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JDSR-60; No. of Pages 8 Japanese Dental Science Review (2010) xxx, xxxxxx

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Review article

New trends in the study of podoplanin as a cell morphological regulator

Yoshihiko Sawa *
Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-Ku, Fukuoka 814-0193, Japan Received 7 August 2009; received in revised form 26 November 2009; accepted 13 January 2010

KEYWORDS
Podoplanin; Cancer

Abstract Podoplanin is a mucin-type glycoprotein rstly identied in podocytes, which is homologous to the type I alveolar cell specic T1a-2 antigen and to the oncofetal antigen M2A recognized by the D2-40 antibody. Podoplanin possesses a platelet aggregation-stimulating domain causes the platelet aggregation on cancer cells by the binding activity to CLEC-2. Podoplanin also contributes to the formation of membrane-actin structures. The increased podoplanin expression is found in squamous cell carcinomas at the invasive edge. It has been reported that the podoplanin induces an actin cytoskeleton rearrangement dependent on the RhoA GTPase activation to phosphorylate ezrin and facilitates an epithelial-mesenchymal transition (EMT) which induces the single cell migration of cancer cells. However, the podoplaninexpressing cancer cells often express E-cadherin and migrate in a collective manner, suggesting that there are podoplanin-induced alternative pathways for the actin cytoskeleton rearrangement independent of the RhoA activation and EMT. The strong expression of podoplanin is present in salivary gland myoepithelial cells, and in enamel epithelia and odontoblasts of the tooth germ for a bell stage. Podoplanin may act as a cell morphological regulator in normal and cancer cells. # 2010 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved.

Contents 1. Expression of podoplanin in the human somatic tissue . . . 1.1. Roles of podoplanin as a podocyte antigen . . . . . . . 1.2. Lymphatic endothelial cell marker, podoplanin. . . . 1.3. Podoplanin expression in the oral and other tissues Expression of podoplanin in cancer . . . . . . . . . . . . . . . . . Molecular characteristics of podoplanin . . . . . . . . . . . . . . 3.1. Transcription of podoplanin gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000 000 000 000 000 000 000

2. 3.

* Tel.: +81 92 801 0411; fax: +81 92 801 4909. E-mail address: ysawa@college.fdc.net.ac.jp. 1882-7616/$ see front matter # 2010 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved. doi:10.1016/j.jdsr.2010.01.003

Please cite this article in press as: Sawa Y. New trends in the study of podoplanin as a cell morphological regulator. Japanese Dental Science Review (2010), doi:10.1016/j.jdsr.2010.01.003

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Y. Sawa 3.2. Molecular biological function of podoplanin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Novel aspects on the event podoplanin induces in cancer cells: EMT or not?. . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000 000 000

1. Expression of podoplanin in the human somatic tissue

1.1. Roles of podoplanin as a podocyte antigen
Podoplanin, also called as E11 antigen/GP36/aggrus, is a mucin-type 38-kDa transmembrane glycoprotein rstly identied as a 43-kd protein of the kidney glomerular epithelial cells, podocytes [1]. Podoplanin is homologous to T1a-2 gene which encodes the type I alveolar cell specic antigen and to the oncofetal antigen M2A recognized by the D2-40 antibody [24]. The role of podoplanin has been studied in a rat model of nephropathy by the puromycin aminonucleoside nephrosis with severe proteinuria. In the kidney with nephropathy, podocyte foot processes are extensive attening and podoplanin is selectively reduced to <30%. It has been thought that podoplanin plays a role in maintaining the shape of podocyte foot processes and glomerular permeability because of morphological alterations of cell shape with selective loss of podoplanin in the nephrosis accompanies proteinuria [1,57].

regulated by IL-3 which expressed in lymphatic but not blood endothelium. The endogenous IL-3 is required to maintain the lymphatic endothelial cell phenotype and appears to induce transdifferentiation of blood endothelium into lymphatic endothelium [16]. Furthermore, recent study showed that the early activation of a Ras homolog gene family, member A (RhoA)-GTPase in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression [18]. In the study for the response of lymphatic vascular systems against the infection, we have been reported that lymphatic endothelium signicantly increases podoplanin expression with lipopolysaccharide and lipoteichoic acid by the recognition mechanisms to pathogen-associated molecular patterns through toll-like receptors [1921].

