Podoplanin (PDPN) is a mucin-type transmembrane protein (36–43 kDa) having homologous in various species including humans, mice, rats, dogs, and hamsters and also well conserved between them. Since its discovery in 1990, several homologous proteins of PDPN were identified and described in different tissues, i.e., as E11 in lymphatic endothelial cells, gp38 in fibroblastic reticular cells of lymphoid organs and thymic epithelial cells, T1α/rTI40 in alveolar type 1 epithelial cells, PA2.26 in skin keratinocytes upon injury, OTS-8 in induced osteoblasts, and as Aggrus, a platelet-aggregating factor. In 1997, finally this molecule was named as podoplanin by Breiteneder-Geleff et al. due to its expression in developing glomeruli in rat kidney and its potential role in flattening of foot processes (podocytes). In 1999, PDPN was accepted as a novel marker of lymphatic endothelial cells (Astarita et al. 2012).
PDPN expression is negatively regulated by the squamous differentiation both in vitro and in vivo as Ohta M et al. demonstrated its decreased expression in the PDPN-positive cells by enhancing squamous cell differentiation by the process of contact normalization, a process that forces the cells to maintain normal non-transformed cell phenotype. Epigenetic mechanism like histone deacetylation has been proposed as another negative regulator of PDPN expression (Ohta et al. 2013).
PDPN and Physiology
It is well known that lymphatic system is derived from venous system, and PDPN is a lymphatic endothelial cell (LEC) marker. In vasculogenesis, PDPN has a crucial role in the separation of lymphatic from the blood circulatory system. All endothelial cells in the cardinal vein of experimental mice uniformly express the hyaluronan receptor LYVE-1 till embryonic day (E) 9.5, when a subset of cells switch on expression of the homeobox transcription factor Prox1, a master regulator of lymphatic endothelial cell differentiation. This triggers the upregulation of several other lymphatic markers including vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin as compared with surrounding blood vascular endothelium. Finally, the interaction of CLEC-2 receptor platelet and PDPN on lymphatic endothelial cells induces platelet aggregation and prevents blood from flowing into new lymphatic vessels budding from the cardinal vein. Furthermore, continued expression of PDPN into adulthood reinforces its importance in maintaining proper lymphatic architecture (Pan et al. 2014).
Apart from lymphatic system, PDPN was thought to be linked with heart development. In mice with podoplanin gene knockout, the embryo showed underdeveloped cardiac structure due to lack of cell migration and epithelial mesenchymal transition which may be regulated by PDPN. Podoplanin is also expressed by mouse keratinocytes during wound healing which indicates its role in tissue regeneration (Astarita et al. 2012).
Since its first expression, the PDPN is found to have a wide range of distribution, i.e., in the bone (osteocytes, osteoblasts, and periosteum), lungs (alveolar type I cells), mesothelial cells, epidermal basal layer cells, central nervous system (choroid plexus epithelial cells, ependyma, meninges), thymus type I epithelial cells, breast (myoepithelial cells), prostate myofibroblasts, lymphoid organs (follicular dendritic cells, stromal reticular cells), immature cells like fetal germ cells and developing Sertoli cells, peripheral nervous system (perineurium), and kidney (glomerular podocytes) (Schacht et al. 2005). In addition, normal oral and paraoral tissues also express PDPN in variable amount. Till now, a little is known about podoplanin expression in human salivary gland tissue. In 2008, Hata et al. have investigated the distribution of podoplanin in mouse major salivary glands. They noticed the selective expression podoplanin in mucus acinar and myoepithelial cells, but rarely in serous acini. The strong expression was also found at the basal portion of intercalated, striated, and interlobular ducts of all major salivary glands. In their study, as the podoplanin detection levels coincide with the distribution of other myoepithelial cell-specific markers like P-cadherin for major salivary glands, authors have suggested podoplanin as a useful marker for myoepithelial cells. Electron microscopic study of myoepithelial cells disclosed the possible role of podoplanin in maintaining myoepithelial cell integrity, by promoting plasma membrane extension through actin cytoskeleton rearrangement and also by its ability to resist the proteases as it is a negatively charged mucin-type protein (Hata et al. 2008). In recent years, the pattern of PDPN expression was exclusively studied in tooth as well as in various stages of tooth development as a thought of reflection from similar podoplanin immunoreactivity in oral epithelium. At the bud stage, podoplanin was found to be expressed both in oral mucous epithelia and in tooth bud, whereas at the cap stage, podoplanin was exclusively expressed on inner and outer enamel epithelia but not in ectomesenchymal cells. In early bell stage, the expression extended to cervical loop enamel epithelia and odontoblasts. Furthermore, Hertwig epithelial sheath and odontoblasts of radicular dentin showed podoplanin immunoreactivity during root formation. In the later stages, odontoblasts exhibited continued intense podoplanin expression at the predentin junction, while no expression was detected in the enamel organ containing ameloblasts. These findings suggest the sustained podoplanin immunoreactivity in both differentiating and differentiated odontoblasts unlike the ameloblasts which show only transient expression during differentiation and secretion. Although the potential role of podoplanin in tooth development is not clear, perceivable podoplanin reactivity of odontoblasts and enamel epithelia strongly suggests contribution of this protein in the cytoskeletal arrangement and the cell adhesive property. So it is also thought that the podoplanin may have a role in formation of odontoblastic fiber or function in anchorage of cells during tooth development (Caetano et al. 2013).
