Title: Osteoprotegerin Contributes to the Metastatic Potential of Cells with a Dysfunctional TSC2 Tumor-Suppressor Gene
Abstract: In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor–related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors. In addition to its effects on bone metabolism, osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family of receptors, promotes smooth muscle cell proliferation and migration and may act as a survival factor for tumor cells. We hypothesized that these cellular mechanisms of OPG may be involved in the growth and proliferation of lymphangioleiomyomatosis (LAM) cells, abnormal smooth muscle-like cells with mutations in one of the tuberous sclerosis complex tumor-suppressor genes (TSC1/TSC2) that cause LAM, a multisystem disease characterized by cystic lung destruction, lymphatic infiltration, and abdominal tumors. Herein, we show that OPG stimulated proliferation of cells cultured from explanted LAM lungs, and selectively induced migration of LAM cells identified by the loss of heterozygosity for TSC2. Consistent with these observations, cells with TSC2 loss of heterozygosity expressed the OPG receptors, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules showed reactivities to antibodies to tumor necrosis factor–related apoptosis-inducing ligand, receptor activator of NF-κB ligand, syndecan-1, and syndecan-2. LAM lung nodules also produced OPG, as shown by expression of OPG mRNA and colocalization of reactivities to anti-OPG and anti-gp100 (HMB45) antibodies in LAM lung nodules. Serum OPG was significantly higher in LAM patients than in normal volunteers. Based on these data, it appears that OPG may have tumor-promoting roles in the pathogenesis of lymphangioleiomyomatosis, perhaps acting as both autocrine and paracrine factors. Osteoprotegerin (OPG; TNFRSF11B), a soluble member of the tumor necrosis factor (TNF) receptor family, is best known as a regulator of bone metabolism that promotes bone formation by inhibiting osteoclast development, thus protecting against osteoporosis.1Schoppet M. Preissner K.T. Hofbauer L.C. RANK ligand and osteoprotegerin paracrine regulators of bone metabolism and vascular function.Arterioscler Thromb Vasc Biol. 2002; 22: 549-553Crossref PubMed Scopus (370) Google Scholar, 2Trouvin A.P. Göeb V. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss.Clin Interv Aging. 2010; 5: 345-354PubMed Google Scholar OPG, acting as a decoy receptor, binds to receptor activator of NF-κB ligand (RANKL), preventing the interaction of RANKL with its receptor RANK, resulting in the inhibition of osteoclast activation and bone resorption. Polymorphisms in the OPG gene have been linked to development of osteoporosis.3Jurado S. Nogues X. Agueda L. Garcia-Giralt N. Urreizti R. Yoskovitz G. Perez-Edo L. Salo G. Carreras R. Mellibovsky L. Balcells S. Grinberg D. Diez-Perez A. 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Gretarsdottir S. Gudbjartsson D.F. Walters G.B. Ingvarsson T. Jonsdottir T. Saemundsdottir J. Center J.R. Nguyen T.V. Bagger Y. Gulcher J.R. Eisman J.A. Christiansen C. Sigurdsson G. Kong A. Thorsteinsdottir U. Stefansson K. Multiple genetic loci for bone mineral density and fractures.N Engl J Med. 2008; 358: 2355-2365Crossref PubMed Scopus (507) Google Scholar Patients with juvenile Paget disease, a rare inherited disease affecting children, show increased bone turnover, leading to skeletal deformity. Mutations in the OPG gene determine the severity of the juvenile Paget disease phenotype,7Brunetti G. Marzano F. Colucci S. Ventura A. Cavallo L. Grano M. Faienza M.F. Genotype-phenotype correlation in Juvenile Paget disease: role of molecular alterations of the TNFRSF11B gene.Endocrine. 