1.3. Podoplanin expression in the oral and other tissues

In the oral tissue the expression of podoplanin is weakly found in mucous epithelial cells at a basal layer and strongly in myoepithelial cells of salivary glands [22] (Fig. 1). The podoplanin expression is rarely found in acini of the parotid gland but clearly found at the basal portion of acini in the submandibular and sublingual glands. The strong expression of podoplanin is also found at the basal portion of the intercalated duct, striated duct and interlobular duct in all major salivary glands. In the parotid gland few myoepithelial cells are on the terminal portion while in the sublingual gland and submandibular gland, approximately 50% and 25% of the terminal portion surface are covered by myoepithelial cells with a number of broad and extensive cellular processes. Myoepithelial cells act as a contractile cell for the saliva secretion and the cell shape varies considerably with the different glands in relation to the physical properties of secretions [23]. Podoplanin would be a marker protein of salivary gland myoepithelial cells because the podoplanin detection levels coincide with the distribution and the cell shape of myoepithelial cells specic for major salivary glands. Podoplanin may play a role in maintaining the shape of myoepithelial cell foot processes to press acinar cells from the outside. The podoplanin expression is also found in the tooth germ tissue [24] (Fig. 2). The expression is strongly found in inner and outer enamel epithelia at the early bell stage while rarely in the enamel organ at the crown stage. Furthermore, the podoplanin expression is rarely found in the mesenchymal cells of dental papilla except for odontoblasts. The strong expression of podoplanin in odontoblasts is sustained during a dentin formative period and diminished at the root formation stage. It has been reported that podoplanin promotes the plasma membrane extension and the actin cytoskeleton rearrangement involved in cell mobility [2527]. Podoplanin may be involved in the cell shape formation of odontoblasts and in the migration of epithelial cells into mesenchymal cells like cancer cells.

1.2. Lymphatic endothelial cell marker, podoplanin

The study for the lymphatic system at a molecular biology level made rapid progress greatly since 1999 when much breakthrough about lymphatic endothelial cell markers, such as desmoplakin, LYVE-1, Prox1, and podoplanin, has been accomplished [811]. The discovery most major in these would be that lymphatic development is independent of the vascular system development, and that lymphatic-specic homeobox gene Prox1 induces the lymphatic endothelial development from embryonic veins and homozygous Prox1-null mouse embryos die because of multiple developmental defects [1115]. Prox1 is a master regulator of the podoplanin expression induced with epidermal growth factor, basic broblast growth factor, tumor necrosis factor alpha, interleukin-3, and transforming growth factor beta. Prox1 enables to reprogram vascular endothelial cells to become podoplanin-expressing lymphatic endothelial cells [1316]. It has been rstly reported that podoplanin is predominantly expressed by lymphatic endothelium in angiosarcomas mixed endothelial phenotypes of blood and lymphatic capillaries [8]. Podoplanin is rst expressed at around E11.0 in Prox1-positive lymphatic progenitor cells [10]. Podoplanin(/) mice die at birth because of the respiratory failure and have defects in lymphatic formation with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels, but not have defects in blood vessel pattern formation. Podoplanin promotes lymphatic endothelial cell adhesion and regulates the lymphatic vasculature formation [10]. The immunoelectron microscopic study has demonstrated that podoplanin is expressed on the luminal side of lymphatic vessels and rarely on the basal side [17]. The podoplanin and Prox1 expression is

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Podoplanin as a morphological regulator

