Biochemical and Biophysical Research Communications
Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells
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.
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Fellow, the Japan Society for the Promotion of Science, Japan.