Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 29;7(13):16070-89.
doi: 10.18632/oncotarget.7445.

Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

Patricia Carrasco-Ramírez  1 , David W Greening  2 , Germán Andrés  3 , Shashi K Gopal  2 , Ester Martín-Villar  1 , Jaime Renart  1 , Richard J Simpson  2 , Miguel Quintanilla  1
Affiliations

Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

Patricia Carrasco-Ramírez et al. Oncotarget. .

Abstract

Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis.

Keywords: exosomes; lymphangiogenesis; microvesicles; podoplanin; tumor progression.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Western blot analysis of protein expression in whole cells and EVs isolated from different cell lines
EVs were isolated as crude EXOs by sequential ultracentrifugation as described in Materials and methods. A. MDCK cells expressing either PDPNeGFP or eGFP. B. HN5 cells expressing endogenous PDPN. C. SK-MEL-28 cells expressing either PDPNeGFP or eGFP. WCL, whole cell lysate.
Figure 2
Figure 2. Confocal immunofluorescence reveals colocalization of PDPN with endosomal protein markers
HN5 cells were labeled at 4°C with anti-PDPN Ab and, after washing, incubated at 37°C for 40 min to analyze the presence of PDPN in early endosomes labeled with anti-EEA1 (upper panel) and 90 min to analyze the presence of PDPN in late endosomes labeled with anti-CD63 (bottom panel). EEA1 and CD63 were detected using specific monoclonal Abs and AlexaFluor 546-conjugated goat anti-mouse IgG (red). PDPN was detected using AlexaFluor 488-conjugated goat anti-rabbit IgG (green). Scale bar, 10 μm.
Figure 3
Figure 3. Cryoelectron microscopy reveals MVB colocalization of PDPN and CD63
PDPN was detected in SK-MEL-28-PDPNeGFP cells with a specific polyclonal Ab and protein A conjugated to 5-nm gold particles (small arrowheads), and CD63 with a specific monoclonal Ab and a goat anti-mouse conjugated to 15-nm gold particles (large arrowheads). Note the presence of PDPN in ILVs of MVBs, although most of PDPN labeling occurs at the plasma membrane (PM). Scale bar, 100 nm.
Figure 4
Figure 4. PDPN immunoelectron microscopy of EVs isolated from SK-MEL-28-PDPNeGFP cells
PDPNeGFP was detected by two distinct rabbit polyclonal Abs directed either against the extracellular domain of PDPN (pdpn) or the intracellular tag GFP (gfp), and protein A conjugated to 5-nm gold particles (middle panels). The presence of CD63, an EXO marker, in EVs expressing PDPNeGFP was determined by double immunogold labeling using rabbit Abs against either PDPN or GFP and a specific monoclonal Ab to CD63. Signal was revealed by using an anti-rabbit Ab conjugated to 15-nm gold particles and an anti-mouse Ab conjugated to 5-nm gold particles (right panels). No signal was detected in control SK-MEL-28-eGFP cells. Scale bar, 100 nm.
Figure 5
Figure 5. Purification and characterization of EXOs released from MDCK-CMV and MDCK-PDPN cells
A. Experimental workflow for MV and EXO purification from MDCK-CMV and MDCK-PDPN cells. B. Western blot analysis of PDPN and Alix expression in fractions 6-9 from OptiPrep density-gradient performed with the crude EXO preparation from MDCK-PDPN cells. Note that significant enrichment of both Alix and PDPN were observed in fraction 8. C. Dynamic Light Scattering of EXOs from MDCK-PDPN and control MDCK-CMV cells purified by OptiPrep density-gradient fractionation. D. Western blot analysis of PDPN expression in MDCK-PDPN cell lysate (WCL) and corresponding MV and EXO fractions derived from the conditioned medium in the presence/absence of 10% FBS (EV-depleted). * indicates a band of ~80 kDa that corresponds to a PDPN non-covalent homodimer identified in normal and tumor tissues [41].
Figure 6
Figure 6. PDPN facilitates increased production and release of EV subtypes
