Immunocytochemical and biochemical detection of the urokinase-type plasminogen activator receptor (uPAR) in the rat tooth germ and in lipid rafts of PMA-stimulated dental epithelial cells

Abstract

Urokinase-type plasminogen activator receptor (uPAR) regulates pericellular proteolysis by binding the serine proteinase urokinase-type plasminogen activator (uPA) that promotes cell surface activating of plasminogen to plasmin. In addition, uPAR as a glycosylphosphatidylinositol (GPI)-anchored signaling receptor affects cell migration, differentiation, and proliferation. The aim of the present study was to monitor the occurrence and distribution pattern of uPAR in cells of the rat molar tooth germ. By means of immunocytochemistry moderate, uPAR immunoreactivity was detected in epithelial cells of the enamel organ and in ameloblasts and odontoblasts. RT-PCR and Western blotting experiments demonstrated the expression of uPAR in phorbol 12-myristate 13-acetate (PMA)-stimulated dental epithelial cells (HAT-7 cells). A substantial part of uPAR was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. However, co-immunoprecipitation experiments showed that uPAR and caveolin-1 do not associate with each other directly. Cell stimulation experiments with PMA indicated that protein kinase C (PKC)-mediated signaling pathways contribute to the expression of uPAR in cells of the enamel organ. The localization of uPAR in membrane rafts provides a basis for further investigations on the role of uPAR-mediated signaling cascades in ameloblasts.