2012; 42: 266-271Crossref PubMed Scopus (19) Google Scholar with the loss of the entire gene or mutations leading to the loss of OPG structure resulting in a severe phenotype.More recently, the role of OPG in vascular cell biological characteristics has been studied. OPG knockout mice have both severe osteoporosis and significant arterial calcification,8Bucay N. Sarosi I. Dunstan C.R. Morony S. Tarpley J. Capparelli C. Scully S. Tan H.L. Xu W. Lacey D.L. Boyle W.J. Simonet W.S. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2111) Google Scholar suggesting that OPG plays a protective role against arterial calcification in mice. OPG serum levels are associated with the severity of cardiovascular disease in humans.9Jono S. Ikari Y. Shioi A. Mori K. Miki T. Hara K. Nishizawa Y. Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease.Circulation. 2002; 106: 1192-1194Crossref PubMed Scopus (452) Google Scholar, 10Tousoulis D. Siasos G. Maniatis K. Oikonomou R. Kioufis S. Zaromitidou M. Paraskevopoulos T. Michalea S. Kollia C. Miliou A. Kokkou E. Papavassiliou A.G. Stefanadis C. Serum osteoprotegerin and osteopontin levels are associated with arterial stiffness and the presence and severity of coronary artery disease.Int J Cardiol. 2012; http://dx.doi.org/10.1016/j.ijcard.2012.05.001Abstract Full Text Full Text PDF Scopus (10) Google Scholar, 11Venuraju S.M. Yerramasu A. Corder R. Lahiri A. Osteoprotegerin as a predictor of coronary artery disease and cardiovascular mortality and morbidity.J Am Coll Cardiol. 2010; 55: 2049-2061Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar OPG levels may be higher either directly, through a proatherosclerotic effect, or indirectly, because of an incomplete compensatory mechanism in which increases in serum OPG levels are seen as a response to RANKL activity.9Jono S. Ikari Y. Shioi A. Mori K. Miki T. Hara K. Nishizawa Y. Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease.Circulation. 2002; 106: 1192-1194Crossref PubMed Scopus (452) Google Scholar, 10Tousoulis D. Siasos G. Maniatis K. Oikonomou R. Kioufis S. Zaromitidou M. Paraskevopoulos T. Michalea S. Kollia C. Miliou A. Kokkou E. Papavassiliou A.G. Stefanadis C. Serum osteoprotegerin and osteopontin levels are associated with arterial stiffness and the presence and severity of coronary artery disease.Int J Cardiol. 2012; http://dx.doi.org/10.1016/j.ijcard.2012.05.001Abstract Full Text Full Text PDF Scopus (10) Google Scholar, 11Venuraju S.M. Yerramasu A. Corder R. Lahiri A. Osteoprotegerin as a predictor of coronary artery disease and cardiovascular mortality and morbidity.J Am Coll Cardiol. 2010; 55: 2049-2061Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar This compensatory effect may also be invoked to explain high serum levels of OPG, sometimes seen in subjects with osteoporosis.12Yano K. Tsuda E. Washida N. Kobayashi F. Goto M. Harada A. Ikeda K. Higashio K. Yamada Y. Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increase serum concentrations in postmenopausal women with osteoporosis.J Bone Miner Res. 1999; 14: 518-527Crossref PubMed Scopus (355) Google ScholarVascular smooth muscle cells express OPG, and aortic smooth muscle cells proliferate in response to OPG.13Corallini F. Gonelli A. D'Aurizio F. di Iasio M.G. Vaccarezza M. Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin.Eur J Histochem. 