PDPN and Tumorigenesis
Cumulative evidence of constitutive expression of PDPN in various tumor model experiments, both in vitro and in vivo, suggests its potential role in tumorigenesis. There are very few research works which support the exact role of podoplanin in cancer initiation. Atsumi N et al. have observed the PDPN-positive cancer cells exhibited stem cell-like properties as they had the ability to repopulate and to generate a heterogeneous cancer cell population in squamous cell carcinoma. Intrinsic mechanism like activation of the SHH signaling pathway by PDPN was thought to have contribution in both initiation and progression of tumor (Atsumi et al. 2008).
The key step for tumor invasion or cell migration is actin cytoskeleton remodeling which leads to the formation of cell protrusions, i.e., filopodia and lamellopodia. There are reports of upregulation of PDPN expression localized to these membrane extensions. The conserved motif of three basic residues in cytoplasmic tail of PDPN binds with ERM family of proteins (ezrin, radixin, and moesin), and furthermore this results in phosphorylation of ERM proteins, which serve as connectors between integral membrane proteins and actin cytoskeleton. Apart from ERM protein function, the activities of Rho-family GTPases, in particular RhoA, are modulated independently by PDPN. Regulation of RhoA activity is causally involved in the pro-migratory phenotype observed in PDPN-expressing cancer cells. Decreased stress fibers and increased filopodia formation in PDPN-positive cell lead to mesenchymal appearance. These changes, in addition to a downregulation of E-cadherin and other epithelial markers, indicative of cells undergoing epithelial mesenchymal transition (EMT) were observed. However, Wicki and Christofori reported an independent pathway on migration of PDPN-overexpressed cancer cells in the absence of a cadherin switch and EMT. Recently, CD44, another type I transmembrane glycoprotein, has been considered as a novel partner of PDPN by regulating its recruitment and helps in directional persistence of motility in PDPN-positive tumor cells.
Till now, podoplanin-mediated platelet aggregation is thought to be another potential mechanism of tumor invasion and metastasis by various mechanisms, i.e., protecting tumor cells during their transit through the bloodstream, mediating adherence to vascular endothelium, evasion from immunosurveillance, and release of various potent bioactive molecules that facilitate tumor cell extravasation and growth at metastatic sites. Various studies support PDPN as a powerful platelet aggregator by binding platelet C-type lectin receptor, CLEC-2, via platelet aggregation-stimulating (PLAG) domain which is a highly conserved amino acid sequence in the PDPN extracellular domain. This interaction induces bidirectional signaling causing both clustering PDPN and platelet aggregation which sequentially promote tumor cell migration and metastasis. In contrast, its interaction with another protein CD9, which belongs to tetraspanin family of protein, reduces the metastatic potential of PDPN-platelet complex. It was shown that interaction of transmembrane domain of CD-9 with PDPN lowered the aggregation of platelet caused by participation of PDPN and CLEC-2 in experimental lung metastases in nude mice model (Wicki and Christofori 2007; Nakazawa et al. 2008).
PDPN and Other Pathology
Upregulation of PDPN expression has been observed in many types of human cancers mainly in squamous cell carcinoma of oropharyngeal region, skin, and lungs. In almost of all cases, PDPN expression was correlated with regional lymph node metastasis, recurrences, and shorter survival. Apart from squamous cell carcinomas, PDPN expression was shown in other tumors like germ cell tumors of the ovary, angiosarcomas, osteosarcoma, mesotheliomas, basal cell carcinomas of the skin, and follicular dendritic cell tumors. PDPN was also found in certain adenocarcinomas, such as colorectal and stomach carcinomas. Increased amounts of podoplanin were also observed in some carcinomas of the central nervous system, such as germinoma-type tumors and glioblastoma multiforme (Ugorski et al. 2016). In addition to carcinomas, PDPN expression was correlated with precancerous conditions mainly in oral cavity. In oral epithelial dysplasia, growing evidence of association of high PDPN expression with increased risk of progression to carcinoma suggests that PDPN could be an effective biomarker for assessment of malignant transformation risk. Precancerous lesions with PDPN expression beyond the basal layer indicate about upward clonal expansion of PDPN positive cells (stem cell clones/tumor-initiating cells) within the epithelium (Swain et al. 2014).
