Immunoelectron microscopic study of podoplanin localization in mouse salivary gland myoepithelium

Abstract

We have recently reported that salivary gland cells express the lymphatic endothelial cell marker podoplanin. The present study was aimed to immunohistochemically investigate the expression of the myoepithelial cell marker α-smooth muscle actin (SMA) on podoplanin-positive cells in mouse parotid and sublingual glands, and to elucidate podoplanin localization in salivary gland myoepithelial cells by immunoelectron microscopic study. The distribution of myoepithelial cells expressing podoplanin and α-SMA was examined by immunofluorescent staining, and the localization of reaction products of anti-podoplanin antibody was investigated by pre-embedded immunoelectron microscopic method. In immunohistochemistry, the surfaces of both the mucous acini terminal portion and ducts were covered by a number of extensive myoepithelial cellular processes expressing podoplanin, and the immunostaining level with anti-podoplanin antibody to myoepithelial cells completely coincided with the immunostaining level with anti-α-SMA antibody. These findings suggest that podoplanin is a salivary gland myoepithelial cell antigen, and that the detection level directly reflects the myoepithelial cell distribution. In immunoelectron microscopic study, a number of reaction products with anti-podoplanin antibody were found at the Golgi apparatus binding to the endoplasmic reticulum in the cytoplasm of myoepithelial cells between sublingual gland acinar cells, and were also found at the myoepithelial cell membrane. These findings suggest that salivary gland myoepithelial cells constantly produce podoplanin and glycosylate at the Golgi apparatus, and transport them to the cell membrane. Podoplanin may be involved in maintaining the homeostasis of myoepithelial cells through its characteristic as a mucin-type transmembrane glycoprotein.

Keywords: myoepithelium; podoplanin; salivary gland; α-smooth muscle actin.

Fig. 1

Whole staining of sublingual gland with anti-podoplanin and anti-α-SMA. Concanavalin A (Con A) was used to visualize the cell membrane of the acinar terminal portion in red. Terminal portion surfaces of mucous acini are covered by a number of extensive myoepithelial cellular processes with green fluorescence immunostaining by anti-podoplanin of which the degree and extent completely coincide with anti-α-SMA immunostaining. Bar=100 µm.

Fig. 2

Comparison of immunostaining by anti-podoplanin and anti-α-SMA between parotid and sublingual glands. In mouse parotid glands the terminal secretory end pieces of acini were densely composed of serous pyramidal cells with basophilic cytoplasm. In sublingual gland the terminal secretory end pieces of acini were composed of large mucus-filled cells with cytoplasm which was less basophilic than that of the parotid glands. The portion around the acinar cells is immunostained by both anti-podoplanin and anti-α-SMA (merged, in yellow). In the sublingual gland mucous acini there are more parts that are immunostained with anti-podoplanin in the sublingual gland mucous acini than in the parotid gland serous acini. The basal side of the ducts is also immunostained with anti-podoplanin in both the parotid and sublingual glands at the same level, and to a stronger extent than with anti-α-SMA (arrowheads, intercalated duct). There are more parts that are immunostained with anti-α-SMA than with anti-podoplanin. Reaction products with anti-α-SMA are also found in the center of the acinar and at the luminal side of the duct where reaction products with anti-podoplanin are not found. Bar=100 µm.

Fig. 3

Immunoelectron microscopy for podoplanin production in the cytoplasm of salivary gland myoepithelial cells. (A) A strong density of immunostaining with anti-podoplanin is found in the region of the cell membrane and in the cytoplasm of myoepithelial cells between sublingual gland acinar cells. Some reaction products of anti-podoplanin are also observed at the basal side in the cytoplasm of the acinar cells. Bar=10 µm. (B) In the higher magnification of the cell highlighted in A (box), a number of reaction products with anti-podoplanin are found at the Golgi apparatus binding to endoplasmic reticulum (arrowheads). Bar=2 µm.

Fig. 4

Immunoelectron microscopy for the membrane localization of podoplanin in salivary gland myoepithelial cells. (A) A strong density of immunostaining with anti-podoplanin is found at the cell membrane and in the cytoplasm of myoepithelial cells between sublingual gland acinar cells. Bar=10 µm. (B) A number of small DAB granules with strong density by immunostaining with anti-podoplanin are found at the myoepithelial cell membrane between sublingual gland acinar cells (arrowheads). Bar=2 µm.