2009; 53: 19-24Crossref PubMed Google Scholar OPG induced both the proliferation and migration of pulmonary artery smooth muscle cells14Lawrie A. Waterman E. Southwood M. Evans D. Suntharalingam J. Francis S. Crossman D. Croucher P. Morrell N. Newman C. Evidence of a role for osteoprotegerin in the pathogenesis of pulmonary artery hypertension.Am J Pathol. 2008; 172: 256-264Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar and human microvascular endothelial cells.15Kobayashi-Sakamoto M. Isogai E. Hirose K. Chiba I. Role of αV integrin in osteoprotegerin-induced endothelial cell migration and proliferation.Microvasc Res. 2008; 76: 139-144Crossref PubMed Scopus (32) Google Scholar The effects of OPG on human microvascular endothelial cells were mediated through integrins αVβ3 and αVβ5 and the extracellular signal–regulated kinase 1/2. OPG can also stimulate monocyte migration; this effect was shown to involve syndecans and phosphatidylinositol-3-OH kinase/Akt, protein kinase C, and tyrosine kinases.16Mosheimer B.A. Kaneider N.C. Feistritzer C. Djanani A.M. Sturn D.H. Patsch J.R. Wiedermann C.J. Syndecan-1 is involved in osteoprotegerin-induced chemotaxis in human peripheral blood monocytes.J Clin Endocrinol Metab. 2005; 90: 2964-2971Crossref PubMed Scopus (75) Google ScholarOPG also has roles in tumor development and metastasis.17Holen I. Shipman C.M. Role of osteoprotegerin (OPG) in cancer.Clin Sci. 2006; 110: 279-291Crossref PubMed Scopus (94) Google Scholar, 18Zauli G. Melloni E. Capitani S. Secchiero P. Role of full-length osteoprotegerin in tumor cell biology.Cell Mol Life Sci. 2009; 66: 841-851Crossref PubMed Scopus (71) Google Scholar OPG can bind TNF-related apoptosis-inducing ligand (TRAIL), blocking TRAIL's apoptotic effects on cancer cells.19Neville-Webbe H.L. Cross N.A. Eaton C.L. Nyambo R. Evans C.A. Coleman R.E. Osteoprotegerin (OPG) produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis.Breast Cancer Res Treat. 2004; 86: 269-279Crossref PubMed Scopus (107) Google Scholar, 20Nyambo R. Cross N.A. Lippitt J.M. Holen I. 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Kuraoka K. Motoshita J. Oda N. Oue N. Yasui W. Expression of osteoprotegerin correlates with aggressiveness and poor prognosis of gastric carcinoma.Virchows Arch. 2003; 443: 146-151Crossref PubMed Scopus (41) Google Scholar Tumor growth and metastasis are also supported by OPG's promotion of endothelial cell survival and angiogenesis.28Malyankar U. Scatena M. Suchland K. Yun T. Clark E. Giachelli C. Osteoprotegerin is an αvβ3-induced, NF-κΒ-dependent survival factor for endothelial cells.J Biol Chem. 2000; 275: 20959-20962Crossref PubMed Scopus (331) Google Scholar, 29Cross S.S. Yang Z. Brown N.J. Balasubramanian S.P. Evans C.A. Woodward J.K. Neville-Webbe H.L. Lippitt J.M. Reed M.W. Coleman R.E. Holen I. Osteoprotegerin (OPG): a potential new role in the regulation of endothelial cell phenotype and tumour angiogenesis.Int J Cancer. 2006; 118: 1901-1908Crossref PubMed Scopus (91) Google Scholar Interestingly, some malignant breast cancer tumors show endothelial OPG expression, whereas neighboring normal endothelium does not express high levels of the protein.29Cross S.S. Yang Z. Brown N.J. Balasubramanian S.P. Evans C.A. Woodward J.K. Neville-Webbe H.L. Lippitt J.M. Reed M.W. Coleman R.E. Holen I. Osteoprotegerin (OPG): a potential new role in the regulation of endothelial cell phenotype and tumour angiogenesis.Int J Cancer. 