Aberrant Nuclear Translocation of E2F1 and Its Association in Cushing's Disease

Abstract

Nonsurgical medical treatments are often performed for Cushing's disease due to high recurrence rates. However, current medical treatment that targets corticotroph adenomas are limited. To develop a treatment that specifically targets corticotrophs in Cushing's disease, it is necessary to identify corticotroph lineage-specific proteins, which are involved in the Cushing's tumor phenotype. We have previously reported that the expression of E2F transcription factor 1 (E2F1), one of the cell cycle regulatory proteins, was increased in corticotrophs in Cushing's disease model mice and was involved in the regulation of POMC gene expression. Phosphorylation of Ser337 of E2F1 (pS337-E2F1) facilitates its binding to the POMC promoter, which was suggested to contribute to elevated POMC expression in corticotrophs. Here, we report that E2F1 expression is specific to the corticotroph lineage in normal human pituitaries and that the E2F1 protein is localized in the cytosol in normal corticotrophs. We show that pS337-E2F1 is localized in the nucleus specifically in Cushing's tumors, while it is localized in the perinuclear cytoplasm in the normal pituitary. This observation demonstrates that pS337 is a marker for Cushing's tumors and suggests that phosphorylation of E2F1 may be a target for developing a novel pharmacological treatment for tumorigenesis and hormone dysregulation of Cushing's disease.

Keywords: Cushing’s disease; E2F1; POMC; corticotroph; pS337-E2F1.

Figure 1.

E2F1 corticotroph-specific expressions. (A) Autopsy-derived normal human pituitaries were immunostained with E2F transcription factor 1 (E2F1), proopiomelanocortin, and diaminopyrolylindole 4,6-diamino, 2-pyrolylindole (DAPI). E2F1 and POMC cytoplasmic colocalization was shown in merged images. (B) Autopsy derived normal human pituitaries were immunostained with E2F1 and pituitary hormones (growth hormone, thyroid-stimulating hormone, prolactin, and follicle-stimulating hormone) and DAPI. E2F1 and pituitary hormones were not colocalized. (C) Autopsy derived normal human testis were immunostained with E2F1 and DAPI. Colocalization of E2F1 and DAPI is shown. Scale bar, 50 µm.

Figure 2.

Perinuclear expression of pS337-E2F transcription factor 1 (E2F1) in human corticotrophs. (A) Autopsy derived normal human pituitaries were immunostained with E2F1, pS337-E2F1, proopiomelanocortin, and diaminopyrolylindole 4,6-diamino, 2-pyrolylindole (DAPI). Scale bar, 20 µm. (B) Autopsy derived normal human pituitaries were immunostained with pS337-E2F1 and lamin A/C (left), and plotted line scans of pS337-E2F1 and lamin A/C were shown on the right. Width of the nucleus were normalized in all cells analyzed, and the highest intensity of fluorescence was described as relative intensity 1.0 (see Materials and Methods). Area within the 2 arrows on the x-axis represents nucleus (N) area, and the area outside of the 2 arrows in the x-axis represents cytoplasm (C) area. Scale bar, 10 µm. (C) Autopsy-derived normal human testis were immunostained with pS337-E2F1, E2F1, and DAPI. Scale bar, 50 µm.

Figure 3.

Nuclear translocation of pS337-E2F transcription factor 1 (E2F1) in corticotroph adenomas. (A) Cushing’s tumors were immunostained with pS337-E2F1 and lamin A/C (left), and plotted line scans of pS337-E2F1 and lamin A/C were shown on the right. Scale bar, 10 µm. (B) Autopsy-derived normal pituitaries were immunostained with proopiomelanocortin, pS337-E2F1, and diaminopyrolylindole 4,6-diamino, 2-pyrolylindole (DAPI; left), and plotted line scans of pS337-E2F1 and DAPI are shown (right). Width of the nucleus were normalized in all cells analyzed, and highest intensity of fluorescence was described as relative intensity 1.0 (see Materials and Methods). Scale bar, 20 µm. (C) Nine samples of Cushing’s tumors (Cs 1-9) were immunostained with proopiomelanocortin, pS337-E2F1, and DAPI (left), and plotted line scans of pS337-E2F1 and DAPI are shown (right). Width of the nucleus were normalized in all cells analyzed, and the highest intensity of fluorescence was described as relative intensity 1.0 (see Materials and Methods). Scale bar, 20 µm. Characterizations of Cushing’s tumors are shown in Table 1. (D) The average of nuclear signal intensities of 3 samples of normal corticotrophs and 9 samples of Cushing’s tumors were plotted. The average of each group is shown.

Figure 4.

Schematic of E2F transcription factor 1 (E2F1) and pS337-E2F1 expressions in normal pituitary, corticotroph adenomas, and E2F1-medicated proopiomelanocortin gene regulations. (A) Schematic image of subcellular localizations of E2F1 and pS337-E2F1 expression in normal human corticotrophs (left) and in Cushing’s tumors (right). (B) Schematic image of pS337-E2F1 binding to the POMC promoter.