Up-Regulation of the Lymphatic Marker Podoplanin, a Mucin-Type Transmembrane Glycoprotein, in Human Squamous Cell Carcinomas and Germ Cell Tumors
2005; Elsevier BV; Volume: 166; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)62311-5
ISSN1525-2191
AutoresVivien Schacht, Soheil S. Dadras, Louise A. Johnson, David G. Jackson, Young‐Kwon Hong, Michael Detmar,
Tópico(s)Lymphatic Disorders and Treatments
ResumoThe mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers. The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers. Lymphatic vessels play an important role in the maintenance of tissue homeostasis1Witte MH Bernas MJ Martin CP Witte CL Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.Microsc Res Tech. 2001; 55: 122-145Crossref PubMed Scopus (189) Google Scholar and in the transport of immune cells,2von Andrian UH Mackay CR T-cell function and migration. Two sides of the same coin.N Engl J Med. 2000; 343: 1020-1034Crossref PubMed Scopus (1237) Google Scholar but they also serve as the primary conduit for malignant tumor cell metastasis to regional lymph nodes.3Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1518) Google Scholar Although there is considerable evidence, obtained in genetic and xenotransplant tumor models, that tumor lymphangiogenesis promotes lymphatic tumor spread,3Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1518) Google Scholar, 4Stacker SA Caesar C Baldwin ME Thornton GE Williams RA Prevo R Jackson DG Nishikawa S Kubo H Achen MG VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med. 2001; 7: 186-191Crossref PubMed Scopus (1074) Google Scholar it has remained controversial whether human tumors might actively induce lymphangiogenesis, and whether the degree of intra- or peritumoral lymphangiogenesis might serve as a prognostic indicator of tumor progression.5Straume O Jackson DG Akslen LA Independent prognostic impact of lymphatic vessel density and presence of low-grade lymphangiogenesis in cutaneous melanoma.Clin Cancer Res. 2003; 9: 250-256PubMed Google Scholar, 6Dadras SS Paul T Bertoncini J Brown LF Muzikansky A Jackson DG Ellwanger U Garbe C Mihm MC Detmar M Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.Am J Pathol. 2003; 162: 1951-1960Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar Several new markers for the specific detection of human lymphatic endothelium versus blood vascular endothelium have been recently identified;7Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar, 8Breiteneder-Geleff S Soleiman A Horvat R Amann G Kowalski H Kerjaschki D Podoplanin—a specific marker for lymphatic endothelium expressed in angiosarcoma.Verh Dtsch Ges Pathol. 1999; 83: 270-275PubMed Google Scholar, 9Wigle JT Oliver G Prox1 function is required for the development of the murine lymphatic system.Cell. 1999; 98: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1272) Google Scholar however, there have been no commercially available antibodies against these lymphatic-specific proteins and, therefore, large-scale studies of tumor lymphangiogenesis are still lacking. The mucin-type transmembrane glycoprotein podoplanin is one of the most highly expressed lymphatic-specific genes in cultured human lymphatic endothelial cells (LECs),10Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar and we have previously shown that podoplanin is a target gene of the homeobox gene Prox1, a master gene that controls the development of lymphatic progenitors from embryonic veins.11Hong YK Harvey N Noh YH Schacht V Hirakawa S Detmar M Oliver G Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.Dev Dyn. 2002; 225: 351-357Crossref PubMed Scopus (442) Google Scholar In vivo expression of podoplanin in lymphatic endothelium was first reported by Wetterwald and colleagues,12Wetterwald A Hoffstetter W Cecchini MG Lanske B Wagner C Fleisch H Atkinson M Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes.Bone. 1996; 18: 125-132Abstract Full Text PDF PubMed Scopus (252) Google Scholar who named it “E11 antigen.” It was further characterized under the name “podoplanin,” because of its low-level expression in kidney podocytes.13Breiteneder-Geleff S Matsui K Soleiman A Meraner P Poczewski H Kalt R Schaffner G Kerjaschki D Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.Am J Pathol. 