Podoplanin associates with CD44 to promote directional cell migration

This article is under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.-- et al.Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.This work was supported by grant SAF2007-63821 from the Spanish Ministry of Science and Innovation (to M.Q.), the Royal Society University Research Fellowship (to M.P.), Medical Research Council (to G.E.J.) EU FP7 T3Net Consortium (GEJ), and Cancer Research UK (to G.E.J. and E.M.V.).Peer reviewe

Regulation of podoplanin/PA2.26 antigen expression in tumour cells. Involvement of calpain-mediated proteolysis

El pdf del articulo es el manuscrito de autor.Podoplanin/PA2.26 antigen is a small transmembrane mucin expressed in different types of cancer where it is associated with increased cell migration, invasiveness and metastasis. Little is known about the mechanisms that control podoplanin expression. Here, we show that podoplanin synthesis can be controlled at different levels. We analyzed podoplanin expression in a wide panel of tumour cell lines. The podoplanin gene (PDPN) is transcribed in cells derived from sarcomas, embryonal carcinomas, squamous cell carcinomas and endometrial tumours, while cell lines derived from colon, pancreatic, ovarian and ductal breast carcinomas do not express PDPN transcripts. PDPN is expressed as two mRNAs of ∼2.7 and ∼0.9 kb, both of which contain the coding sequence and arise by alternative polyadenylation. Strikingly, in most of the cell lines where PDPN transcripts were found, no podoplanin or only very low levels of the protein could be detected in Western blot. Treatment of several of these cell lines with the calpain inhibitor calpeptin resulted in podoplanin accumulation, whereas lactacystin, a specific inhibitor of the proteasome, had no effect. In vitro experiments showed that podoplanin is a substrate of calpain-1. These results indicate that at least in some tumour cells absence or reduced podoplanin protein levels are due to post-translational calpain-mediated proteolysis. We also report in this article the identification of a novel podoplanin isoform that originates by alternative splicing and differs from the standard form in lacking two cytoplasmic residues (YS). YS dipeptide is highly conserved across species, suggesting that it might be functionally relevant. © 2008 Elsevier Ltd. All rights reserved.This work was supported by grant SAF2007-63821 from the Spanish Ministry of Education and Science.Peer Reviewe

Podoplanin binds ERM proteins to activate RhoA and promote epithelial-mesenchymal transition

13 pages, 8 figures, 1 table.Podoplanin is a small membrane mucin expressed in tumors associated with malignant progression. It is enriched at cell-surface protrusions where it colocalizes with members of the ERM (ezrin, radixin, moesin) protein family. Here, we found that human podoplanin directly interacts with ezrin (and moesin) in vitro and in vivo through a cluster of basic amino acids within its cytoplasmic tail, mainly through a juxtamembrane dipeptide RK. Podoplanin induced an epithelial-mesenchymal transition in MDCK cells linked to the activation of RhoA and increased cell migration and invasiveness. Fluorescence time-lapse video observations in migrating cells indicate that podoplanin might be involved in ruffling activity as well as in retractive processes. By using mutant podoplanin constructs fused to green fluorescent protein we show that association of the cytoplasmic tail with ERM proteins is required for upregulation of RhoA activity and epithelial-mesenchymal transition. Furthermore, expression of either a dominant-negative truncated variant of ezrin or a dominant-negative mutant form of RhoA blocked podoplanin-induced RhoA activation and epithelial-mesenchymal transition. These results provide a mechanistic basis to understand the role of podoplanin in cell migration or invasiveness.This work was supported by grants: SAF2004-04902 from the Ministry of Education and Science (MEC), GR/SAL/0871/2004 from the Autonomous Community of Madrid (CAM) and RTICCC CO3/10 from the ‘Instituto de Salud Carlos III’ (FIS) of Spain. E.M.-V. and M.M.Y. were the recipients of a postgraduate I3P fellowship from the Spanish Research Council (CSIC) and an MEC predoctoral fellowship, respectively.Peer reviewe