2006; 118: 1901-1908Crossref PubMed Scopus (91) Google ScholarLymphangioleiomyomatosis (LAM) cells are abnormal neoplastic smooth muscle-like cells, with mutations in one of two tuberous sclerosis complex tumor-suppressor genes (TSC1 or TSC2). TSC1 (encoding hamartin) and TSC2 (tuberin) form a complex that regulates the serine/threonine kinase, mammalian target of rapamycin.30Orlova K.A. Crino P.B. The tuberous sclerosis complex.Ann N Y Acad Sci. 2010; 1184: 87-105Crossref PubMed Scopus (307) Google Scholar Mutations in TSC1/TSC2 lead to uncontrolled mammalian target of rapamycin activity, resulting in increased cell proliferation and size.30Orlova K.A. Crino P.B. The tuberous sclerosis complex.Ann N Y Acad Sci. 2010; 1184: 87-105Crossref PubMed Scopus (307) Google Scholar These LAM cells form nodules covered with type II pneumocytes, with surrounding areas of cystic destruction in the lungs of patients with LAM. In addition to the cystic destruction of lung parenchyma, LAM, a rare multisystem disease affecting women,31Steagall W.K. Taveira-DaSilva A.M. Moss J. Clinical and molecular insights into lymphangioleiomyomatosis.Sarcoidosis Vasc Diffuse Lung Dis. 2005; 22: S49-S66PubMed Google Scholar is characterized by lymphatic abnormalities and abdominal tumors (eg, angiomyolipomas). LAM cells can metastasize, as LAM cells from lung lesions and angiomyolipomas in the same patient have the same TSC2 mutation.32Carsillo T. Astrinidis A. Henske E.P. Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.Proc Natl Acad Sci U S A. 2000; 97: 6085-6090Crossref PubMed Scopus (536) Google Scholar Consistent with their migratory behavior, LAM cells have been isolated from blood and other body fluids of patients with LAM.33Crooks D.M. Pacheco-Rodriguez G. DeCastro R.M. McCoy Jr., J.P. Wang J.A. Kumaki F. Darling T. Moss J. Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis.Proc Natl Acad Sci U S A. 2004; 101: 17462-17467Crossref PubMed Scopus (150) Google Scholar, 34Cai X. Pacheco-Rodriguez G. Fan Q.-Y. Haughey M. Samsel L. El-Chemaly S. Wu H.-P. McCoy J.P. Steagall W.K. Lin J.-P. Darling T.N. Moss J. Phenotypic characterization of disseminated cells with TSC2 loss of heterozygosity in patients with lymphangioleiomyomatosis.Am J Respir Crit Care Med. 2010; 182: 1410-1418Crossref PubMed Scopus (58) Google Scholar LAM cells have characteristics of both smooth muscle cells, such as reactivity with antibodies to smooth muscle actin and desmin, and of melanocytes, with reactivity with HMB45,35Adema G.J. de Boer A.J. van't Hullenaar R. Denijn M. Ruiter D.J. Vogel A.M. Figdor C.G. Melanocyte lineage-specific antigens recognized by monoclonal antibodies NKI-beteb, HMB-50, and HMB-45 are encoded by a single cDNA.Am J Pathol. 1993; 143: 1579-1585PubMed Google Scholar an antibody recognizing gp100, a melanosomal protein.36Darling T.N. Pacheco-Rodriguez G. Gorio A. Lesma E. Walker C. Moss J. Lymphangioleiomyomatosis and TSC2-/- cells.Lymphat Res Biol. 2010; 8: 59-69Crossref PubMed Scopus (21) Google Scholar, 37Ferrans V.J. Yu Z.-X. Nelson W.K. Valencia J.C. Tatsuguchi A. Avila N.A. Riemenschn W. Matsui K. Travis W.D. Moss J. Lymphangioleiomyomatosis (LAM): a review of clinical and morphological features.J Nippon Med Sch. 2000; 67: 311-329Crossref PubMed Scopus (115) Google Scholar, 38Matsumoto Y. Horiba K. Usuki J. Chu S.C. Ferrans V.J. Moss J. Markers of cell proliferation and expression of melanosomal antigen in lymphangioleiomyomatosis.Am J Respir Cell Mol Biol. 1999; 21: 327-336Crossref PubMed Scopus (115) Google ScholarIn this study, we investigated the effect of OPG on the neoplastic smooth muscle cell-like LAM cells. OPG promoted proliferation of cells grown from explanted LAM lungs and specifically induced LAM cell migration. Three OPG receptors, RANKL, syndecan-1, and syndecan-2, were detected on LAM cells and LAM