Figure 1 Podoplanin expression in the mouse oral tissues. (A and D) Serial sections of the tongue tissue. Reaction products of antipodoplanin with green uorescence are found in the mucosal epithelial cells at a basal layer (asterisk), myoepithelial cells of lingual glands (arrowheads), and lymphatic vessels (arrow). (B and E) Serial sections of parotid gland. Podoplanin expression is rarely found in the terminal secretory end pieces of acini composed of serous pyramidal cells but clearly found in the striated and intercalated ducts (SD, IC) composed of columnar cells with acidopillic cytoplasm. (C and F) Serial sections of sublingual gland. Podoplanin expression is clearly found in terminal secretory end pieces composed of mucous-lled acinar cells and in the intercalated ducts (IC) having a narrow lumen surrounded by cuboidal cells. Bar, 0.1 mm.

Figure 2 Podoplanin expression in the mouse tooth germ tissue. (A and D) Serial sections of the molar tooth germ at the bell stage. Reaction products of anti-podoplanin with green uorescence are observed in the cervical loops where the outer and inner enamel epithelia join at apical sides of the tooth germ (arrowheads), and in the odontoblast layer (arrow). The podoplanin expression is partially weakly found in the enamel organ containing ameloblasts (asterisk) but not found in the mesenchymal cells of dental papilla except for odontoblasts. (B and E) Cervical loop. Higher magnication of (a). Reaction products with anti-podoplanin are observed strongly at plasma membrane of inner and outer enamel epithelia (arrowheads) and in odontoblasts (arrow). The podoplanin expression is not found in the mesenchymal cells of dental papilla (asterisk). (C and F) Odontoblast layer. Higher magnication of (b). Reaction products with antipodoplanin are observed strongly at plasma membrane in odontoblasts (arrow) but not in the mesenchymal cells of dental papilla. The podoplanin expression is weakly found in the enamel organ containing ameloblasts (asterisk). Bar, 0.1 mm.
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4 There has been also reports for the podoplanin expression in osteocytes and osteoblasts [28], mesothelial cells [4,29], epidermal basal layer cells [4,10], choroid plexus epithelial cells, thymus type I epithelial cells, myoepithelial cells, prostate myobroblasts, follicular dendritic cells, and immature cells like fetal germ cells and developing Sertoli cells [1,4,10,2934]. Elucidation of the common biological importance in these somatic tissues is required.

Y. Sawa showing that podoplanin expression in cancer-associated broblasts is associated with decreased colorectal carcinoma cell invasion [52]. Furthermore, over-expression of podoplanin in 66% of squamous cell carcinoma of the lung is reported [53]. The expression may reect the most immature status in the differentiation process of lung squamous cell carcinoma with lower biological aggressiveness because patients with podoplanin-positive tumors resulted in signicantly better survival than those with podoplanin-negative tumors [54]. It must be elucidated why the biological signicances of podoplanin expression in cancer and cancer-associated broblasts are directed to two contradictory prognosis factors.