1997; 151: 1141-1152PubMed Google Scholar However, podoplanin is homologous to T1alpha, which was found to encode an antigen that is expressed at the apical surface of alveolar type I cells in rat lung.14Dobbs LG Williams MC Gonzalez R Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells.Biochim Biophys Acta. 1988; 970: 146-156Crossref PubMed Scopus (218) Google Scholar, 15Rishi AK Joyce-Brady M Fisher J Dobbs LG Floros J VanderSpek J Brody JS Williams MC Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives.Dev Biol. 1995; 167: 294-306Crossref PubMed Scopus (181) Google Scholar Expression of podoplanin has also been detected in the choroid plexus in the rat brain and the ciliary epithelium in the rat eye.16Williams MC Cao Y Hinds A Rishi AK Wetterwald A T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats.Am J Respir Cell Mol Biol. 1996; 14: 577-585Crossref PubMed Scopus (128) Google Scholar Other podoplanin homologs include OTS-8,17Nose K Saito H Kuroki T Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3–E1) and expressed constitutively in ras-transformed cells.Cell Growth Differ. 1990; 1: 511-518PubMed Google Scholar RTI40,18Gonzalez RF Dobbs LG Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein.Biochim Biophys Acta. 1998; 1429: 208-216Crossref PubMed Scopus (43) Google Scholar gp38,19Farr AG Berry ML Kim A Nelson AJ Welch MP Aruffo A Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues.J Exp Med. 1992; 176: 1477-1482Crossref PubMed Scopus (144) Google Scholar canine gp40,20Zimmer G Lottspeich F Maisner A Klenk HD Herrler G Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin-Darby canine kidney cells (type I).Biochem J. 1997; 326: 99-108Crossref PubMed Scopus (38) Google Scholar human gp36,21Zimmer G Oeffner F Von Messling V Tschernig T Groness HJ Klenk HD Herrler G Cloning and characterization of gp36, a human mucin-type glycoprotein preferentially expressed in vascular endothelium.Biochem J. 1999; 341: 277-284Crossref PubMed Scopus (73) Google Scholar and murine PA2.26.22Gandarillas A Scholl FG Benito N Gamallo C Quintanilla M Induction of PA2.26, a cell-surface antigen expressed by active fibroblasts, in mouse epidermal keratinocytes during carcinogenesis.Mol Carcinog. 1997; 20: 10-18Crossref PubMed Scopus (85) Google Scholar However, little is understood about the biological function of podoplanin. Recently, we found that mice deficient in podoplanin develop congenital lymphedema and that they have defects in lymphatic vessel, but not blood vessel, pattern formation.23Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (569) Google Scholar Moreover, our in vitro studies indicated that podoplanin is involved in mediating cell motility by promoting rearrangement of the actin cytoskeleton.23Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (569) Google Scholar In this study, we aimed to identify an anti-human podoplanin antibody suitable for immunostains of archival paraffin-embedded human tissues, and to comprehensively characterize the cell type-specific expression of podoplanin in normal tissues and its potential involvement in tumor progression. We show that the commercially available antibody D2-40, originally raised against an unidentified M2A protein derived from germ cell tumors,24Marks A Sutherland DR Bailey D Iglesias J Law J Lei M Yeger H Banerjee D Baumal R Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours.Br J Cancer. 1999; 80: 569-578Crossref PubMed Scopus (245) Google Scholar specifically recognizes human podoplanin and that it can be used for routine immunohistochemical studies of tumor lymphangiogenesis. Using normal human tissue arrays, we found that podoplanin is also expressed by bile duct cells of the liver, peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependyma cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in tissue arrays of normal mouse tissues. Importantly, podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles and by dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas (SCCs) studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in human archival tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis and its role in tumor progression. Immunofluorescence stainings were performed on 6-μm cryostat sections of neonatal human foreskin or on 6-μm paraffin sections of human malignant melanoma as described previously,6Dadras SS Paul T Bertoncini J Brown LF Muzikansky A Jackson DG Ellwanger U Garbe C Mihm MC Detmar M Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.Am J Pathol. 