Effect of E-cadherin expression on hormone production in rat anterior pituitary lactotrophs in vitro

Abstract

Cadherins are a family of transmembrane glycoproteins that mediate cell-to-cell adhesion. A change in cadherin type in cells, i.e., cadherin switching, induces changes in the character of the cell. Recent studies of the developing rat adenohypophysis found that primordial cells co-expressed E- and N-cadherins, but that hormone-producing cells lost E-cadherin and ultimately possessed only N-cadherin. In the present study, we examined the roles of cadherin switching in cytogenesis of anterior pituitary cells by observing prolactin mRNA and protein expression in lactotrophs that were transformed with an E-cadherin expression vector. In hormone-producing cells that were transfected with a pIRES2-ZsGreen1 plasmid with a full-length E-cadherin cDNA (rE-cad-IZ) insert in primary culture, we detected E- and N-cadherins on plasma membrane and E-cadherin in cytoplasm. In these rE-cad-IZ-transfected cells, in situ hybridization revealed prolactin mRNA signals that were at a level identical to that in control cells, while prolactin protein was barely detectable using immunocytochemistry. The mean signal intensity of prolactin protein in rE-cad-IZ-transfected cells was approximately one fourth that in intact cells and in null-IZ-transfected cells (P<0.01). These results suggest that the expression of E-cadherin does not affect prolactin mRNA transcription; rather, it reduces prolactin protein content, presumably by affecting trafficking of secretory granules.

Keywords: E-cadherin; anterior pituitary; hormone production; prolactin; transfection.

Fig. 1

Immunohistochemistry of E-cadherin and N-cadherin in transformed anterior pituitary cells. Anterior pituitary cells were transfected with pIRES2-ZsGreen1 plasmid with a null-insert, null-IZ (A, C, E and G, in the identical field), and the same plasmid with cDNA for the entire region of rat E-cadherin, rE-cad-IZ (B, D, F and H, in the identical field). A and B: Differential interference contrast images of transformed cells. C and D: Merged images of immunocytochemistry for E-cadherin (light blue), N-cadherin (red), and fluorescence from ZsGreen1 (green). E and F: Immunoreaction of N-cadherin. G and H: Immunoreaction of E-cadherin. Immunocytochemistry for E-cadherin and a phase-contrast image of transfected hormone-producing cells sorted by fluorescence-activated cell sorting are also shown in I and J. I: Purified hormone-producing cells were transfected with null-IZ. J: Purified hormone-producing cells were transfected with rE-cad-IZ. The differential interference contrast image is merged. The differential interference contrast image is merged. Arrows and arrowheads indicate cells transfected with rE-cad-IZ and null-IZ, respectively. A small arrow shows the boundary of cells transfected with rE-cad-IZ. Bar=5 µm.

Fig. 2

In situ hybridization and immunocytochemistry of transfected anterior pituitary cells. Anterior pituitary cells in primary culture were transfected with null-IZ (A, C, E and G, in the identical field) and with rE-cad-IZ (B, D, F and H, in the identical field). A and B: Differential interference contrast images of transformed cells. C and D: Merged images of immunoreaction of prolactin protein (red), in situ hybridization signal for prolactin mRNA (light blue), and fluorescence from ZsGreen1 (C and D). E and F: Immunoreaction of prolactin. G and H: In situ hybridization signals for prolactin mRNA. Arrows and arrowheads show cells transfected with rE-cad-IZ and null-IZ, respectively. Bar=5 µm.

Fig. 3

Fluorescence intensities of immunocytochemistry for prolactin protein in prolactin mRNA-positive cells. Fluorescence intensities were quantified from intact rE-cad-IZ-transfected cells and null-IZ-transfected cells. Each group consisted of 30 prolactin mRNA-positive cells. The fluorescence intensities are expressed as the ratio to the mean value of the intact group. Open and solid columns represent the fluorescence intensity of prolactin immunoreactivity and prolactin mRNA, respectively. Data are mean±S.E. *: statistically significant (P<0.01 by the Bonferroni test).