A, B. Quantification of MVs (A) and EXOs (B) released by MDCK, MDCK-CMV and MDCK-PDPN cells. MVs and crude EXOs were isolated from the same number (2×106) of seeded cells, as indicated in the workflow presented in Figure 5A. After CM collection, cells were counted and used for normalization. EVs isolated from the CM were resuspended in the same volume (50 μl) of PBS, quantified and represented as a proportion per 106 cells. In panel B, a Western blot of Alix expression, as a protein marker of EXOs, is presented. The same volume (20 μl) of the crude EXO fractions was loaded onto each lane. C. Quantification of EXOs released by HN5 in response to PDPN knockdown. Two specific shRNAs (sh3, sh4) and shRNA control (sc) were used to deplete endogenous PDPN from HN5 cells. In the upper panel, a Western blot showing PDPN downregulation in HN5 cells in which GAPDH was used as a control of protein loading is presented. Aliquots of total cell lysates containing equivalent amount of proteins (30 μg) were loaded. In the lower panel, the Western blot shows diminished expression of CD63 as a measure of EXOs released by PDPN-knockdown cells. The same volume (20 μl) of crude EXO fractions were loaded. Results are expressed as the mean of three independent experiments α s.e.m. **p < 0.01 (A, B); *p < 0.05 (C).
Figure 7
Figure 7. Proteomic profiling of MDCK cells and EXOs following PDPN overexpression
A. Two-way Venn diagram of proteins commonly and uniquely identified in MDCK-CMV and MDCK-PDPN cells and EXOs. B. Heatmap representing proteins for which an increase (red) or decrease (blue) in protein expression was observed in MDCK-PDPN EXOs compared to control MDCK-CMV EXOs. For EXOs, differentially expressed upregulated (upper) and downregulated (bottom) proteins are shown.
Figure 8
Figure 8. Comparative Western blot analysis of protein expression in cells and EXOs from MDCK and HN5 cell systems
A. E-cadherin (E-CD) was downregulated while mesenchymal N-cadherin (N-CD) was upregulated in MDCK-PDPN cells and EXOs. Annexin A7 and flotillin 1 were upregulated in MDCK-PDPN EXOs. Both MDCK-PDPN and MDCK-CMV EXOs contained actin and CD44 and absence of the Golgi marker TGN46. B. Annexin A7 and flotillin 1 were downregulated in EXOs derived from PDPN knockdown HN5-sh3 cells while CD9 levels remained unchanged.
Figure 9
Figure 9. MDCK-PDPN and MDCK-CMV-released EXOs stimulate in vitro angiogenesis
Representative micrographs A. and quantitative evaluation B. of the formation of closed capillary-like structures by HUVECs seeded on Matrigel-coated wells untreated (Control) or treated with MDCK-CMV and MDCK-PDPN crude EXOs (40 μg/ml). Data are expressed as the number of closed tubes per field. Bar, 150 μm. **p < 0.01. A representative experiment out of three is presented.
Figure 10
Figure 10. MDCK-PDPN-released EXOs stimulate in vitro lymphangiogenesis
A. Quantitative evaluation of the length of tubes per field formed by HLECs seeded on Matrigel-coated wells untreated (Control) or treated with MDCK-CMV and MDCK-PDPN crude EXOs (40 μg/ml) for 2 h and 4 h. A representative experiment out of two is presented. B, C. Representative micrographs (B) and quantitative evaluation of the number of closed capillary-like structures per field (C) formed by HLECs seeded on Matrigel-coated wells untreated (Control) or treated with MDCK-CMV and MDCK-PDPN crude EXOs (40 μg/ml) for 6 h. EXOs were preincubated with mAb NZ1 (0.5 μg/ml and 1 μg/ml) recognizing the extracellular domain of PDPN or control IgG (1 μg/ml), as indicated, for 1h at 4°C. Bar, 100 μm. *p < 0.05. A representative experiment out of two is presented.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Astarita JL, Acton SE, Turley SJ. Podoplanin: emerging functions in development, the immune system, and cancer. Front Immunol. 2012;3:283. - PMC - PubMed
    1. Renart J, Carrasco-Ramirez P, Fernandez-Munoz B, Martin-Villar E, Montero L, Yurrita MM, Quintanilla M. New insights into the role of podoplanin in epithelial-mesenchymal transition. Int Rev Cell Mol Biol. 2015;317:185–239. - PubMed
    1. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM, Munoz-Guerra M, Cruces J, Quintanilla M. Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas. Int J Cancer. 2005;113:899–910. - PubMed
    1. Wicki A, Lehembre F, Wick N, Hantusch B, Kerjaschki D, Christofori G. Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton. Cancer Cell. 2006;9:261–272. - PubMed
    1. Yuan P, Temam S, El-Naggar A, Zhou X, Liu DD, Lee JJ, Mao L. Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer. 2006;107:563–569. - PubMed