Enhanced LH action in transgenic female mice expressing hCGbeta-subunit induces pituitary prolactinomas; the role of high progesterone levels

Abstract

The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) beta-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P(4)) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P(4) levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P(4) treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P(4) was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P(4). If extrapolated to humans, and given the importance of endogenous P(4) and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas.

Figure 1

Immunohistochemical evaluation of Ki-67 expression and density of proliferating cells in the pituitary glands of wild-type (WT) and transgenic (TG) female mice. Representative immunohistochemical images of Ki-67 staining are shown in 6-month-old WT, and 4-, 6-, and 12-month-old TG mouse samples. The arrowheads indicate Ki-67-positive cells. A large part of the 12-month-old TG sample comprises a macroprolactinoma with numerous Ki-67-positive cells. Scale bars are 100 μm, and the insets are larger magnifications of the same samples (scale bar=25 μm). Numeric data on density of proliferating cells/mm² in the pituitary glands are presented on the right side. The WT values are the mean of the 4-, 6-, and 12-month-old mice. Different superscript letters indicate that these groups differ significantly (P<0.05); three to seven mice were analyzed per group (mean±s.e.m.), and at least two sections were calculated per specimen. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-10-0016.

Figure 2

Pituitary weights and serum PRL concentrations in the WT and TG mice, and their responses to hormonal manipulations in the antagonist treatment (A and B) and hormone treatment (C and D) experiments, as explained in Table 1. Pituitary weights (A and C) and serum PRL levels (B and D) of WT control (open bars) and TG (filled bars) mice were measured (mean±s.e.m.; 5–16 mice/group). In A and B, the data are obtained from 6-month-old mice gonadectomized (Gx) or sham-operated (C) at 2 months of age, followed by treatment between 2 and 6 months of age with bromocriptine (Br), mifepristone (Mf), tamoxifen (Tx), or Mf+Tx combination. In addition, one group of mice received short Tx treatment between 2 weeks and 2 months (sTx; to abolish the peripubertal E₂ peak), and in one group, the latter was followed by a 2–6-month-treatment with Mf (sTxMf). In C and D, the data are obtained from 4.5-month-old mice with Gx at 2 months of age, followed by 2.5-month treatments with vehicle (Gx), E₂ (Gx+E₂), P₄ (Gx+P₄), or their combination (Gx+E₂P₄). For marking of statistical differences, each bar is provided by superscript letters. If all letters between two bars are different (e.g. ab and cd), then they differ significantly at P<0.05. If any of the superscript letters of two bars is the same (e.g. ab and bc), then there is no statistically significant difference between them.

Figure 3

Pituitary expression of Ccnd1, E2f1, Hmga2, CDK4, and RB in WT and TG female mice. (A–C) depict the levels of Ccnd1, E2fi, and Hmga2 respectively as measured by qRT-PCR, in WT (open bars) and TG (filled bars) mice at 2, 4, 6, and 12 months of age (mean±s.e.m. of 4–6 mice/group). Bars with different superscript letters differ significantly (P<0.05). (D) Shows representative micrographs of CCND1, CDK4, and RB immunohistochemistry of WT and TG mice at the ages (mo) indicated. The arrows indicate nuclei positive for the respective stainings. Note the weak nuclear staining of CCND1 and CDK4 in WT mice. Similar expression patterns were observed with CCND1 and CDK4. The bottom row of panels presents RB expression, which shows strong nuclear staining in WT but weak in TG mice, especially in older ages. The scale bars are 25 μm in the insets. Full colour version of this figure available via http://dx.doi.org/10.1677/ERC-10-0016.

Figure 4

Pituitary expression levels of selected genes in WT and TG mice, and their responses to hormonal manipulations. The genes studied were Ccnd1 (A), E2f1 (B), Hmga2 (C), Pgr (D), Gal (E), and Nog (F). For treatments and abbreviations, see Table 1, antagonist treatments. The results shown are mean±s.e.m. of 3–5 mice per group. Bars with different superscript letters differ significantly (P<0.05).

Figure 5

Effects of E₂, P₄, and their combinations on cell proliferation and signaling in primary mouse pituitary cells and rat somatomammotroph GH3 cells. (A) Expression of Ccnd1 mRNA in primary pituitary cells isolated from 5-month-old TG mice and stimulated for 24 h as indicated. The results are mean±s.e.m. of five individual experiments. Different letters indicate significant differences (P<0.05) between the treatment groups. (B) Effects of 48 h P₄ stimulation on GH3 cell proliferation when co-treated with E₂ and/or mifepristone (Mf). The mean cell number in controls was taken as 100%, and those of the other treatments were related to this figure (mean±s.e.m. of 5–6 independent experiments). *P<0.05, **P<0.01 versus basal plus 100 pM E₂; ^†P<0.05, ^††P<0.01 vs 0.1 nM P₄; ^‡P<0.05, ^‡‡P<0.01 vs 1 nM P₄; ^§P<0.01 vs 0.1 and 1 nM P₄+1000 pM E₂+10 μM Mf.