Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway

Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health

Soluble guanylyl cyclase beta1 subunit targets epithelial-to-mesenchymal transition and downregulates Akt pathway in human endometrial and cervical cancer cells

Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and β1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCβ1 subunit has not been addressed. The aim of the present work was to study the impact of sGCβ1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCβ1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCβ1, unlike sGCα1. Overexpression of sGCβ1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCβ1-driven effects were independent of sGC enzymatic activity. sGCβ1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and β-catenin and increased the expression of E-cadherin. sGCβ1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3β and c-Raf. Our results show for the first time that sGCβ1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways

Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary

Cadmium stimulates anterior pituitary lactotroph proliferation.

<p>Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 96 h. Cell growth was determined by ICC measuring 24 h-BrdU incorporation. Lactotrophs were identified by prolactin-specific antibody and cell nuclei were stained by DAPI. Pictures are representative of three independent experiments performed in triplicate. Bars represent the mean ± SE of BrdU-labeling index expressed as positive BrdU lactrotroph / total lactotroph cell number x100. ANOVA followed by Tukey-Kramer’s test, **p<0.001 vs. control (N=3). </p

Cadmium increases gene expression of proliferation markers in anterior pituitary cells.

<p>Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2. Gene expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values of cyclins D1 and D3 after 72 h (A) or <i>c-fos</i> after 8-24 h (B) normalized to GAPDH expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.001 vs. control (N=3). </p

Cadmium exposure increases ERα

<p><b>protein expression in anterior pituitary cells</b>. Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 24h. A representative western blot is shown. Bars represent the mean ± SE of densitometric values of full-length ERα (open bars) and ERα46 (black bars) normalized to β-actin expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.01, ***p<0.001 vs. respective control; #p<0.05, ###p<0.001 vs. Cd (N=3).</p

E2 receptors (ERs) mediates cadmium effect on cyclins D1 and D3 and PRL mRNA expression.

<p>Anterior pituitary cells cultures were first incubated with 100 nM ICI 182,780 (ICI) for 20 min and then incubated with vehicle (control) or 10 nM Cd for 72 h. Cyc D1 and D3 (A) and PRL (B) mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, **p<0.01 vs. control, ##p<0.01 vs. Cd (N=3).</p

Cadmium increased 23 kDa prolactin (PRL) protein expression in anterior pituitary cells in culture.

<p>Anterior pituitary cells were incubated with 10 nM Cd or vehicle (control) for 8 h. Protein expression was measured by western blot. Bars represent mean ± SEM of PRL densitometric values normalized to β-actin and are expressed as percent of control. **P<0.01, Student’s ‘t’ test (N=3).</p

Additive effect of cadmium and E2 co-treatment on ERα mRNA expression.

<p>Anterior pituitary cell cultures were treated with vehicle (control), 10 nM Cd or 1 nM E2 or 10 nM Cd plus 1 nM E2 for 8 h or 24 h. ERα mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH. ANOVA followed by Tukey-Kramer’s test, *p<0.05, </p><p>** p<0.01, *** p<0.001 vs. Control; #p<0.05 vs. Cd; <b>^</b>p<0.05 vs. E2 (N=3).</p><p></p