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The influence of 17beta-estradiol on annexin 1 expression in the anterior pituitary of the female rat and in a folliculo-stellate cell line.

Davies E, Omer S, Morris JF, Christian HC - J. Endocrinol. (2007)

Bottom Line: Administration of 17beta-estradiol (1 microg/100 g) significantly (P<0 x 01) increased anterior pituitary ANXA1 expression in the ovariectomized animals.Treatment of TtT/GF cells, a folliculo-stellate cell line, with 17beta-estradiol (1 x 8-180 nM) increased ANXA1 mRNA expression and increased the amount of ANXA1 protein externalized in response to a dexamethasone stimulus.These results indicate that 17beta-estradiol stimulates ANXA1 expression in the anterior pituitary and in vivo an adrenal factor contributes to the mechanism of action.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.

ABSTRACT
Annexin 1 (ANXA1) is a Ca2+- and phospholipid-binding protein that plays an important role as a mediator of glucocorticoid action in the host-defence and neuroendocrine systems. Sex differences in hypothalamo-pituitary-adrenal (HPA) axis activity are well documented and a number of studies have demonstrated that gonadal steroids act as regulators of HPA activity. The aim of this study was to investigate the effect of ovariectomy and 17beta-estradiol replacement, and estrous cycle stage, on anterior pituitary ANXA1 content. The amount of anterior pituitary ANXA1 determined by western blotting varied with estrous cycle stage with a peak at estrus declining to a trough at proestrus. Ovariectomy resulted in a significant (P<0 x 05) decrease in anterior pituitary ANXA1 content. Administration of 17beta-estradiol (1 microg/100 g) significantly (P<0 x 01) increased anterior pituitary ANXA1 expression in the ovariectomized animals. In contrast, there was no change in pituitary ANXA1 content in response to 17beta-estradiol in adrenalectomized and adrenalectomized/ovariectomized rats. Treatment of TtT/GF cells, a folliculo-stellate cell line, with 17beta-estradiol (1 x 8-180 nM) increased ANXA1 mRNA expression and increased the amount of ANXA1 protein externalized in response to a dexamethasone stimulus. These results indicate that 17beta-estradiol stimulates ANXA1 expression in the anterior pituitary and in vivo an adrenal factor contributes to the mechanism of action.

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(A) Effect of 17β-estradiol (E2; 1·8–180 nM, 24 h) and dexamethasone (Dex; 0·1 μM, 24 h) on the amount of ANXA1 mRNA in TtT/GF cells measured by quantitative PCR. Values expressed as means±s.e.m., n=3 experiments *P<0·01 versus negative control, ANOVA. (B) Effect of E2 (1·8–180 nM, 24 h) on the amount of ANXA1 protein in TtT/GF cells. Western blot: lanes 1 and 2, control; lanes 3 and 4, 1·8 nM E2-treated; lanes 5 and 6, 18 nM E2-treated; lanes 7 and 8, 180 nM E2-treated. (C) and (D) Integrated densitometry data of the effects of (C) increasing concentrations of E2 (0·18–1800 nM, 24 h) and (D) duration of treatment with 18 nM E2 (3–24 h) on the amount of ANXA1 in TtT/GF cells. Open columns, vehicle control; filled columns, 17β-estradiol-treated. Values expressed as means±s.e.m., n=3 experiments; no significant differences measured versus control, ANOVA.
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fig5: (A) Effect of 17β-estradiol (E2; 1·8–180 nM, 24 h) and dexamethasone (Dex; 0·1 μM, 24 h) on the amount of ANXA1 mRNA in TtT/GF cells measured by quantitative PCR. Values expressed as means±s.e.m., n=3 experiments *P<0·01 versus negative control, ANOVA. (B) Effect of E2 (1·8–180 nM, 24 h) on the amount of ANXA1 protein in TtT/GF cells. Western blot: lanes 1 and 2, control; lanes 3 and 4, 1·8 nM E2-treated; lanes 5 and 6, 18 nM E2-treated; lanes 7 and 8, 180 nM E2-treated. (C) and (D) Integrated densitometry data of the effects of (C) increasing concentrations of E2 (0·18–1800 nM, 24 h) and (D) duration of treatment with 18 nM E2 (3–24 h) on the amount of ANXA1 in TtT/GF cells. Open columns, vehicle control; filled columns, 17β-estradiol-treated. Values expressed as means±s.e.m., n=3 experiments; no significant differences measured versus control, ANOVA.