2. Expression of podoplanin in cancer

Podoplanin up-regulation has been reported in squamous cell carcinomas of the skin, lung, esophagus, cervix, larynx and oral cavity whereas very low levels or a complete absence of podoplanin protein is found in cell lines and adenocarcinomas [35,36,33]. There are reports about the podoplanin expression directed to two different biological signicances for tumor aggressiveness. In generative organs, the expression of podoplanin as an immunohistochemical marker to cancer has been rstly reported for seminoma of which about 90% are podoplanin positive [3739]. Podoplanin is highly expressed in primary and metastatic seminoma. In testicular germ-cell tumors of young adults derived from a pre-invasive intratubular lesion, carcinoma in situ, podoplanin overexpresses in the tumors compared to the normal adult testis [11]. Podoplanin is also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors [4,11]. In brain tumors, podoplanin expression is correlated to malignancy and the expression extremely occurs in a high rate in pilocytic astrocytomas, glioblastomas, and germinomas. Podoplanin expression is markedly higher in glioblastomas than in anaplastic astrocytomas [4042]. It is also reported that podoplanin expression is strongly induced in epidermal squamous cell carcinomas because podoplanin is the fos target gene in skin carcinogenesis [43]. Podoplanin expression extremely occurs in a high rate in epithelioid mesotheliomas [4447]. In oral squamous cell carcinomas, podoplanin expression is present in about 50% tumors at the invasive front. The 60% of oral tongue cancers express high levels of podoplanin and the patients have a statistically signicantly higher rate of lymph node metastasis. Patients with lymph node metastasis and high-level podoplanin show the shortest disease-specic survival than other patients. Furthermore, podoplanin frequently expresses in oral leukoplakia which is a heterogeneous oral lesion with an increased oral cancer risk. These reports may suggest that podoplanin is involved in oral tumorigenesis and serves as a predictor for the risk for oral cancer development, lymph node metastasis and poor clinical outcome [4850]. It appears that podoplanin plays a tumor biological role in not only cancer cells but also cancer-associated broblasts. There are two different reports about signicances of podoplanin expression in cancer-associated broblasts. One suggests that podoplanin expression in cancer-associated broblasts is associated with poor prognosis for the lung adenocarcinoma [51]. The other suggests that podoplanin expression in stromal broblasts could have a protective role against colorectal carcinoma cell invasion and is a signicant indicator of a good prognosis in patients with advanced colorectal carcinoma. It is supported by biological analysis

3. Molecular characteristics of podoplanin

3.1. Transcription of podoplanin gene
Podoplanin consists of 162 amino acids with 9 amino acids of extracellular domain, and is regulated with the gene constructed by 34.2 kbp with 8 exons and four transcript variants [9]. It has been reported that epidermal growth factor, basic broblast growth factor and tumor necrosis factor could induce podoplanin expression in MCF7 breast cancer cells [25,33,55], and transforming growth factor-beta induces the expression in human brosarcoma [56]. Podoplanin transcription enhances in cells having a chromatin with strongly methylated CpG sites at the promoter region of podoplanin gene. Putative binding sites for transcription factor AP-2, AP4, C/EBP, and NF-1 exist in podoplanin promoter sequence [57]. The podoplanin gene 50 anking region lacks a consensus TATA box whereas a GATA box (GATAAA) is localized 31 nucleotides upstream of the main mRNA transcription initiation site. The podoplanin gene 50 -anking region has a high GC content and revealed the existence of several potential Sp1 transcription factor sites. Sp1 and Sp3 occupy the same or overlapping sites of the podoplanin promoter because Sp1 and Sp3 are not bound coordinately to the DNA stretches. Over-expressions of Sp1 and Sp3 independently increase podoplanin transcription in human osteoblastic osteosarcoma cell line MG63 and osteosarcoma cell line Saos-2 which insufciently recruits nuclear Sp for transcriptional activation, and the promoter controlling podoplanin transcription in MG63 exhibits 30-fold more activity than in Saos-2. The MG63 cell has a chromatin with strongly methylated CpG sites at a 464 bp upstream of the distal promoter region of podoplanin gene but the same sites of Saos-2 cell chromatin are hypomethylated, and a treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A results in the transcriptional downregulation of podoplanin mRNA in MG63 cells. These suggest that the highly methylation of chromatin at the promoter region of podoplanin gene aids to the cell-type specic up-regulation of podoplanin gene transcriptional activity [57].