Recently, many investigations have emphasized on PDPN expression in individual pathologic conditions originating from tooth forming organs in addition to its orthologic expression. Varied immunostaining pattern of PDPN was detected in odontogenic cysts and tumors. In odontogenic cyst like radicular cyst, follicular cyst, and orthokeratinized odontogenic cysts, immunoreaction with anti-PDPN antibody was limited only to the basal cell layer, thus suggesting the contribution of PDPN in the expansion of these nonmalignant odontogenic cystic lesions. Numerous investigations have focused on reclassification of keratocystic odontogenic tumor (KCOT) from cyst to tumor status by using clinic-pathological parameters; however, there is no conclusive immunohistochemical marker that can be used to delineate odontogenic cysts from tumors. An exclusive study on KCOT by Okamoto et al. suggested PDPN can be used as a reliable marker in justifying its neoplastic behavior. Enhanced expression of PDPN was especially evident in the cell membrane and cytoplasm of most of the cells in the basal and suprabasal layers, areas of budding basal cell proliferation, epithelial nests, and peripheral cells of daughter cysts of KCOT. A recent study also reported similar continuous linear immunoreactivity of basal epithelial cells for PDPN in nevoid basal cell carcinoma syndrome-associated KCOT as observed in sporadic cases. This peculiar pattern of immunostaining suggests possible contribution of PDPN in the local invasiveness, well-known recurrences, and the neoplastic nature of the KCOT (Okamoto et al. 2010). In an extensive study on PDPN expression in benign epithelial odontogenic tumors with or without ectomesenchyme, Caitano et al. observed intense expression exclusively in the epithelium of these odontogenic tumors. The ectomesenchyme showed weak or absence of PDPN expression with the exception of active odontoblasts in ameloblastic fibro-odontoma. Among the epithelial tumors like amelobastoma, adenomatoid odontogenic tumor, KCOT, and calcifying epithelial odontogenic tumor, significant cytoplasmic and membranous PDPN expression was seen in peripheral neoplastic cells, whereas PDPN-positive central cells were found in lesions like plexiform ameloblastoma, adenomatoid odontogenic tumor, and ameloblastic fibroma. Many mature and quiescent structures like acanthomatous metaplasia, ghost cells, and calcification foci unanimously showed negative PDPN expression. In tumors like odontomas, epithelial cells adjacent to ghost cells were stained with anti-PDPN antibodies which indicate altered cytoskeletal remodeling in pre-ghost cells that result in rapid degeneration. These peculiar staining patterns can be correlated with tentative role of PDPN in the process of cellular proliferation, differentiation, and collective cell migration. The distribution of PDPN in various odontogenic cysts and tumors suggests that its expression is upregulated by interactions between odontogenic epithelium and mesenchyme; it could also be related to epithelial-mesenchymal transition, in which odontogenic epithelial cells lose their polarity and cohesiveness and acquire migratory features under the influence of stromal-derived growth factors (Caetano et al. 2013).
Apart from its physiologic expression in salivary glands, very few researches have been carried out for immunolocalization of PDPN in pathologic entities of the same. Enhanced expression in inflammatory condition like sialoadenitis suggests its possible association with inflammation or tissue regeneration. In benign tumor like pleomorphic adenoma, the exclusive expression of PDPN was noticed in epithelioid- and spindle-cell types neoplastic myoepithelial cells (Tsuneki et al. 2013). The exact explanation for this peculiar distribution is still not known, whereas in case of Warthin tumor, the results of PDPN expression help in better understanding of the pathogenesis of the tumor. Numerous subcapsular PDPN-positive lymphatic vessels revealing a sinus-like structure confirm the tumor origin in regional lymph nodes as these subcapsular sinuses are a crucial morphological element of lymph nodes. In malignant tumors like adenoid cystic carcinoma, a close correlation between PDPN expression and distant metastasis and disease-free survival was observed.
Nevertheless, further studies with larger sample sizes are needed to validate the detailed mechanisms underlying the relationship between PDPN expression and tumorigenesis and clinical outcomes of different salivary gland neoplasms (Wu et al. 2012).
Since its discovery, altered expression of PDPN in diverse population of tissues indicates its multifaceted role in both embryologic development and tumorigenesis. Many authors have suggested about the putative role of PDPN in critical steps of carcinogenesis and its correlation with biological behavior of various tumors through various clinical and experimental studies. Having well-documented roles like induction of cancer cell migration and significant contribution in metastasis along with its specificity in evaluating lymphovascular invasion, PDPN could be proposed as a novel diagnostic and prognostic biomarker. Still there are some unexplored aspects like the exact role of podoplanin in EMT and tumor microenvironment, which need to be clarified to fully elucidate the pathophysiological function of this wonder molecule.
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