The expression of Wnt4 is regulated by estrogen via an estrogen receptor alpha-dependent pathway in rat pituitary growth hormone-producing cells

Abstract

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone beta (TSHbeta)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway.

Keywords: Wnt signaling; estrogen; estrus cycle; growth hormone; pituitary gland.

Fig. 1

Absorption testing. One-milliliter aliquots of anti-Wnt4 antibody solution (2.5 µg/mL) were reacted with (A) 0 µg, (B) 0.004 µg, (C) 0.02 µg, (D) 0.1 µg or (E) 0.5 µg of synthetic Wnt4 peptide (Abcam) and then the reacted solution was applied to sections of rat normal pituitary gland. Original magnification ×400; Bar=100 µm. Note the dose-dependent decrease in staining by absorption.

Fig. 2

Colocalization of Wnt4 with pituitary hormones in pituitary glands of sham-operated rats. Expression of Wnt4 and pituitary hormones shown by double immunofluorescence staining with confocal microscopy, stained for Wnt4 (green) and (A) GH (red), (B) TSHβ, (C) PRL, (D) ACTH, (E) LHβ or (F) αGSU. Nuclei were stained with TOTO-3 (blue). Wnt4 was expressed in almost all growth hormone (GH)-producing cells, while only a few of Wnt4-positive cells were also expressed in thyroid stimulating hormone (TSH)β-producing cells (see arrow). Original magnification ×630; Bar=20 µm.

Fig. 3

Colocalization of estrogen receptor alpha (ERα) with GH- or TSHβ-producing cells in pituitary glands of sham-operated rats. Expression of ERα and (A) GH- and (B) TSHβ-producing cells shown by double immunohistochemical staining, stained for ERα (brown) and GH- and TSHβ-producing cells (blue).

Fig. 4

Effect of estrogen on Wnt4 and Wnt5a mRNA expression levels in rat pituitary glands. Expression levels of (A) Wnt4 and (C) Wnt5a mRNA in 4-weeks ovariectomized rats injected with estrogen for 7 d were measured by real-time reverse transcription polymerase chain reaction (RT-PCR), then normalized to Gapdh as an internal standard. Data are expressed as fold changes from the sham-operated control group. Each value represents the mean±SD of six animals per group. *P<0.01, **P<0.001. (B) Expression of Wnt4 protein was analyzed by immunohistochemical staining. Original magnification ×400. Bar=100 µm.

Fig. 5

Wnt4 and Wnt5a mRNA levels during the estrus cycle. Expression levels of (A) Wnt4 and (B) Wnt5a mRNA in pituitary in the different stages of estrus cycle were measured by real-time RT-PCR, then normalized to Gapdh as an internal standard. Data are expressed as fold change from proestrus. Each value represents the mean±SD of eight animals per group. *P<0.01, **P<0.001. (C) The concentration of plasma estrogen in Sprague Dawley rats peaks in proestrus [36, 37].

Fig. 6

Time-dependent effect of E2 on Wnt4 mRNA expression in rat somatotrophic pituitary tumor (MtT/S) cells. Cells were treated with E2 at 10⁻⁹ M for 2, 6 and 10 hr. Expression levels of Wnt4 mRNA in MtT/S cells were measured by real-time RT-PCR, then normalized to Gapdh as an internal standard. Each value represents the mean±SD of three per group. *P<0.05.

Fig. 7

The pure ER antagonist ICI 182,780 reverses the effects of E2 on the expression of Wnt4 mRNA in MtT/S cells. Cells were incubated with 10⁻⁹ M E2 and/or 10⁻⁸ M to 10⁻⁶ M ICI 182,780 for 2 hr. Expression levels of Wnt4 mRNA in MtT/S cells were measured by real-time RT-PCR, then normalized to Gapdh as an internal standard. Data are expressed as fold changes from vehicle control. Each value represents the mean±SD of seven per group. *P<0.001.

Fig. 8

Detection of ERα and ERβ mRNA expression in MtT/S cells by RT-PCR analysis. Lane 1, normal rat anterior pituitary gland (positive control); lane 2, MtT/S cells; lane 3, no template cDNA (negative control). Top panel: ERα expression was detected as a 134 bp fragment. Middle panel: ERβ expression was detected as a 262 bp fragment. Bottom panel: Gapdh expression was detected as a 136 bp fragment. All products were amplified for 30 cycles.