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

E2 decreased sGC α1 protein expression in a PI3k-dependent pathway involving non-nuclear ER but does not modify sGC β1 after 3 h of incubation.

<p>Pituitary cells in culture were incubated with vehicle (control) or 1 nM estrogen dendrimer conjugate (EDC), unable to trespass cellular membrane for 3 h with or without 50 µM LY294002 (LY), a PI3K inhibitor, 30 min before treatment. Protein expression was evaluated by western blot. (<b>A, B</b>) <i>Top</i>, representative western blots. <i>Bottom</i>, Corresponding average densitometric values. Bars represent mean ± SE of α1 (open bars) and β1 (black bars) protein densitometric values normalized to β-actin, as percent of control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05 vs. respective controls; ΔΔ<i>P</i><0.01 <i>vs</i>. E2; #<i>P</i><0.05 <i>vs</i>. EDC.</p

E2 treatment increases AUF1p37 mRNA expression in anterior pituitary gland.

<p>Pituitary cells in culture were incubated for 6 h with vehicle (control) or with 1 nM E2. Exon 2 and exon 7 from AUF1 (A) or expression of a conservated domain of AUF1 (B) were evaluated by semi-quantitative PCR. <i>Top</i>, a representative PCR. <i>Bottom</i>, average densitometric values. Bars represent mean ± SE of relative units corresponding to densitometric values of exon 2 and exon 7 (A) and AUF1 conservated domain (B) normalized to β-actin, expressed as percent of control (n = 3). Student's ‘<i>t</i>’ test, **<i>P</i><0.01 <i>vs</i>. control.</p

E2 actions on sGC subunits expression depended on <i>de novo</i> transcription.

<p>Pituitary cells in culture were incubated with vehicle (control) or 1 nM E2 for 6 h with or without 2 µM actinomycin D (Act D), a transcription inhibitor, 30 min before treatment. (A), mRNA expression was evaluated by PCR. <i>Top</i>, a representative PCR. <i>Bottom</i>, Average densitometric values. Bars represent mean ± SE of relative units corresponding to α1 (open bars) and β1 (black bars) mRNA densitometric values normalized to β-actin, as percent of control (n = 3). (B), protein expression was evalulated by western blot. <i>Top</i>, a representative western blot. <i>Bottom</i>, Average densitometric values. Bars represent mean ± SE of relative units corresponding to α1 (open bars) and β1 (black bars) protein densitometric values normalized to β-actin, as percent of control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. respective controls; Δ<i>P</i><0.05 <i>vs</i>. E2.</p

Primers used for semi-quantitative RT-PCR assays.

<p>Primers used for semi-quantitative RT-PCR assays.</p

E2 actions on sGC α1 and β1 mRNAs were mediated by nuclear ER after 6 h of incubation.

<p>Pituitary cells in culture were incubated with vehicle (control) or 1 nM E2 or with 1 nM E2-conjugated to BSA (E2-BSA) unable to trespass cell membrane during 6 h. sGC α1 and β1 mRNAs were evaluated by PCR. <i>Top</i>, a representative PCR. <i>Bottom,</i> average densitometric values. Bars represent mean ± SE of relative units, corresponding to α1 (open bars) and β1 (black bars) densitometric values normalized to β-actin, and are expressed as percent of the control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. respective controls.</p

E2 actions on sGC α1 and β1 mRNAs depended on <i>de novo</i> translation.

<p>Pituitary cells in culture were incubated for 8 h with vehicle (control) or 1 nM E2 with or without 10 µg/mL cycloheximide (CHX), a translation inhibitor. mRNA expression was evaluated by semiquantitative PCR. <i>Top</i>, A representative PCR. <i>Bottom</i>, average densitomentric values. Bars represent mean ± SE of relative units corresponding to densitometric values of α1 (open bars) and β1 (black bars) normalized to β-actin, expressed as percent of control (n = 3). ANOVA followed by Tukey's test, **<i>P</i><0.01 <i>vs</i>. respective controls; Δ<i>P</i><0.05, ΔΔ<i>P</i><0.01 <i>vs</i>. E2.</p

E2 actions on sGC α1 and β1 proteins were mediated by nuclear ER after 6 h of incubation.

<p>Pituitary cells in culture were incubated for 6 h with vehicle (control) 1 nM E2 or with 1 nM estrogen dendrimer conjugate (EDC) unable to trespass cell membrane, or with 1 nM dendrimer alone (D). sGC α1 and β1 proteins were evaluated by western blot. <i>Top</i>, a representative western blot. <i>Bottom,</i> average densitometric values. Bars represent mean ± SE of relative units corresponding to α1 (open bars) and β1 (black bars) densitometric values normalized to β-actin, and are expressed as percent of the control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05 <i>vs</i>. respective controls.</p