Aryl-hydrocarbon receptor activity modulates prolactin expression in the pituitary

Abstract

Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotrope and lactotrope adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferation and endogenous pituitary hormone expression in the GH3 rat somatolactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction in pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggest that environmental dioxins can exert endocrine disrupting effects at the pituitary.

Figure 1. β-naphthoflavone alone and with α-naphthoflavone activated Cyp1a1 and suppressed AhR expression

Real time quantitative reverse transcriptase PCR (qRT-PCR) showed significantly higher levels of Cyp1a1 at 1 μM β-naphthoflavone and both 100 nM and 1 μM β-naphthoflavone plus 100 nM α-naphthoflavone (1A). AhR was significantly reduced at 10 nM and 100 nM β-naphthoflavone and at all doses of β-naphthoflavone plus α-naphthoflavone in cultured GH3 cells (1B). Levels of ERα (Esr1) and ERβ (Esr2) mRNA were also mildly affected, based on treatment (1C). mRNA values normalized to beta-actin, n=3.

Figure 2. Growth hormone is unaffected but prolactin is suppressed by β-naphthoflavone

Growth hormone mRNA levels remained largely unchanged, however Prl was significantly reduced with all doses of β-naphthoflavone and of β-naphthoflavone plus α-naphthoflavone in cultured GH3 cells (2A). mRNA values normalized to beta-actin, n=3.

Figure 3. AhR suppressed TGFβ mRNA expression

A significant reduction in TGFβ mRNA occurred at 10 nM and 100 nM β-naphthoflavone and all doses of β-naphthoflavone plus α-naphthoflavone in cultured GH3 cells. mRNA values normalized to beta-actin, n=3.

Figure 4. Prolactin and gonadotropin subunit mRNA expression were reduced in Ahr^−/− females

qRT-PCR performed on isolated female P3 pituitaries from Ahr^−/− and wildtype littermates shows a significant decrease in Prl (p=0.0002) (A), as well as Cga (p=0.0321), Lhb (p=0.0035), and Fshb (p=0.02) (C). Gh levels remained unchanged (A), n=3 individual pituitaries of each genotype. qRT-PCR performed on isolated female P90-100 pituitaries from Ahr^−/− and wildtype littermates shows no change in Gh or Prl at this age. (B) n≥5 individual pituitaries for each genotype. All mRNA values were normalized to Gapdh.

Figure 5. AhR activation with β-naphthoflavone did not affect the proliferative capacity of GH3 pituitary cells

β-naphthoflavone at 1 and 10 μM did not alter the percentage of GH3 cells in G1, S, or G2 phases of the cell cycle in either standard (5A) or charcoal dextran treated serum, phenol red free medias (5B). Staining occurred by propidium iodide, and cell cycle analyzed by FACS, n=3.