Mentions: In order to explore the possibility of direct actions of 17β-estradiol on ANXA1 expression in FS cells, the effects of graded concentrations of 17β-estradiol (0·18–1800 nM) on ANXA1 mRNA and protein content in the TtT/GF FS cell line were investigated. Initial immunofluorescence microscopy studies localized the subcellular distribution of ERα and ERβ in TtT/GF cells (Fig. 4). In control- and 17β-estradiol-treated (24 h, 180 nM) conditions, strong immunofluorescent staining for ERα (Fig. 4A–F) and ERβ (Fig. 4G–L) was localized to the nucleus with weaker immunofluorescence extended throughout the cytoplasm. No non-specific immunolabeling was detected in absorbed antibody controls (Fig. 4M and N). The effects of incubating TtT/GF cells for 24 h with increasing concentrations of 17β-estradiol (0·18–1800 nM) on the amount of ANXA1 mRNA and protein are shown in Fig. 5. Real-time PCR assay of ANXA1 mRNA in TtT/GF cells confirmed that the positive control stimulus dexamethasone (0·1 μM, 24 h) produced a significant (P<0·05) increase in ANXA1 mRNA and revealed that 17β-estradiol (1·8–180 nM, 24 h) caused a concentration-dependent increase in ANXA1 mRNA (Fig. 5A). In contrast, western blot analysis demonstrated no significant effect of graded concentrations of 17β-estradiol (0·18–1800 nM, 24 h; Fig. 5B and C) and no significant effect of 17β-estradiol (18 nM) treatment for various periods (0–24 h; Fig. 5D) on ANXA1 protein content in TtT/GF cells. However, pretreatment of TtT/GF cells with 17β-estradiol (100 nM, 24 h) significantly (P<0·05) increased the amount of surface ANXA1 detected in response to dexamethasone-stimulated (0·1 μM, 3 h) externalization (Fig. 6A, lanes 7 and 8 17β-estradiol and dexamethasone-treated versus lanes 3 and 4 control- and dexamethasone-treated; Fig. 6D), whereas the amount of intracellular ANXA1 measured was unaffected (Fig. 6C and E). No actin ir bands were detected in cell surface EDTA washes (Fig. 6B) confirming cell integrity. In subsequent experiments, the effect of co-treatment of TtT/GF cells with 17β-estradiol (18 nM) and dexamethasone (0·1 μM) for 24 h on ANXA1 content was measured (Fig. 7). Western blot analysis confirmed that dexamethasone alone significantly (P<0·05) increased ANXA1 content, whereas 17β-estradiol treatment (18 nM) alone had no significant effect (Fig. 7A, lanes 1 and 2 control versus lanes 3 and 4 dexamethasone-treated versus lanes 5 and 6 17β-estradiol-treated). However, co-treatment of TtT/GF cells with 17β-estradiol (18 nM) and dexamethasone (0·1 μM) for 24 h significantly (P<0·05) enhanced ANXA1 content compared with dexamethasone treatment alone (Fig. 7A, lanes 7 and 8 dexamethasone and 17β-estradiol-treated versus lanes 3 and 4 dexamethasone alone). However, no change was observed in the amount of β-actin detected (Fig. 7B).


The influence of 17beta-estradiol on annexin 1 expression in the anterior pituitary of the female rat and in a folliculo-stellate cell line.

Davies E, Omer S, Morris JF, Christian HC - J. Endocrinol. (2007)