lung nodules. Furthermore, LAM cells produced OPG, and OPG levels were elevated in serum from patients with LAM compared with healthy volunteers, suggesting both autocrine and paracrine effects of OPG in LAM.Materials and MethodsStudy PopulationResearch was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute, Bethesda, MD (protocols 95-H-0186 and 96-H-0100). All participants gave written informed consent. Patients with LAM, for serum protein measurements, had a mean age of 50.5 ± 1.1 years and included 71 whites, four Asians, four African Americans, and one Hispanic. Healthy volunteers were seen at the NIH and were screened for pulmonary health by medical history and physical examination. The exclusion criteria for participants in the healthy volunteer group were as follows: aged <18 or >80 years, serum test result positive for HIV or hepatitis virus, and inability to perform reliable pulmonary function tests. The principal investigator (J.M.) reviewed applicants with minor health problems on a case-by-case basis. Healthy volunteers for the serum protein measurements had a mean age of 41.4 ± 2.0 years and included 20 whites, five Asians, four African Americans, one Hispanic, and one of unknown ethnicity. Concentrations of OPG, RANKL, and TRAIL in serum were quantified using kits from Alpco Diagnostics (Windham, NH) and Pierce Biotechnology (Rockford, IL).Tissue Samples and Cell CultureLung tissue was collected from patients with LAM undergoing transplantation, and cells were grown, as previously described,39Pacheco-Rodriquez G. Steagall W.K. Crooks D.M. Stevens L.A. Hashimoto H. Li S. Wang J.A. Darling T.N. Moss J. TSC2 loss in lymphangioleiomyomatosis cells correlated with expression of CD44v6, a molecular determinant of metastasis.Cancer Res. 2007; 67: 10573-10581Crossref PubMed Scopus (51) Google Scholar in mesenchymal stem cell medium (Lonza, Walkersville, MD). Briefly, lung sections obtained at transplant were placed on plastic dishes with mesenchymal stem cell medium containing 0.05 U/mL penicillin, 0.05 μg/mL streptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum. When colonies of cells formed, the tissue sections were removed and cells were allowed to grow for 3 to 7 days before trypsinization and transfer to new dishes. This procedure resulted in a heterogeneous mixture of cells that have functional TSC2 and those with TSC2 loss of heterozygosity (LOH). These cultures have been previously characterized.39Pacheco-Rodriquez G. Steagall W.K. Crooks D.M. Stevens L.A. Hashimoto H. Li S. Wang J.A. Darling T.N. Moss J. TSC2 loss in lymphangioleiomyomatosis cells correlated with expression of CD44v6, a molecular determinant of metastasis.Cancer Res. 2007; 67: 10573-10581Crossref PubMed Scopus (51) Google Scholar, 40Ikeda Y. Taveira-DaSilva A.M. Pacheco-Rodriguez G. Steagall W.K. El-Chemaly S. Gochuico B.R. May R.M. Hathaway O.M. Li S. Wang J.A. Darling T.N. Stylianou M. Moss J. Erythropoietin-driven proliferation of cells with mutations in the tumor suppressor gene TSC2.Am J Physiol Lung Cell Mol Physiol. 2011; 300: L64-L72Crossref PubMed Scopus (11) Google Scholar, 41Pacheco-Rodriguez G. Kumaki F. Steagall W.K. Zhang Y. Ikeda Y. Lin J.-P. Billings E.M. Moss J. Chemokine-enhanced chemotaxis of lymphangioleiomyomatosis cells with mutations in the tumor suppressor TSC2 gene.J Immunol. 