (MAPK) pathway. These pathways impact crucial cellular processes, including, proliferation, survival, and differentiation. LAM cells grow in the affected lung interstitium as well as in LAM nodules in the walls of cysts. In the current study, prolactin signal transduction pathways were assessed in lymphangioleiomyomatosis lung nodules from human lung explants. In addition, prolactin effects on proliferation and signaling were quantified in tuberin-deficient and tuberin-expressing rat cells. Prolactin levels in lymphangioleiomyomatosis patient sera were correlated with clinical status. We observed that prolactin and its receptor were produced by LAM cells in the lung nodule. Higher levels of prolactin and prolactin receptor mRNA were found in lymphangioleiomyomatosis lesions than in vascular smooth muscle cells. Activation of intracellular signaling pathways involved in prolactin signaling was seen in LAM lesions. In particular, JAK/STAT and MAP kinase pathways appeared to be activated with phospho-JAK2, phospho-STAT3 and phospho-p44/42 detected by immunohistochemical methods. Further, prolactin stimulated signaling pathways and proliferation of Tsc2-/- cells. Tsc2-/- Eker rat embryonic fibroblasts expressed more prolactin receptor than did Tsc2+/+ cells. Prolactin activated STAT1, STAT3, p44/42 and p38 MAPK pathways and promoted proliferation of Tsc2-/- cells more than it did Tsc2+/+ cells. Alternative splicing of the prolactin receptor differed in TSC2+/+ and TSC2-/- cells, with greater amounts of a long form of the prolactin receptor found in the TSC2-/- cells. A prolactin receptor antagonist, S179D-PRL, increased the amount of the short form of the prolactin receptor. The short form of the prolactin receptor is associated with reduced cell proliferation in a number of systems. In support of the potential clinical relevance of prolactin in the progression of LAM, higher prolactin levels in sera of lymphangioleiomyomatosis patients were associated with a faster rate of decline in FEV1 and a history of more pneumothoraces. These findings suggest that PRL signaling may be important in Tsc2-/- cells and contribute to LAM pathogenesis in an autocrine/paracrine manner. Lymphangioleiomyomatosis occurring sporadically (S-LAM) or in patients with tuberous sclerosis complex (TSC), results from abnormal proliferation and dissemination of smooth muscle-like LAM cells exhibiting mutations or loss of heterozygosity (LOH) of a TSC gene. LAM cells from lung nodules, AMLs, and lymph nodes of the same patient showed identical TSC2 mutations and LOH patterns, consistent with metastatic spread among organs. Further supporting this model, LAM cells were identified in donor lungs following transplantation and could be isolated from blood, urine, and chyle of LAM patients, consistent with LAM cell dissemination via body fluids. Identification of LAM cells in blood by LOH was aided by fluorescence-activated cell sorting (FACS) to remove of non-LAM cells after immunostaining with antibodies against leukocyte common antigen (CD45) and glycophorin A (CD235a), a protein present on LAM cells in lung nodules. In our previous studies, we were able to isolate LAM cells from only 60% of patients, and thus could not answer questions such as whether sporadic LAM was primarily TSC2 driven, whether LAM cells in different body fluids showed similar LOH patterns, or whether LAM cells could be isolated from bronchoalveolar lavage fluid (BALF). We, therefore, looked for cell surface molecules unique to TSC2-/-cells and used these findings to separate LAM cells with TSC2 LOH from BALF, urine, chyle, and blood. We have shown that CD44v6 is expressed by LAM cells in lung nodules and is present on LAM cells grown from explanted lungs. This splice variant of the hyaluronic acid receptor is believed to be involved in tumor metastasis and progression. we observed that the tetraspanin CD9, a highly expressed gene identified by microarray analysis of TSC2-/- cells from TSC skin lesions, and CD44v6 identified LAM cells with TSC2 LOH from BALF, urine, and chylous effusions. Our objectives in the present study were to identify molecular markers useful for isolating LAM cells from body fluids and determine the frequency of TSC1 or TSC2 LOH. Candidate cell surface markers were identified using gene microarray analysis of human TSC2-/- cells. Cells from bronchoalveolar lavage fluid (BALF), urine, chylous effusions, and blood were sorted based on reactivity with antibodies against these proteins (e.g., CD9, CD44v6) and analyzed for LOH using TSC1- and TSC2-related microsatellite markers and single nucleotide polymorphisms in the TSC2 gene. CD44v6+CD9+ cells from BALF, urine, and chyle showed TSC2 LOH in 80%, 69%, and 50% of patient samples, respectively. LAM cells with TSC2 LOH were detected in over 90% of blood samples. LAM cells from different body fluids of the same patients, showed, in most cases, identical LOH patterns, that is, loss of alleles at the same microsatellite loci. In a few S-LAM patients, LAM cells from different body fluids differed in LOH patterns. No S-LAM patients with TSC1 LOH were identified, suggesting that TSC2 abnormalities are responsible for the vast majority of S-LAM cases, and that TSC1-disease may be subclinical. Our data support a common genetic origin of LAM cells in most S-LAM patients, consistent with a metastatic model. They also suggest, however, that LAM cells in different sites, independently of their potential origin, or those within the same site could exhibit genetic and phenotypic heterogeneity.

Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2010
Total Cost
$2,413,413
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Gupta, Nishant; Lee, Hye-Seung; Ryu, Jay H et al. (2018) The NHLBI LAM Registry: Prognostic Physiologic and Radiologic Biomarkers Emerge From a 15-Year Prospective Longitudinal Analysis. Chest :
Le, Kang; Steagall, Wendy K; Stylianou, Mario et al. (2018) Effect of beta-agonists on LAM progression and treatment. Proc Natl Acad Sci U S A 115:E944-E953
Larsen, Thomas C; Gopalakrishnan, Vissagan; Yao, Jianhua et al. (2018) Optimization of a secondary VOI protocol for lung imaging in a clinical CT scanner. J Appl Clin Med Phys 19:271-280
Taveira-DaSilva, Angelo M; Julien-Williams, Patricia; Jones, Amanda M et al. (2018) Rates of change in FEV1 and DLCO as potential indicators for mTOR inhibitor therapy in premenopausal lymphangioleiomyomatosis patients. Eur Respir J 51:
Gupta, Nishant; Johnson, Simon R; Moss, Joel et al. (2018) Reply to Yanagisawa: Treatment of Pulmonary Lymphangioleiomyomatosis during Pregnancy. Am J Respir Crit Care Med 197:1507-1508
Glasgow, Connie G; Pacheco-Rodriguez, Gustavo; Steagall, Wendy K et al. (2018) CA-125 in Disease Progression and Treatment of Lymphangioleiomyomatosis. Chest 153:339-348
Oyerinde, Oyetewa; Buccine, Danielle; Treichel, Alison et al. (2018) Fibrous cephalic plaques in tuberous sclerosis complex. J Am Acad Dermatol 78:717-724
Pacheco, Gustavo G; Jones, Amanda M; Yao, Jianhua et al. (2018) Mounier-Kuhn Syndrome Mimicking Lymphangioleiomyomatosis. Chest 153:e19-e23
Sapp, Julie C; Johnston, Jennifer J; Driscoll, Kate et al. (2018) Evaluation of Recipients of Positive and Negative Secondary Findings Evaluations in a Hybrid CLIA-Research Sequencing Pilot. Am J Hum Genet 103:358-366
Lamattina, Anthony M; Taveira-Dasilva, Angelo; Goldberg, Hilary J et al. (2018) Circulating biomarkers from the phase I trial of sirolimus and autophagy inhibition for patients with lymphangioleiomyomatosis (SAIL). Chest :

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