(A) Effect of 17β-estradiol (E2; 1·8–180 nM, 24 h) and dexamethasone (Dex; 0·1 μM, 24 h) on the amount of ANXA1 mRNA in TtT/GF cells measured by quantitative PCR. Values expressed as means±s.e.m., n=3 experiments *P<0·01 versus negative control, ANOVA. (B) Effect of E2 (1·8–180 nM, 24 h) on the amount of ANXA1 protein in TtT/GF cells. Western blot: lanes 1 and 2, control; lanes 3 and 4, 1·8 nM E2-treated; lanes 5 and 6, 18 nM E2-treated; lanes 7 and 8, 180 nM E2-treated. (C) and (D) Integrated densitometry data of the effects of (C) increasing concentrations of E2 (0·18–1800 nM, 24 h) and (D) duration of treatment with 18 nM E2 (3–24 h) on the amount of ANXA1 in TtT/GF cells. Open columns, vehicle control; filled columns, 17β-estradiol-treated. Values expressed as means±s.e.m., n=3 experiments; no significant differences measured versus control, ANOVA.
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fig5: (A) Effect of 17β-estradiol (E2; 1·8–180 nM, 24 h) and dexamethasone (Dex; 0·1 μM, 24 h) on the amount of ANXA1 mRNA in TtT/GF cells measured by quantitative PCR. Values expressed as means±s.e.m., n=3 experiments *P<0·01 versus negative control, ANOVA. (B) Effect of E2 (1·8–180 nM, 24 h) on the amount of ANXA1 protein in TtT/GF cells. Western blot: lanes 1 and 2, control; lanes 3 and 4, 1·8 nM E2-treated; lanes 5 and 6, 18 nM E2-treated; lanes 7 and 8, 180 nM E2-treated. (C) and (D) Integrated densitometry data of the effects of (C) increasing concentrations of E2 (0·18–1800 nM, 24 h) and (D) duration of treatment with 18 nM E2 (3–24 h) on the amount of ANXA1 in TtT/GF cells. Open columns, vehicle control; filled columns, 17β-estradiol-treated. Values expressed as means±s.e.m., n=3 experiments; no significant differences measured versus control, ANOVA.
Mentions: In order to explore the possibility of direct actions of 17β-estradiol on ANXA1 expression in FS cells, the effects of graded concentrations of 17β-estradiol (0·18–1800 nM) on ANXA1 mRNA and protein content in the TtT/GF FS cell line were investigated. Initial immunofluorescence microscopy studies localized the subcellular distribution of ERα and ERβ in TtT/GF cells (Fig. 4). In control- and 17β-estradiol-treated (24 h, 180 nM) conditions, strong immunofluorescent staining for ERα (Fig. 4A–F) and ERβ (Fig. 4G–L) was localized to the nucleus with weaker immunofluorescence extended throughout the cytoplasm. No non-specific immunolabeling was detected in absorbed antibody controls (Fig. 4M and N). The effects of incubating TtT/GF cells for 24 h with increasing concentrations of 17β-estradiol (0·18–1800 nM) on the amount of ANXA1 mRNA and protein are shown in Fig. 5. Real-time PCR assay of ANXA1 mRNA in TtT/GF cells confirmed that the positive control stimulus dexamethasone (0·1 μM, 24 h) produced a significant (P<0·05) increase in ANXA1 mRNA and revealed that 17β-estradiol (1·8–180 nM, 24 h) caused a concentration-dependent increase in ANXA1 mRNA (Fig. 5A). In contrast, western blot analysis demonstrated no significant effect of graded concentrations of 17β-estradiol (0·18–1800 nM, 24 h; Fig. 5B and C) and no significant effect of 17β-estradiol (18 nM) treatment for various periods (0–24 h; Fig. 5D) on ANXA1 protein content in TtT/GF cells. However, pretreatment of TtT/GF cells with 17β-estradiol (100 nM, 24 h) significantly (P<0·05) increased the amount of surface ANXA1 detected in response to dexamethasone-stimulated (0·1 μM, 3 h) externalization (Fig. 6A, lanes 7 and 8 17β-estradiol and dexamethasone-treated versus lanes 3 and 4 control- and dexamethasone-treated; Fig. 6D), whereas the amount of intracellular ANXA1 measured was unaffected (Fig. 6C and E). No actin ir bands were detected in cell surface EDTA washes (Fig. 6B) confirming cell integrity. In subsequent experiments, the effect of co-treatment of TtT/GF cells with 17β-estradiol (18 nM) and dexamethasone (0·1 μM) for 24 h on ANXA1 content was measured (Fig. 7). Western blot analysis confirmed that dexamethasone alone significantly (P<0·05) increased ANXA1 content, whereas 17β-estradiol treatment (18 nM) alone had no significant effect (Fig. 7A, lanes 1 and 2 control versus lanes 3 and 4 dexamethasone-treated versus lanes 5 and 6 17β-estradiol-treated). However, co-treatment of TtT/GF cells with 17β-estradiol (18 nM) and dexamethasone (0·1 μM) for 24 h significantly (P<0·05) enhanced ANXA1 content compared with dexamethasone treatment alone (Fig. 7A, lanes 7 and 8 dexamethasone and 17β-estradiol-treated versus lanes 3 and 4 dexamethasone alone). However, no change was observed in the amount of β-actin detected (Fig. 7B).

Bottom Line: Administration of 17beta-estradiol (1 microg/100 g) significantly (P<0 x 01) increased anterior pituitary ANXA1 expression in the ovariectomized animals.Treatment of TtT/GF cells, a folliculo-stellate cell line, with 17beta-estradiol (1 x 8-180 nM) increased ANXA1 mRNA expression and increased the amount of ANXA1 protein externalized in response to a dexamethasone stimulus.These results indicate that 17beta-estradiol stimulates ANXA1 expression in the anterior pituitary and in vivo an adrenal factor contributes to the mechanism of action.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.

ABSTRACT
Annexin 1 (ANXA1) is a Ca2+- and phospholipid-binding protein that plays an important role as a mediator of glucocorticoid action in the host-defence and neuroendocrine systems. Sex differences in hypothalamo-pituitary-adrenal (HPA) axis activity are well documented and a number of studies have demonstrated that gonadal steroids act as regulators of HPA activity. The aim of this study was to investigate the effect of ovariectomy and 17beta-estradiol replacement, and estrous cycle stage, on anterior pituitary ANXA1 content. The amount of anterior pituitary ANXA1 determined by western blotting varied with estrous cycle stage with a peak at estrus declining to a trough at proestrus. Ovariectomy resulted in a significant (P<0 x 05) decrease in anterior pituitary ANXA1 content. Administration of 17beta-estradiol (1 microg/100 g) significantly (P<0 x 01) increased anterior pituitary ANXA1 expression in the ovariectomized animals. In contrast, there was no change in pituitary ANXA1 content in response to 17beta-estradiol in adrenalectomized and adrenalectomized/ovariectomized rats. Treatment of TtT/GF cells, a folliculo-stellate cell line, with 17beta-estradiol (1 x 8-180 nM) increased ANXA1 mRNA expression and increased the amount of ANXA1 protein externalized in response to a dexamethasone stimulus. These results indicate that 17beta-estradiol stimulates ANXA1 expression in the anterior pituitary and in vivo an adrenal factor contributes to the mechanism of action.

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