3.2. Molecular biological function of podoplanin

Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and is a high content of sialic acid [57,58]. About the molecular biological characterization of podoplanin there are detailed studies by Kato and coworkers [58,59]. The C-type lectin-like receptor CLEC-2

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Podoplanin as a morphological regulator (CLEC1B) is the receptor for podoplanin which mediates the platelet aggregation on cancer cells without blood coagulation factors and plasma components. Podoplanin consists of an extracellular domain having rich serine and threonine residues, a transmembrane domain, and a cytoplasmic tail for protein kinase C and cAMP phosphorylation [59,60]. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain (EDDVVTPG) in an extracellular domain. In the PLAG domain only the Thr52 is O-glycosylated with disialyl-core1 (NeuAc-a2-3Galb1-3 (NeuAca2-6) GalNAca1O-Thr) to induce platelet aggregation and the mutation of threonine residues in the PLAG domain reduces the platelet aggregation. The binding of podoplanin to the specic receptor CLEC-2 is dependent on sialic acid on O-glycans in the PLAG domain by the lectin activity and plays a key role for the platelet aggregation (Fig. 3). It is thought that the interaction between podoplanin and CLEC-2 is one of the factor regulates tumor invasion and metastasis because both the platelet aggregation and lung metastasis of mouse colon carcinoma are inhibited by 8F11 monoclonal antibody for podoplanin [53,5862]. There are three tandem repeats of the PLAG domain which are conserved in podoplanin homologues from rat, hamster, dog, bovine, human and mouse. The podoplanin homologues conserve the segment of EDxxVTPG in the extracellular domains critical for their platelet aggregation-inducing activities. Either the rst or last PLAG domain is critical for the platelet aggregationinducing activity. Bovine podoplanin has a sporadic deletion mutation in the rst PLAG domain and lacks platelet aggre-

5 gation-inducing activity [62,63]. The podoplanin is a main factor for platelet aggregation induced by the glioblastoma cell line LN319 and a NZ-1 antibody for the PLAG domain completely inhibits podoplanin-induced platelet aggregation because of the neutralization between podoplanin and CLEC2 [58,61]. In an experimental mouse model podoplanin promoted hematogenous metastasis to the lung and the antipodoplanin antibody NZ-1 suppressed both the podoplaninCELC-2 interaction and podoplanin-induced pulmonary metastasis [59,61,64,65]. Possibility of the clinical application on the neutralization of podoplanin-CLEC-2 interaction inducing hematogenous metastasis should be settled early.

3.3. Novel aspects on the event podoplanin induces in cancer cells: EMT or not?
Analysis of cancer cell migration and invasion mechanisms caused by podoplanin expression is directed to two contradictory reports although both reports accord about the role of podoplanin which mediates remodeling of the actin cytoskeleton. One suggests that podoplanin induces an actin cytoskeleton rearrangement by epithelial-mesenchymal transition (EMT) based on the binding activity with plasma membrane protein ERM: ezrin, radixin and moesin to activate RhoA [27]. The other suggests that podoplanin-induced migration and invasion could occur in the absence of EMT because of neither ERM up-regulation nor E-cadherin down-regulation in podoplanin-positive cancer [55]. In fact, some cancer cell migration and invasion depend on an active remodelling of the actin cytoskeleton linked with membrane proteins via ERM [66] (Fig. 1). Ezrin is a cytoplasmic linker protein which is expressed in epithelial cell surface structures like microvilli but not usually in mesenchymal cells [67]. Ezrin with the phosphorylated tyrosine (pTyr)-353 protects epithelial cells against apoptosis and determines cell survival by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway because the pTyr-353 residue binds to the regulatory subunit of PI3-kinase. Ezrin-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway and functions in the development of membraneactin structures critical for cell shape and motility [6870]. Podoplanin co-localizes with ERM at actin-rich microvilli and the podoplanin over-expression in MCF7 cells, MDCK cells, and HaCaT keratinocytes leads to a marked increase in phosphorylation of ezrin [25,27,48]. Podoplanin directly binds to ezrin at three basic residue RKR in the cytoplasmic tail amino acid RKMSGRYSP and up-regulates a Ras homolog gene family, member A (RhoA)-GTPase activity resulting in the ezrin phosphorylation by Rho-associated coiled coil-containing protein kinase 1 (ROCK1) [27]. The increased cellsurface distribution of phosphorylated ezrin promotes the actin cytoskeleton rearrangement by the development of membrane-actin structures because phosphorylated ezrin is a cytoplasmic linker protein intermediating the connection of membrane proteins at the NH2-terminal domain and Factin at the COOH-terminal domain. Therefore, podoplanin indirectly promotes an actin cytoskeleton rearrangement by the RhoA activation. Furthermore, podoplanin binds to phospholyrated ezrin at the cytoplasmic tail and directly promotes the stabilization of the phosphorylated ezrin mediating an anchorage of F-actin to the plasma membrane.