Podoplanin is a substrate of presenilin-1/γ-secretase

Podoplanin (PDPN) is a mucin-like transmembrane glycoprotein that plays an important role in development and cancer. Here, we provide evidence that the intracellular domain (ICD) of podoplanin is released into the cytosol following a sequential proteolytic processing by a metalloprotease and γ-secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP, 50-63 kDa) constitutively express two C-terminal fragments (CTFs): a ∼33 kDa membrane-bound PCTF33, and a ∼29 kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with γ-secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by γ-secretase depends on presenilin-1 (PS1) function: cells expressing a dominant negative form of PS1 (PS1 D385N), and mouse embryonic fibroblasts (MEFs) genetically deficient in PS1, but not in PS2, show higher levels of PCTF33 expression with respect to wild-type MEFs. Furthermore, transfection of PS1 deficient MEFs with wild-type PS1 (PS1 wt) decreased PCTF33 levels. N-terminal amino acid sequencing of the affinity purified PICD revealed that the γ-secretase cleavage site was located between valines 150 and 151, but these residues are not critical for proteolysis. We found that podoplanin CTFs are also generated in cells expressing podoplanin mutants harboring heterologous transmembrane regions. Taken together, these results indicate that podoplanin is a novel substrate for PS1/γ-secretase. © 2013 Elsevier Ltd. All rights reserved.This work was supported by grants SAF2010-19152 from the Spanish Ministry of Economy and Competitiveness and S2010/BMD-2359 (Skin Model-CM) from the Community of Madrid. M.M.Y. and G.d.C. were funded during part of this work by the Formación de Personal Investigador and Juan de la Cierva programs, respectively. EMV is funded by the Spanish Association for Cancer Research Foundation (AECC).Peer Reviewe

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.This work was supported by grants SAF2007-63821 and SAF2010-19152 from the Spanish Ministry of Science and Innovation. The support of Cancer Research UK to EM-V is also acknowledged. BF-M was the recipient of a predoctoral fellowship from the CSIC.Peer reviewe

Characterization of human PA2.26 antigen (T1α-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas

El pdf del artículo es la versión post-print.We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1α-2, podoplanin), a small mucin-type trans-membrane glycoprotein originally identified as a cell-surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E-cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell-surface protrusions and reduced Ca2+-dependent cell-cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion. © 2004 Wiley-Liss, Inc.Funded by: Fondo de Investigaciones Sanitarias. Grant Numbers: FIS-01/1125, FIS-02/1025; Ministerio de Ciencia y Tecnología. Grant Number: SAF2001-2361; FIS and Fundación Carolina.Peer Reviewe

New insights into the role of podoplanin in epithelial–mesenchymal transition

Podoplanin is a small mucin-like transmembrane protein expressed in several adult tissues and with an important role during embryogenesis. It is needed for the proper development of kidneys and lungs as well as accurate formation of the lymphatic vascular system. In addition, it is involved in the physiology of the immune system. A wide variety of tumors express podoplanin, both in the malignant cells and in the stroma. Although there are exceptions, the presence of podoplanin results in poor prognosis. The main consequence of forced podoplanin expression in established and tumor-derived cell lines is an increase in cell migration and, eventually, the triggering of an epithelial–mesenchymal transition, whereby cells acquire a fibroblastoid phenotype and increased motility. We will examine the current status of the role of podoplanin in the induction of epithelial–mesenchymal transition as well as the different interactions that lead to this program.Work in our laboratory is financed by grants SAF2013-46183-R from the Ministery of Economy and Competitiveness (MINECO), and S2010/BMD-2359 from the Comunidad Autónoma de Madrid. PC-R holds a predoctoral contract from the Ministery of Economy and Competitiveness. EM-V is the recipient of a postdoctoral research contract from the Scientific Foundation of Asociación Española Contra el Cáncer (AECC). MMY was supported by a contract from the Ministery of Economy and Competitiveness.Peer reviewe

Prolonged lenalidomide maintenance therapy improves the depth of response in multiple myeloma.

