Morphological and Functional Analyses of the Tight Junction in the Palatal Epithelium of Mouse

Abstract

Tight junction (TJ) is one of the cell-cell junctions and known to have the barrier and fence functions between adjacent cells in both simple and stratified epithelia. We examined the distribution pattern, constitutive proteins, and permeability of TJ in the stratified squamous epithelium of the palatal mucosa of mice. Ultrastructural observations based on the ultrathin section and freeze-fracture methods revealed that poorly developed TJs are located at the upper layer of the stratum granulosum. The positive immunofluorescence of occludin (OCD), claudin (CLD)-1 and -4 were localized among the upper layer of the stratum granulosum showing a dot-like distribution pattern. And CLD-1 and -4 were localized among the stratum spinosum and the lower part of stratum granulosum additionally showed a positive reaction along the cell profiles. Western blotting of TJ constitutive proteins showed OCD, CLD-1, -2, -4, and -5 bands. The permeability test using biotin as a tracer revealed both the areas where biotin passed through beyond OCD positive points and the areas where biotin stopped at OCD positive points. These results show that poor TJs localize at the upper layer of the stratum granulosum of the palatal epithelium, and the TJs are leaky and include at least CLD-1 and -4.

Keywords: claudin; occludin; palatal epithelium; stratum granulosum; tight junction.

Fig. 1.

Structure of the mouse palatal epithelium. The palatal epithelium is composed of stratum basale, spinosum, granulosum, and corneum. The stratum basale was observed as a single layer, and the stratum spinosum consisted of several layers of cubic or cylindrical cells. The stratum granulosum showed flattened cell layers, and included fine granules in the cytoplasm. The stratum corneum was relatively thick. A toluidine blue stained semi-thin section. Bar = 20 μm.

Fig. 2.

Electron micrographs at the upper layer of stratum granulosum. (A) TJ is shown as a cell membrane fused point (an arrow) in the stratum granulosum by conventional electron microscopic observation. A thin section stained with uranium and lead citrate. (B) Lanthanum nitrate penetrated the intercellular spaces between neighboring cells of the stratum granulosum. TJ was identified by the interruption of lanthanum penetration (an arrow). The lanthanum impregnation method. (C) The freeze-fracture method revealed TJ strands (arrowheads) on the P face. The strands were poorly distributed, and the discontinuous sites of strands were frequently observed. Bars = 250 nm.

Fig. 3.

Immunohistochemistry of TJ constitutive proteins. The immunofluorescence of OCD (A), CLD-1 (C), and -4 (D) were localized between the upper layers of the stratum granulosum showing dot-like fluorescence (arrowheads). In addition, CLD-1 (C) and CLD-4 (D) showed network-like positive reaction from the lower layer of the stratum granulosum to the upper layer of the stratum spinosum. CLD-2 (F) and CLD-5 (G) were positive in the salivary glands and the endothelium of blood vessel respectively, but were negative in the palatal epithelium. Background staining levels were checked by omitting the primary antibodies (B: rabbit anti-goat IgG labeled with Alexa Fluor 488, E: goat anti-rabbit IgG labeled with Alexa Fluor 488). SC: stratum corneum, SB: stratum basale, Blue: nuclei stained with Hoechst. Bars = 25 μm (A, B, C, D, E), 50 μm (F), 10 μm (G).

Fig. 4.

Western blotting of the palatal mucosa. OCD, CLD-1, -2, -4, -5 were positive, but CLD-3 was negative in the palatal mucosa.

Fig. 5.

Permeability of the palatal epithelium with the use of biotin as a tracer. Biotin penetrated between the intercellular spaces from the stratum basale toward the stratum corneum in the palatal epithelium. The sites where biotin was stopped at the OCD positive sites (arrowheads) were observed. On the other hand, the sites where biotin passed through the OCD positive sites were also shown (arrow). SC: stratum corneum, Red: biotin, Green: OCD, Blue: nuclei. Bar = 25 μm.