Figure 3 Podoplanin-induced actin cytoskeleton re-arrangement. Podoplanin induces the activation of a RAS homolog gene family, member A (RhoA) GTPase and a Rho-associated coiled coil-containing protein kinase 1 (ROCK1) to phosphorylate ezrin. Phospholyrated ezrin acts as a cytoplasmic linker protein, which intermediately connects F-actin to membrane proteins including podoplanin, and develops membrane-actin structures at the cellsurface. Podoplanin directly binds to phospholyrated ezrin at the cytoplasmic tail and indirectly promotes an actin cytoskeleton re-arrangement by stabilizing the phosphorylated ezrin mediating an anchorage of F-actin to the plasma membrane.

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6 Such a series of events in podoplanin-positive cancer cells may facilitate EMT which plays a role for the plasma membrane extension of epithelial cell surface structures like microvilli, and for the cancer cell migration and invasion [2527]. The cadherin switch is one of the EMT events in the transition from adenoma to carcinoma and some carcinomas correlate with EMT based on the down-regulation of the cell cell adhesion protein E-cadherin [71,72]. It is necessary to lose E-cadherin expression and express N-cadherin instead at the progression from adenoma to carcinoma in the wellstudied multistep tumor progression model of the Rip1Tag2 mouse pancreatic beta-cell carcinogenesis [73]. However, the forced podoplanin expression in beta-cell tumors of Rip1Tag2 mice induces the carcinoma formation without EMT and no ERM up-regulation occurs in podoplanin-positive cancer [55]. Furthermore, the podoplanin-expressing cancer cells often express E-cadherin even in advanced stages and the cancer cells tend to migrate in a collective manner. It seems that podoplanin could induce an actin cytoskeleton rearrangement independent of the RhoA activation and promote the invasion activity without a cadherin switch [55]. In squamous cell carcinoma, the podoplanin expression is frequently restricted to the invasive front and in stem cell marker CD44 and p63 positive tumor cells less than 50% simultaneously expresses podoplanin. Podoplanin-positive cells are localized more peripherally in the tumor nests than CD44 and p63 positive cells, showing that tumor cells are aligned in the hierarchical distribution pattern. Patients having podoplanin-positive tumors with the hierarchical pattern resulted in signicantly better survival than those having podoplanin-negative tumors. The three hierarchical expressions of podoplanin, CD44 and p63 in the squamous cell carcinoma may reect the most immature status of tumor with lower biological aggressiveness [54]. The human squamous cell carcinoma cell line A431 contains podoplaninpositive cells which generate podoplanin positive and negative cells [74]. Podoplanin-negative cells rarely generate podoplanin-positive cells. Furthermore, podoplanin positive A431 cells share sonic hedgehog and CD44 expression with stem cells in normal squamous epithelium. These studies may suggest the role of podoplanin as a candidate of tumorinitiating cell marker in squamous cell carcinoma. The elucidation about tumor biological signicance of podoplanin should be dependent on the kinetics with modular proteins to podoplanin.

Y. Sawa
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Please cite this article in press as: Sawa Y. New trends in the study of podoplanin as a cell morphological regulator. Japanese Dental Science Review (2010), doi:10.1016/j.jdsr.2010.01.003