Lenalidomide is an immunomodulatory drug approved for maintenance treatment in newly diagnosed multiple myeloma, and it has been shown to improve progression-free survival (PFS) and, in several studies, overall survival. Nevertheless, the impact of prolonged treatment with lenalidomide on the kinetics of minimal residual disease (MRD) and its prognostic impact have not been studied in depth. To obtain better knowledge in this regard, we retrospectively analyzed 139 patients who received lenalidomide maintenance in real-world clinical practice and whose MRD levels were observed during the treatment period by multiparametric flow cytometry or next-generation sequencing with a sensitivity of at least 10-4. Lenalidomide maintenance correlated with an increased depth of the disease response, with 38.1% of patients achieving maximal response during maintenance. Moreover, 34.3% of patients who were MRD positive after induction treatment achieved MRD-negative status during maintenance and ultimately had improved PFS. Sequential MRD assessments identified patients with progressively decreasing MRD levels who also had better PFS outcomes, compared with patients not showing a decreasing pattern of MRD. These results support the role of maintenance therapy, not only to sustain, but also to increase the depth of disease response with a PFS benefit. In addition, MRD monitoring during maintenance identifies patients with better prognosis and may help in their clinical management

Unión Ibero-Americana, Año XXIX, Núm. 10

171 páginasTEXTO.- La Fiesta de la Raza en la Unión Ibero-Americana.- Carta del Ecmo. Sr. Presidente de la República Argentina, Sr. Duque de Ripalda.- Discurso pronunciado el día 12 de octubre por el Excmo. Sr. Marqués de Lema, Ministro de Estado.- Profecía, por E. Castelar.- El descubrimiento de América, por Salvador Falla.- Cable del Ecuador.- La Fiesta de la Raza, por J. de D. Méndez y Mendoza.- Carta del Excmo. Sr. Presidente de la República del Uruguay.- Nuestro Presidente.- De la Geográfica mexicana.- Honrando a España en tierra de Guatemala, por J. Francisco López Escobar.- Desde Chile, por Marcial Martínez.- Discurso pronunciado por el Excmo. Sr. D. Ra fael Conde y Luque.- Oración.- Carta del Excmo. Sr. Presidente de la República del Salvador.- Trébol de gloria (poesía), por Alfredo Gómez Jaime.- La Fiesta de la Raza, por Carlos R. Tobar.- 12 octubre de 1492, por Felipe Tejera.- Discurso del Excmo. Sr D. Juan Antonio Cavestany.- Carta de la Presidencia de la República de Cuba, por Rafael Montoro y Valdés.- La Fiesta de la Raza y la Prensa.- De El Social (semanario bonaerense).- Gloria a España (poesía), por Víctor M. Rendón.- Discurso del Excmo. Sr D. Luis Palomo. Enseña que tremoló Cristóbal Colón el 12 de octubre de 1492, por A. P. G.- Epitalamio (poesía), por Amado Nervo.- Carta del Excmo. Sr. Ministro de Instrucción Pública de Bolivia.- El por qué de la Fiesta de la Raza, por el Excelentísimo Sr. D. Manuel de Saralegui y Medina.- Auto episcopal.- A España (poesía), por Enrique Teenzier.- Carta del Excmo. Sr. Ministro de Instrucción Pública de Panamá.- De una culta Profesora costarricense (Angela Baldares).- Las carabelas de Colón, por Juan J. Canas.- Discurso pronunciado por el Excmo. Sr. D. Rafael María de Labra y Cadrana.- Del Secretario de la Academia mexicana de la Lengua (José López-Portillo y Rojas).- Los conquistadores, por J. Barrio y Bravo.