2003; 162: 1951-1960Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 10Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar using the mouse monoclonal antibody D2-40 (Signet, Dedham, MA), rabbit polyclonal antibodies against the lymphatic markers LYVE-17Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar and Prox125Karkkainen MJ Haiko P Sainio K Partanen J Taipale J Petrova TV Jeltsch M Jackson DG Talikka M Rauvala H Betsholtz C Alitalo K Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.Nat Immunol. 2004; 5: 74-80Crossref PubMed Scopus (1089) Google Scholar (kindly provided by Dr. K. Alitalo, University of Helsinki, Helsinki, Finland), CD34, CD31 (BD Pharmingen, San Diego, CA), and corresponding secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR). Nuclei were counterstained with 20 μg/ml of Hoechst bisbenzimide (Molecular Probes). Additional immunohistochemical stains were performed on tissue arrays of normal mouse (MaxArray mouse tissue microarray slides; Zymed, San Francisco, CA) and human tissues (MaxArray human normal tissue microarray slides, Zymed), human skin tumors (IMH-323; Imgenex, San Diego, CA) and ovary tumors (IMH-347, Imgenex) as described previously.6Dadras SS Paul T Bertoncini J Brown LF Muzikansky A Jackson DG Ellwanger U Garbe C Mihm MC Detmar M Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.Am J Pathol. 2003; 162: 1951-1960Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar Briefly, the primary antibodies D2-40 or LYVE-1 were applied, followed by incubation with conjugated anti-mouse or anti-rabbit immunoglobulin using the 3-amino-9-ethylcabazole peroxidase kit (Vector Laboratories, Burlingame, CA). The D2-40 antibody only stains human tissues, but not mouse tissues. For mouse tissues, the hamster monoclonal antibody 8.1.1 (Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA) was used. We have previously shown that this antibody, originally raised against a mouse gp38 antigen,19Farr AG Berry ML Kim A Nelson AJ Welch MP Aruffo A Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues.J Exp Med. 1992; 176: 1477-1482Crossref PubMed Scopus (144) Google Scholar specifically recognizes podoplanin expressed by lymphatic vessels in wild-type mice, but not in podoplanin-deficient mice.23Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (569) Google Scholar The 8.1.1 antibody does not recognize human podoplanin. Sections were examined by using a Nikon E-600 microscope (Nikon, Melville, NY) and images were captured with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Immortalized rat L6 myoblasts26Kanai M Goke M Tsunekawa S Podolsky DK Signal transduction pathway of human fibroblast growth factor receptor 3. Identification of a novel 66-kDa phosphoprotein.J Biol Chem. 1997; 272: 6621-6628Crossref PubMed Scopus (124) Google Scholar were maintained in Dulbecco's modified Eagle's medium that contained 10% fetal bovine serum, 2 mmol/L l-glutamine, and antibiotics (Life Science, Grand Island, NY). The human podoplanin coding sequence was cloned into the pCMV6-XL5 vector (Origene, Rockville, MD). Rat myoblasts were transfected either with the full-length human podoplanin cDNA or with the pCMV6-XL5 vector alone using the SuperFect transfection reagent (Qiagen, Chatsworth, CA). Specific binding of D2-40 antibody to human podoplanin was investigated by immunofluorescence staining of the transiently transfected cells after paraformaldehyde fixation (Fluka, Buchs, Germany). For the amplification of full length human podoplanin, RNA was isolated from cultured human dermal microvascular endothelial cells (Promocell, Heidelberg, Germany) using a Qiagen RNeasy kit, according to the manufacturer's instructions (Qiagen, Valencia, CA). First strand synthesis was performed by oligo-dT priming using 3 μg of total RNA. The podoplanin coding sequence was amplified from 2 μl of this product by polymerase chain reaction with the primers hPodo156FHindIII (GTCAGCAGGAAGCTTCCAGGAGAGCAACAACTCAAC) and hPodo570RBamHI (TCGGCTCCGGATCCACTGTTGACAAACCATCTTTCTC)using Pfu DNA polymerase (Stratagene, La Jolla, CA). After digestion with HindIII and BamHI, the product was cloned into the