Expression of the novel integrin-related protein TIED in human tumor cells and mouse development

Abstract

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Integrins are dynamic cell adhesion molecules involved in cell motility and tumor metastasis. We previously described a novel integrin-like molecule termed TIED, after sequencing a 2.5 kb cDNA predicted to encode a secreted protein of 51.4 kDa. TIED contains 10 repeats with extensive homology to the epidermal growth factor (EGF)-like motif present in 4 copies within integrin β subunits. Given the modular nature of these repeats, and the known activity of the motif in modulating integrin ligand-binding and signalling, our hypothesis is that TIED plays a role in regulating integrin-mediated cell adhesion during normal development and tumor growth and progression. We used northern blotting and reverse transcription followed by polymerase chain reaction (RT-PCR) to quantitate TIED mRNA levels in tumor cell lines and normal mouse tissues. TIED mRNA was detected by RT-PCR and northern blot in human U-2 OS, HOS and MG-63 osteosarcoma cell lines, but the Saos-2 osteosarcoma line was negative in RT-PCR. In addition, RT-PCR revealed TIED mRNA in human H28, 211H and H2052 mesothelioma cell lines, but not in MCF-7 or MDA-MB-435 breast nor HT-29 colon carcinoma lines. We are currently exploring the functions of TIED in osteosarcoma cells by the complementary approaches of overexpressing TIED cDNA in Saos-2 cells and reducing endogenous TIED expression in U-2 OS cells by RNA interference. TIED mRNA was not detectable by northern blot analysis of RNA from various adult mouse tissues. However, TIED was readily detected by RT-PCR of RNA isolated from brain and thymocytes of 2 week-old mice. Furthermore, northern blot analysis of RNA isolated from mouse embryos at various stages of development showed TIED mRNA expression at days E6.5 and E8.5, but not at later stages. In order to analyse TIED protein expression and structure, an expression vector encoding full-length human TIED with a C-terminal c-myc-derived peptide was transfected into human HEK293 cells. Western blots probed with the anti-c-myc 9E10 antibody demonstrated a 35 kDa band both in cell extracts and cell culture medium, although the fusion protein was expected to be 54 kDa. Thus the TIED-c-myc fusion protein was secreted but may be cleaved or partially degraded. A 10 amino acid peptide was synthesized and used as an immunogen to raise rabbit polyclonal antibodies. Preliminary results indicate that these sera recognize a 55 kDa protein in U-2 OS cell extracts and cell culture medium. TIED protein expression in tumor cells and in the developing mouse will be verified, pending validation of these antibodies. The transient presence of mouse TIED mRNA early in development, as well as in the maturing thymus, suggest a role for TIED in modulating integrin activity and signalling during embryogenesis and/or T cell development. The full extent of the role of TIED in tumor growth and progression remains to be determined.