2009; 182: 1270-1277Crossref PubMed Scopus (43) Google ScholarLOH AnalysisKg8 or D16S3395 microsatellite markers from chromosome 16 were amplified with appropriate primer pairs [Kg8 forward (5′-CTCCCAGGGTGGAGGAAGGTG-3′) and Kg8 reverse (5′-Fam-GCAGGCACAGCCAGCTCCGAG-3′) or D16S3395 forward (5′-CTAACCCTCAGCAGAGTTCTG-3′) and D16S3395 reverse (5′-Fam-CCTGGCAGTAAGTCCTGAAA-3′)], and products were analyzed on a 3100 Genetic Analyzer (Applied Biosystems, Carlsbad, CA). LOH was quantified as previously described.33Crooks D.M. Pacheco-Rodriguez G. DeCastro R.M. McCoy Jr., J.P. Wang J.A. Kumaki F. Darling T. Moss J. Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis.Proc Natl Acad Sci U S A. 2004; 101: 17462-17467Crossref PubMed Scopus (150) Google Scholar Briefly, the heights of the two alleles in the sample were determined (L1 and L2), as were the heights of the alleles of the control (N1 and N2). QLOH is defined as (L1/L2)/(N1/N2), where L1 is the diminished allele. If the sample had a QLOH score <0.6, then it had LOH.Definition of a LAM CellIn this study, we are defining a LAM cell as a cell with a mutation or LOH of TSC2. Although many protein markers have been suggested to be expressed by LAM cells, none has been shown to be exclusive to LAM cells.36Darling T.N. Pacheco-Rodriguez G. Gorio A. Lesma E. Walker C. Moss J. Lymphangioleiomyomatosis and TSC2-/- cells.Lymphat Res Biol. 2010; 8: 59-69Crossref PubMed Scopus (21) Google Scholar Therefore, we chose to use the genetic definition of TSC2 LOH to specifically distinguish LAM cells. No one has reported a homogeneous LAM cell culture from lung; all in vitro studies using LAM lung cell cultures were performed on heterogeneous mixtures that have both LAM cells (TSC2 LOH) and non-LAM cells (TSC2 functional). The heterogeneous mixtures of cells grown from LAM lung contain 20% to 40% TSC2 LOH LAM cells by fluorescent in situ hybridization.39Pacheco-Rodriquez G. Steagall W.K. Crooks D.M. Stevens L.A. Hashimoto H. Li S. Wang J.A. Darling T.N. Moss J. TSC2 loss in lymphangioleiomyomatosis cells correlated with expression of CD44v6, a molecular determinant of metastasis.Cancer Res. 2007; 67: 10573-10581Crossref PubMed Scopus (51) Google Scholar In general, these LAM cells are undetectable by PCR analysis of LOH because the altered allele of the LAM cell is obscured by the alleles of the non-LAM cells, unless fractionation or selection is performed to remove some of the non-LAM cells. Separation of populations of cells by fluorescence-activated cell sorting (FACS) using the marker CD44v639 has been shown to enrich for and allow detection of LAM cells having TSC2 LOH.ProliferationA total of 2000 cells per well of four heterogeneous mixtures of cells grown from four different LAM lungs, pulmonary artery smooth muscle (PASM) cells, or normal human fibroblasts were plated in 96-well plates and incubated overnight at 37°C with 10, 50, or 100 ng/mL of OPG, RANKL, or TRAIL. Cell growth was measured using a colorimetric assay (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD). Data were reported as a percentage of the appropriate vehicle-containing control, and the LAM data were the average of the results from four mixtures of cells.Chemotaxis AssaysChemotaxis was assayed in 24-well plates with 8-μm pores (Chemicon, San Diego, CA), using 1 × 106 cells per well. Heterogeneous mixtures of cells were added to the upper chamber; 10% fetal bovine serum, 100 ng/mL OPG, or chemokine ligand (CCL) 2 was added to serum-free medium in the bottom chamber, and cells were permitted to migrate from the upper chamber overnight at 37°C. DNA was prepared from migrated cells (QIAamp DNA Mini Kit; Qiagen, Valencia, CA), and LOH analysis was performed.FACS AnalysisApproximately 1 × 106 cells were incubated with indicated antibodies: fluorescein isothiocyanate (FITC) mouse anti-human CD44v6 (clone VFF-7; Invitrogen, Carlsbad, CA), phycoerythrin mouse anti-human TRAIL (clone 75402; R&D Systems, Minneapolis, MN), Alexa Fluor 647 rat anti-mouse RANKL (clone IK22-5; BD Biosciences, San Jose, CA), phycoerythrin rat anti-human syndecan-1 (clone 359103; R&D Systems), and allophycocyanin rat anti-human syndecan-2 (clone 305515; R&D Systems). Cell sorting was performed on a MoFlo Flow Cytometer (Beckman Coulter, Hialeah, FL). DNA was prepared (QIAamp DNA Mini Kit; Qiagen) from the subpopulations, and LOH analysis was performed.OPG in Cell SupernatantsSerum-starved PASM cells and three heterogeneous mixtures of cells from LAM lungs (each from a different patient) (100,000 cells per well of a 6-well plate) were incubated overnight with serum-free medium, with or without 100 ng/mL CCL2 or 100 ng/mL TNF-α, or complete medium, and culture supernatants were collected. OPG concentrations in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA; Alpco Diagnostics) and normalized with the final number of cells in each well. PASM cells were tested six times, as were each of the mixtures of cells. Data are reported as a percentage of the serum-free medium control. A LAM-specific average was calculated.RT-PCRLaser-capture microdissection was performed on frozen LAM lung tissue samples. LAM cells (5000 to 7000 spots) were collected with the VERITAS microdissection system (Arcturus Engineering, Mountain View, CA). RNA was prepared (Arcturus Picopure RNA Isolation Kit; Applied Biosystems). First-strand cDNA was primed with oligo dT and random primers (Superscript First-Strand Synthesis System for RT-PCR; Invitrogen) and PCR amplified with primers OPGCS (5′-Hex-CTGGATTTGGAGTGGTGCAAGC-3′) and OPGCAS (5′-TGTTTCCGGAACATATGTTGTCGTG-3′). Cells from two heterogeneous mixtures were spun down and lysed in cell lysis buffer (Signosis, Inc., Sunnyvale, CA). OPG gene fragments were produced using the One Step RT-PCR Kit (Qiagen) and primers OPGCS and OPGCAS. Products were analyzed on a 3100 Genetic Analyzer. The plots show PCR product peaks. The 186-bp peak represents OPG RNA. Amplification of genomic DNA would result in a peak >4000 bp because primer pair OPGCS binds to a sequence in exon 2, whereas OPGCAS binds to a sequence in exon 3. Amplification of actin was performed as a positive control.IHC AssaysImmunohistochemical (IHC) assays were performed on formalin-fixed, paraffin-embedded sections. Slides were incubated with rabbit polyclonal antibody against OPG (H-249; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) overnight, then washed, incubated with biotinylated goat anti-rabbit antibody (HRP/DAB Detection System; Spring Bioscience, Fremont, CA), and washed again before development with 3,3′-diaminobenzidine tetrahydrochloride, and counterstained with hematoxylin. A negative control was treated with normal rabbit IgG instead of primary antibody. Slides were inspected with a Nikon DXM-1200 charge-coupled device microscope (Nikon, Inc., Melville, NY), and results were analyzed with Nikon ACT-1 software version 2.Fluorescent IHC AssaysSlides for fluorescent IHC assays were prepared from formalin-fixed, paraffin-embedded or frozen LAM lung tissue sections (10 or 20 μm thick). Normal human lung slides were obtained from Imgenex (San Diego, CA). Dual labeling for immunofluorescence evaluated colocalization of a monoclonal antibody reaction with OPG (labeled with Texas Red; R&D Systems) paired with HMB45 (FITC; Dako, Carpinteria, CA) or antibodies against either CD31 (FITC; Abcam, Cambridge, MA) or podoplanin (FITC; R&D Systems). The characterization of OPG recep