- Sutchi-Quezzali (poesía), por Francisco Gavidia.- Carta del Excmo. Sr. Ministro de Relaciones Exteriores e Instrucción pública, Nicaragua.- A la madre patria España, en el aniversario del descubrimiento de América, por Aquiles B. Oribe.- De El Liberal (de Barcelona).- Carta del Excmo. Sr. Presidente de la República de Venezuela.- Fiesta de la Raza: Memoria leída por el Excelentísimo Sr. D. Luis de Armiñán, Secretario general de la Unión Ibero-Americana, en la sesión celebrada el 12 de octubre de 1915.- Carta del Excmo. Sr. Presidente de la República del Perú.- De La Época (Decano de la prensa rnadrileña).- La reespañolización de América, por P. M. Vélez.- El festival de la Unión Ibero-Americana, por Bartolomé Tavera Acosta.- Pensando en el porvenir, por Emilio Gutiérrez de Quintanilla.- De España y América (revista ilustrada de Cádiz).- Carta del Excmo. Sr. Presidente de la República Dominicana.- Justicia a la raza hispana, por Felipe Yurrita.- América, por Carlos Guido Spano.- Ofrenda al Día de la Raza, por Juan Rodríguez López.- Carta del Excmo. Sr. Presidente de la República de Guatemala.- Un mundo por una frase, por Francisco Tosta García.- El Clero español y la Fiesta de la Raza: Carta del Ilmo. Sr. Obispo de Vich.- De El Porvenir (diario de Cartagena).- Carta del Sr. Rector de la Universidad de Santa Fe.- Algo más que líricos, por Andrés Pando.- EI Quijote del próximo Centenario.- Preparativos para la Fiesta de la Raza en América.- Epopeya de gloria: Al inmortal Colón, por Rafael Abellán.- La fiesta de la Raza en las provincias españolas. GRABADOS.- Cristóbal Colón. (Reproducción fiel, hecha por Galván, de la tabla al óleo conservada en la Biblioteca Nacional (Madrid).- Presidencia de la sesión celebrada en la Unión Ibero-Americana para solemnizar la Fiesta de la Raza el 12 de octubre.- Excmo. Sr. Duque de Ripalda, Marqués de Lema, Ministro de Estado.- Excmo. Sr. D. Faustino Rodríguez San Pedro, Senador, ex Ministro, Presidente de la Unión ibero-Americana.- Busto de Isabel la Católica: Monumento inaugurado en Guatemala, en conmemoración de la Fiesta de la Raza, el día 12 de octubre.- Excmo. Sr. D. Rafael Conde y Luque, Rector de la Universidad Central, Presidente de la Comisión Ejecutiva de la Unión ibero-Americana. Excmo. Sr. D. Juan Antonio Cavestany, Senador de la Real Academia Española.- Excmo. Sr. D. Luis Palomo, Senador, Presidente de la Comisión Permanente de Enseñanza de la Unión Ibero-Americana.- Excmo. Sr. D. Manuel de Saralegui, de la Real Academia Española, Director de la Revista de la Unión Ibero-Americana.- Excmo. Sr. D. Rafael María de Labra, Senador. Presidente del Ateneo de Madrid.- Excmo. Sr. D. Luis de Armiñán. Diputado a Cortes, Secretario general de la Unión Ibero-Americana.- Retrato de Miguel de Cervantes Saavedra.- Idem de D. Andrés Bello.- Granada.- La manifestación pasando frente a la estatua de Isabel la Católica.- Entrada en el Ayuntamiento de la procesión cívica.- Santander: La manifestación cívica a su paso por la Ribera.- Alicante: El Alcalde, acampañado de los Concejales, Claustro de Profesores y Comisiones en la entrega de un manifiesto al Gobernador.- Idem: Procesión cívica.- Vigo: La procesión cívica de la Fiesta de la Raza.- Logroño: La Comisión organizadora de la Fiesta de la Raza en aquella capital.- Los exploradores en la Fiesta de la Raza el 12 de octubre.- Acto inaugural del nuevo domicilio de la Casa de América, de Barcelona, celebrado el 12 de octubre