PDPN Human

What is PDPN and what is its molecular structure?

PDPN is a mucin-type transmembrane sialoglycoprotein with a molecular weight of approximately 36-43 kDa that is encoded by the PDPN gene in humans . Structurally, it is a type-I integral membrane, heavily O-glycosylated glycoprotein with diverse distribution in human tissues . The protein is relatively well conserved between species, with homologues present in humans, mice, rats, dogs, and hamsters .

To study PDPN's structure:

• Recombinant protein expression systems can be utilized to produce the extracellular domain (ePDPN) for structural analysis

• X-ray crystallography and NMR spectroscopy provide detailed structural information

• Glycosylation patterns can be analyzed through mass spectrometry and glycoprotein-specific staining techniques

What are the primary physiological functions of PDPN?

While the specific functions of PDPN are still being elucidated, several important roles have been identified:

• PDPN plays a critical role in the proper formation of linkages between the cardiovascular and lymphatic systems during development

• It mediates effects on cell migration and adhesion through interaction with various binding partners

• During development, it facilitates blood and lymphatic vessel separation by binding to CLEC1B, triggering platelet activation and/or aggregation

• In lymph nodes, PDPN controls fibroblastic reticular cells (FRCs) adhesion to the extracellular matrix and regulates actomyosin contraction

• In neural tissue, PDPN is involved in processes including development and angiogenesis

For experimental investigation of these functions, gene knockout/knockdown models, co-culture systems with PDPN-expressing and PDPN-deficient cells, and interaction assays with identified binding partners are commonly employed.

How is PDPN expression distributed across human tissues?

PDPN shows a diverse distribution pattern across human tissues. Current expression data indicates:

• High expression in lymphatic endothelial cells (serving as a specific lymphatic marker)

• Expression in kidney podocytes (hence the name "podoplanin")

• Presence in lung alveolar cells

• Expression in neural tissue in both mouse and human samples

• Found in other cell types including follicular dendritic cells, reticular cells, mesothelial cells, testicular germ cells, and ovarian cells

Methodologically, tissue expression patterns can be analyzed through:

• Immunohistochemistry using specific anti-PDPN antibodies like D2-40 or the 5B3 monoclonal antibody

• RNA sequencing data from databases like The Human Protein Atlas

• Single-cell RNA sequencing for cell-type specific expression profiling

• In situ hybridization for localization of PDPN mRNA

What methods are most effective for detecting PDPN in experimental settings?

Several validated methods for PDPN detection include:

• Immunohistochemistry (IHC): Using monoclonal antibodies such as D2-40 or 5B3 for tissue sections

• Flow cytometry: For detecting PDPN on live cells, with antibodies like humLpMab-23 demonstrating high affinity (KD values between 4.7-5.4 × 10−9 M)

• Western blotting: For protein detection in cell or tissue lysates

• ELISA: For quantitative measurement of PDPN levels

• Immunofluorescence microscopy: For co-localization studies, particularly useful as PDPN has been shown to co-localize with nestin

When selecting detection methods, researchers should consider that PDPN is heavily glycosylated, which may affect antibody binding efficiency. The 5B3 monoclonal antibody has demonstrated excellent specificity in ELISA, western blot, and immunohistochemistry experiments, with an affinity constant of 2.94 × 108 L/mol .

What are the known interaction partners of PDPN?

PDPN interacts with several proteins to mediate its diverse functions:

• CLEC-2 (C-type lectin 2): Expressed on platelets and hematopoietic cells, this interaction is crucial for blood/lymphatic vessel separation during embryonic development

• CD9: Interaction attenuates platelet aggregation induced by PDPN

• ERM proteins (Ezrin, Radixin, Moesin): Promotes epithelial-mesenchymal transition (EMT) by triggering RHOA activation

• CD44: Promotes directional cell migration in epithelial and tumor cells

• LGALS8 (Galectin-8): May participate in connecting lymphatic endothelium to surrounding extracellular matrix

To study these interactions experimentally:

• Co-immunoprecipitation assays

• Proximity ligation assays

• FRET/BRET analysis for real-time interaction monitoring

• Yeast two-hybrid screening to identify novel binding partners

• Surface plasmon resonance for binding kinetics analysis

How does PDPN contribute to cancer progression and metastasis?

