Differential localization of laminin γ2 and integrin β4 in primary cultures of the rat gingival epithelium

Objectives: The aim of this study was to investigate the differential immunolocalization of laminin γ2 and integrin β4 in primary cultures of the rat gingival epithelium. Methods: The gingival epithelium was obtained from Sprague‐Dawley rats and was cultured in serum‐free keratinocyte growth medium (DK‐SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno‐gold labeling for laminin γ2 and integrin β4 were employed. CLSM images for laminin and integrin were analyzed in horizontal (x–y axis) and in vertical (x–z axis) sections. Results: Both laminin γ2 and integrin β4 were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin γ2 was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin β4 was localized diffusely in the cytoplasm. F‐actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x–z axis images obtained by CLSM, laminin γ2 was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin β4 existed in the area where F‐actin was labeled surrounding the membrane. Immuno‐electron microscopy showed that 10nm colloidal gold particles indicating laminin γ2 were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin β4 was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. Conclusions: In primary cultures of the rat gingival epithelium, both laminin γ2 and integrin β4 may be produced by the epithelium, and irregular rings of laminin γ2 are formed in areas where gingival cells adhere to the extracellular matrix.