Endocrine Abstracts (2008) 16 P749

ProEGF cytoplasmic domain (proEGFcyt)-mediated up-regulation of SNAP25 decreases cathepsin-L secretion and elastinolytic activity in human thyroid carcinoma cells.

Aleksandra Glogowska1, Astrid Kehlen4, Ekkehard Weber5, Marek Los2, Cuong Hoang-Vu6, Thomas Klonisch1 & Thomas Klonisch3

1Department of Human Anatomy and Cell Science, Winnipeg, MB, Canada; 2Manitoba Institute of Cell Biology Cancer Care Manitoba, Winnipeg, MB, Canada; 3Medical Microbiology and Infectious Diseases, Winnipeg, MB, Canada; 4Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany; 5Physiological Chemistry, Martin Luther University Halle-Wittenberg, Halle, Germany; 6Clinics of Surgery, Martin Luther University Halle-Wittenberg, Halle, Germany.

The cytoplasmic domains of EGF-like ligands have important biological functions. Stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 over-expressing the cytoplasmic domain of proEGF (FTC-133-proEGFcyt) demonstrated a transcriptional up-regulation of the lysosomal hydrolases cathepsin- (cath-) B and -D and alterations in the processing of cath-L protein. Cath-L has strong elastinolytic activity and was the only of the three cathepsins to be secreted by FTC-133 transfectants. FTC-133-proEGFcyt clones displayed a markedly reduced ability to migrate through elastin matrices when compared with mock transfectants (empty plasmid) or a natural proEGFsplice mutant construct. Decreased migration through elastin matrix coincided with a reduction of cath-L secretion in FTC-133-proEGFcyt clones. When incubated with a specific cath-L inhibitor, FTC-133-proEGFsplice and mock transfectants showed a similar reduction in elastinolytic activity implicating cath-L to be largely responsible for the elastinolytic activity in our elastin micration assays. Down-regulation of cath-L in FTC-133-proEGFcyt coincided with an upregulation of the t-SNARE component SNAP25 as determined by specific siRNA knock-down of SNAP25. Incubation of FTC-133-proEGFcyt with soluble EGF reduced SNAP25 protein levels in proEGFcyt transfectants and, thus, reversed the inhibitory actions of proEGFcyt on elastinolytic migration. This antagonistic EGF action was mediated by the EGFR. In summary, we have identified proEGFcyt as a novel regulator of the function of the t-SNARE membrane-vesicle fusion complex involved in the exocytosis/secretion of elastinolytic cath-L. This mechanism facilitates the suppressive role of proEGFcyt domain on thyroid carcinoma cell elastinolytic activity and invasiveness. Partly supported by Krebshilfe.

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