Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells

https://doi.org/10.1016/j.bbrc.2013.03.057Get rights and content

Highlights

  • Heat shock protein (HSP) A9 is a novel interaction molecule with podoplanin.

  • HSPA9 is secreted by oral squamous cell carcinoma cells.

  • HSPA9 interacts with podoplanin on the cell membrane.

  • HSPA9 and podoplanin cooperate in invasion of oral squamous cell carcinoma cells.

Abstract

In previous studies, we have shown several lines of evidence that podoplanin (PDPN) plays an important role in cell adhesion via its association with extracellular components in neoplastic conditions, though there has been no trial to search for PDPN-interaction molecules in the extracellular milieu. To screen for those molecules, we performed proteomics-based analysis using liquid chromatography-tandem mass spectrometry followed by co-immunoprecipitation for PDPN in ZK-1, an oral squamous cell carcinoma (SCC) cell system whose cell membrane molecules were cross-linked with each other in their extracellular compartments, and we identified heat shock protein (HSP) A9 as one of the extracellular PDPN bound molecules. Effects of transient PDPN knockdown by siRNA in ZK-1 were also comparatively examined for cellular behaviors in terms of HSPA9 expression and secretion. Finally, HSPA9 expression modes were immunohistochemically visualized in oral SCC tissue specimens. HSPA9 was secreted from ZK-1 cells, and the expression and secretion levels of HSPA9 gene and protein were well coordinated with those of PDPN. Immunohistochemically, HSPA9 and PDPN were co-localized in ZK-1 cells and oral SCC foci, especially in the peripheral zone. In conclusion, the results indicate that HSPA9 secreted by oral SCC cells interacts with PDPN on their cell surface in an autocrine manner and regulates their growth and invasiveness.

Introduction

Podoplanin (PDPN), a mucin-type transmembrane sialoglycoprotein, was originally identified in glomerular visceral epithelial cells (podocytes) [1] and has been utilized as one of the most representative markers of lymphatic endothelium [2]. In recent years, PDPN expression in parenchymal cells has been confirmed in various kinds of benign and malignant tumors such as thymoma [3], central nervous system germ cell tumors [4], and lung squamous cell carcinoma (SCC) [5]. We have also revealed the characteristic immunolocalization of PDPN in close association with neoplastic stromata in oral epithelial dysplasia, carcinoma in situ and SCC [6], [7], as well as in salivary gland tumors [8] and odontogenic tumors [9]. In addition, we demonstrated that PDPN tethers oral SCC cells to hyaluronan-rich extracellular matrices in collaboration with CD44 and hence suggested the function of PDPN in communication with extracellular matrix (ECM) elements [7]. Based on the results from our latest investigation, it was necessary for us to identify any possible candidate molecules which interact with PDPN at its extracellular compartment.

The extracellular domain of PDPN, which is rich in Ser and Thr, contains multiple potential O-glycosylation sites [10]. C-type lectin-like receptor 2 (CLEC-2) [11], galectin-8 [12], and CD44 [13] have been shown to interact with PDPN at the extracellular domain in different cellular events like tumor cell-induced platelet aggregation [11], lymphatic endothelial cell adhesion [12], and directional tumor cell migration [13]. However, these are cell membrane molecules which have not originated from the ECM side. The purpose of the present study was to search for PDPN-interacting molecules by means of proteomics-based analysis and confirm ECM interaction using the same oral SCC cell system which was used in our recent study [7].

Section snippets

Cell systems and reagents

The oral SCC cell system (ZK-1) was established from SCC arising in the tongue [15]. ZK-1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco), 50 μg/ml streptomycin, and 50 IU/ml penicillin (Gibco) [7]. They were incubated at 37 °C in a humidified 5% carbon dioxide/95% air atmosphere.

Antibodies

A mouse monoclonal antibody against human PDPN (D2-40, IgG1) was obtained from Dako (Glostrup, Denmark).

Identification of HSPA9 as a candidate interacting molecule with PDPN

To identify novel interacting proteins of human PDPN in oral SCC, we performed immunoprecipitation using ZK-1 cells which endogenously express PDPN [7]. We found a protein band with a Mr mass of 70 kDa in SDS–PAGE gels stained with silver impregnation (Fig. 1(A), left panel, asterisk) among those co-immunoprecipitated with PDPN. Western blotting with anti-PDPN antibody confirmed that the protein was not a variant of PDPN (Fig. 1(A), right panel). The 70 kDa band was identified to be HSPA9 by

Discussion

In the present study, we demonstrated for the first time the extracellular shedding of HSPA9 from oral SCC cells. Secondly, by using cross-linking treatments [17], the interaction between HSPA9 and PDPN in the cell surface was confirmed. Thirdly, the gene and protein expression levels as well as the secretion levels of HSPA9 were specifically correlated with those of PDPN. Finally, the co-localization of HSPA9 and PDPN on the cell surface was also confirmed in surgical specimens of oral SCC.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and for JSPS Fellows, with additional funding from the Iwadare Scholarship Foundation, Japan.

References (37)

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Fellow, the Japan Society for the Promotion of Science, Japan.

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