PDPN has been extensively studied in cancer research with several mechanisms identified:

• PDPN is often upregulated in various cancers including squamous cell carcinomas, malignant mesothelioma, and brain tumors

• It serves as a specific lymphatic vessel marker, with lymphangiogenesis levels correlating with poor prognosis in cancer patients

• In cancer-associated fibroblasts (CAFs), PDPN upregulation has been associated with poor prognosis

• In squamous cell carcinomas, PDPN plays a key role in cancer cell invasiveness by controlling invadopodia formation and promoting efficient extracellular matrix (ECM) degradation

• It modulates RHOC activity to activate ROCK1/ROCK2 and LIMK1/LIMK2 pathways while inactivating CFL1, leading to invadopodia stability and maturation

Methodological approaches to study these mechanisms include:

• 3D invasion assays with PDPN-expressing versus knockdown cells

• ECM degradation assays using fluorescently labeled matrix proteins

• In vivo metastasis models with PDPN-manipulated cancer cells

• Analysis of patient samples for correlation between PDPN expression patterns and clinical outcomes

• Co-culture models of cancer cells with PDPN-positive CAFs

What is the role of PDPN in fibroblasts within the tumor microenvironment?

Research has revealed complex interactions between PDPN-expressing fibroblasts and tumor progression:

• Podoplanin-positive cancer-associated fibroblasts (CAFs) positively correlate with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion, and poor patient outcomes in breast cancer

• Ectopic expression of podoplanin significantly increases the migration capabilities of fibroblasts (as demonstrated in MSU1.1 and Hs 578Bst fibroblast cell lines)

• PDPN expression in fibroblasts affects the formation of pseudo tubes by endothelial cells, with podoplanin-rich fibroblasts resulting in endothelial cell capillary-like networks characterized by significantly lower numbers of nodes and meshes

• Rather than directly affecting cancer cell migration and invasion, PDPN may facilitate fibroblast movement into tumor stroma, creating a favorable microenvironment for tumor progression by increasing CAF numbers

Experimental approaches to investigate these interactions include:

• Co-culture systems with PDPN-expressing fibroblasts and cancer cells

• 3D organoid models incorporating multiple cell types

• Conditional knockout models targeting PDPN specifically in fibroblasts

• In vitro tube formation assays with endothelial cells and PDPN-manipulated fibroblasts

• RNA-seq analysis of PDPN-positive versus negative fibroblasts to identify secreted factors

What methods are optimal for studying PDPN's role in lymphangiogenesis?

Given PDPN's important role as a lymphatic marker, several specialized approaches are used:

• Immunohistochemical analysis using anti-PDPN antibodies for lymphatic vessel density quantification in tissues

• 3D lymphatic vessel formation assays using lymphatic endothelial cells with PDPN manipulation

• PDPN knockout/knockdown studies in lymphatic endothelial cells to assess functional impacts

• In vivo lymphangiogenesis models with fluorescent labeling of lymphatic vessels

• Co-culture systems to study interactions between PDPN-expressing cells and lymphatic endothelium

Researchers should be aware that PDPN's diagnostic utility stems from its specificity as a lymphatic vessel marker, with lymphangiogenesis levels correlating with poor prognosis in cancer patients . For accurate assessment, multiple PDPN-positive vessel quantification methods should be employed, preferably using automated image analysis systems to reduce observer bias.

How are anti-PDPN antibodies developed and what are their research applications?

Development of anti-PDPN antibodies follows several strategies:

• Generation of recombinant extracellular PDPN (ePDPN) fusion proteins in expression systems like E. coli, as demonstrated with ePDPN-His and GST-ePDPN fusion proteins

• Immunization protocols using purified fusion proteins with adjuvants like QuickAntibody-Mouse5W

• Hybridoma technology for monoclonal antibody production, as exemplified by the 5B3 cell line generating anti-PDPN mAb

• Humanization of mouse antibodies by fusing the variable domain CDRs with human immunoglobulin constant domains, as shown with humLpMab-23

• Engineering of defucosylated antibodies (like humLpMab-23-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)

These antibodies have multiple research applications:

• Diagnostic immunohistochemistry for identifying lymphatic vessels and PDPN-expressing tumors

• Flow cytometry for cell sorting and quantification of PDPN-positive populations

• Therapeutic targeting of PDPN-expressing cancer cells

• Blocking experiments to study PDPN's functional roles

• In vivo imaging of PDPN-expressing tissues

What is known about PDPN's role in neurological diseases and what experimental approaches are used to study it?