HindIII/BamHI-digested IgFc vector pCDM7Ig,27Prevo R Banerji S Ferguson DJ Clasper S Jackson DG Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.J Biol Chem. 2001; 276: 19420-19430Crossref PubMed Scopus (415) Google Scholar to yield a construct encoding podoplanin fused at the COOH terminus to the Fc region of human IgG1.7Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar For expression and purification of the podoplanin-Fc fusion protein, the expression vector was transfected into human 293T cells using the calcium phosphate method. Transfectants were grown in serum-free UltraCHO medium (Bio-Whittaker, Walkersville, MD) for 3 days before harvesting culture supernatants. The fusion protein was purified by affinity chromatography on a column of 1 ml of protein A Sepharose (Sigma, St. Louis, MO). Fractions containing the fusion protein were neutralized and the purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of the mouse monoclonal antibody D2-40 to immobilized podoplanin-Fc fusion protein was tested in 96-well ELISA plates (Nalge Nunc, Rochester, NY). Plates were coated with 5 μg of either human podoplanin-Fc or human LYVE-1-Fc.7Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar After overnight incubation, wells were washed and blocked using 1% bovine serum albumin (Sigma) and 0.05% Tween (Sigma). D2-40 antibody was applied at a series of dilutions from 1:50 to 1:50,000, each in triplicates and incubated for 1 hour. Unbound antibody was removed by washing with phosphate-buffered saline; bound antibody was detected by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) followed by O-phenylenediamine substrate (Sigma), and the absorbance was measured at 490 nm in a microplate reader (Bio-Rad, Hercules, CA). The following small inhibitory RNA (siRNA) oligonucleotides were synthesized by Dharmacon (Lafayette, CO): R1, 5′-GCGAAGACCGCUAUAAGUCdTdT-3′ and R2, 5′-AAGAUGGUUUGUCAACAGUdTdT-3′.23Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (569) Google Scholar Primary human LECs10Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar were transfected or not with siRNA oligonucleotides (500 nmol) or with equimolar concentrations of control plasmid vector by using the Nucleofector kit (Amaxa, Cologne, Germany) according to the manufacturer's instructions. Cells were harvested at 4 days after transfection. For Western analyses, cell lysates were obtained as described11Hong YK Harvey N Noh YH Schacht V Hirakawa S Detmar M Oliver G Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.Dev Dyn. 2002; 225: 351-357Crossref PubMed Scopus (442) Google Scholar and 30 ng of protein per sample were immunoblotted with the D2-40 antibody. For detection of podoplanin-Fc fusion protein, 200 ng of human podoplanin-Fc and human LYVE-1-Fc7Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar as a control were electrophoresed on a polyacrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were incubated with D2-40 (0.5 μg/ml) and were developed by using a chemiluminescent detection kit (Pierce). Immunofluorescence staining of rat myoblasts that were transiently transfected with a human podoplanin overexpression vector, with the monoclonal antibody D2-40 revealed strong cytoplasmic labeling of podoplanin-transfected cells (Figure 1B) whereas control vector-transfected cells were unstained (Figure 1A). The D2-40 antibody specifically detected a human podoplanin-Fc fusion protein, but not a LYVE-1 fusion protein, as assessed by Western blotting (Figure 1C). ELISAs further demonstrated specific binding of the D2-40 antibody to the immobilized human podoplanin-Fc fusion protein, whereas we only found low nonspecific binding to the human LYVE-1-Fc fusion protein (Figure 1D) that also contains glycosylated sialoglycoproteins with O-linked carbohydrate structures. Because the D2-40 antibody recognizes human but not mouse podoplanin, its specificity could not be further tested in lymphatic cells obtained from podoplanin-deficient mice that we previously described.23Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (569) Google Scholar Therefore, we studied siRNA-mediated knock-down of podoplanin in human LECs. We found that siRNA-mediated podoplanin knockdown resulted in reduced podoplanin detection by D2-40, by 66% and 36% (Figure 1E), as compared with control LECs. Immunofluorescence