PDPN's involvement in neurological diseases is an emerging research area:

• PDPN is involved in several physiological and pathological processes in the brain, including development and angiogenesis

• Neurological disorders constitute a major cause of disability and death worldwide (16.8% of total deaths), with a 36% increase in associated deaths over the past 25 years

• Vasculopathy, inflammation, and immune abnormalities play important roles in neurological diseases, with PDPN potentially involved in these processes

• PDPN expression has been detected in neural tissue in both mouse and human samples

Methodological approaches for investigating PDPN in neurological contexts include:

• Immunohistochemical analysis of PDPN expression in normal versus diseased neural tissue

• Primary neural cell cultures with PDPN manipulation

• In vitro blood-brain barrier models to study PDPN's vascular effects

• Conditional knockout models targeting PDPN in specific neural cell populations

• Cerebrospinal fluid analysis for PDPN as a potential biomarker

How does PDPN modulate cell migration and what signaling pathways are involved?

PDPN influences cell migration through several mechanisms:

• In fibroblasts, ectopic expression of podoplanin significantly increases migration capability, as demonstrated in MSU1.1 and Hs 578Bst fibroblast cell lines

• Through MSN or EZR (ezrin) interaction, PDPN promotes epithelial-mesenchymal transition (EMT) leading to EZR phosphorylation and triggering RHOA activation, resulting in increased cell migration and invasiveness

• Interaction with CD44 promotes directional cell migration in epithelial and tumor cells

• In keratinocytes, PDPN induces changes in cell morphology, including elongated shape, numerous membrane protrusions, major reorganization of the actin cytoskeleton, increased motility, and decreased cell adhesion

• PDPN controls invadopodia stability and maturation through modulation of RHOC activity, activating ROCK1/ROCK2 and LIMK1/LIMK2 while inactivating CFL1

Experimental techniques to study these pathways include:

• Live cell imaging with fluorescently labeled cytoskeletal components

• RHOA/RHOC activity assays in PDPN-manipulated cells

• Phosphorylation analysis of EZR and other downstream targets

• 2D and 3D migration assays with pathway inhibitors

• Quantitative analysis of membrane protrusion dynamics

What techniques are most effective for analyzing PDPN's effects on extracellular matrix interactions?

Several specialized techniques help elucidate PDPN's role in ECM interactions:

• Invadopodia formation assays using fluorescently labeled ECM components to visualize degradation

• Atomic force microscopy to measure cell-ECM adhesion forces in PDPN-expressing versus control cells

• Traction force microscopy to assess mechanical forces exerted by cells on the ECM

• ECM remodeling assays measuring collagen contraction by PDPN-expressing fibroblasts

• Co-immunoprecipitation studies to identify PDPN interactions with ECM receptors like integrins

These approaches are particularly relevant given that PDPN modulates invadopodia stability and maturation leading to efficient ECM degradation in tumor cells , and controls fibroblastic reticular cells (FRCs) adhesion to the ECM . Additionally, through binding with LGALS8 (Galectin-8), PDPN may participate in connecting lymphatic endothelium to the surrounding ECM .

What experimental models are available for studying PDPN functions in different tissues?

Researchers can employ various models to investigate tissue-specific PDPN functions:

In vitro models:

• Primary cell cultures from different PDPN-expressing tissues (lymphatic endothelium, neural cells, fibroblasts)

• Cell lines with endogenous or manipulated PDPN expression

• 3D organoid models incorporating multiple cell types

• Co-culture systems (e.g., fibroblasts with endothelial or cancer cells)

In vivo models:

• Global PDPN knockout mice (note: PDPN is required for normal lung cell proliferation and alveolus formation at birth)

• Conditional tissue-specific PDPN knockout models

• PDPN reporter mice for lineage tracing studies

• Xenograft models using PDPN-manipulated human cells

Clinical samples:

• Tissue microarrays from various PDPN-expressing normal and pathological tissues

• Patient-derived organoids

• Single-cell analysis of tissues to identify PDPN-expressing cell populations

When selecting appropriate models, researchers should consider tissue-specific PDPN functions, such as its role in lymphatic vessels, neural tissue, cancer-associated fibroblasts, and various epithelial tissues.