double stains of normal human skin with the D2-40 antibody and with antibodies against the lymphatic-specific hyaluronan receptor LYVE-17Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar or the lymphatic homeobox protein Prox128Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (748) Google Scholar revealed complete overlap of immunoreactivity, confirming specific podoplanin expression by lymphatics, but not by blood vessels (Figure 2; A to F). Double immunostains with D2-40 and with an antibody against the blood vascular-specific marker CD3410Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar further demonstrated mutually exclusive expression of podoplanin and CD34 by cutaneous lymphatic vessels and blood vessels, respectively (Figure 2; G to I). Occasionally, focal expression of podoplanin was also detected on basal epidermal keratinocytes (Figure 2A). We next studied whether podoplanin might also serve as a specific marker for lymphatic vessels in other human organs, in addition to the skin, and whether other cell types might also express podoplanin, using human multiple tissue arrays. In all human organs examined, podoplanin was detected on LECs that also expressed the lymphatic-specific hyaluronan receptor LYVE-1 (see below and data not shown). Moreover, basal epithelial keratinocytes of the skin, cervix, and esophagus also showed focal podoplanin expression (Figure 3; A to C). Surprisingly, strong podoplanin expression was detected on myoepithelial cells of the breast glands and of salivary glands, as well as on myofibroblasts of the prostate (Figure 3; D to F). Moreover, podoplanin-positive stromal reticular cells and follicular dendritic cells were found in the follicular germinal centers of lymphoid organs, including the thymus, tonsils, and lymph nodes (Figure 3; G to I). Staining of serial sections for the follicular dendritic cell marker CD21 (Figure 3M) and for podoplanin (Figure 3N) confirmed that CD21-positive follicular dendritic cells express podoplanin. In the lung, podoplanin was expressed by alveolar type I cells (Figure 3J), as well as by LECs that were also stained in the colon (Figure 3K) and the ovary (Figure 3L). In contrast, blood vascular endothelial cells were negative for podoplanin. All organs examined, except for the central nervous system, showed strong labeling of LECs with the anti-podoplanin antibody 8.1.1 (Figure 4, A to L; Table 1), as confirmed by staining of serial sections for LYVE-1 (data not shown). Myoepithelial cells of the salivary glands and fibromyocytes of the testis (Figure 4, C and F) also expressed podoplanin. In the central nervous system, podoplanin was expressed by ependymal cells lining the ventricles (Figure 4D), by choroid plexus cells (Figure 4D), and by meningeal cells (Figure 4I). In the peripheral nervous system, perineural cells expressed podoplanin, as detected in spinal nerve roots (Figure 4E) and in peripheral nerves of the skin, the tongue, and the skeletal muscle (data not shown). In agreement with the findings in human tissues, stromal reticular cells and dendritic reticular cells of the follicular germinal centers in the spleen and in lymph nodes (Figure 4, G and H), as well as osteocytes (Figure 4I) and alveolar type I cells (Figure 4J) also expressed podoplanin. Remarkably, the germinal epithelium and granulosa cells of primary and secondary ovarian follicles showed strong podoplanin expression (Figure 4L), whereas podoplanin was only weakly expressed by tertiary follicles and was absent from corpora lutea (for summary of results obtained in human and murine tissues, see Table 1). No major differences of staining patterns were observed between human and mouse tissues.Table 1Expression of Podoplanin in Normal Tissues (Combined Results of Human and Murine Tissues)OrganCell typeSkinFocal expression in basal keratinocytes; lymphatic endotheliumLungType I alveolar cells; lymphatic endothelium; pleuraKidneyGlomerular podocytes; parietal epithelial cells of Bowman's capsule; lymphatic endotheliumLiverBile ducts; lymphatic endothelium; peritoneumEsophagusBasal keratinocytes; lymphatic endotheliumIntestineLymphatic endothelium; peritoneumCentral nervous systemChoroid plexus; ependyma; meningesPeripheral nervous systemPerineurium; lymp
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