Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland Jimena P. Cabilla 1 *, Silvana I. Nudler 1 , Sonia A. Ronchetti 1 , Fernanda A. Quinteros 1 , Mercedes Lasaga 2 , Beatriz H. Duvilanski 1 1 Departamento de Quı ´mica Biolo ´ gica, Instituto de Quı ´mica y Fı ´sico-quı ´mica Biolo ´ gicas, Facultad de Farmacia y Bioquı ´mica, Universidad de Buenos Aires, Buenos Aires, Argentina, 2 Instituto de Investigaciones en Reproduccio ´ n, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Abstract 17b-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, a and b, that catalyses cGMP formation. a1b1 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 a1 and decreasing b1 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, a1 mRNA and protein expression is increased while b1 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 b1 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 a1 or b1 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 a1 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. Citation: Cabilla JP, Nudler SI, Ronchetti SA, Quinteros FA, Lasaga M, et al. (2011) Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland. PLoS ONE 6(12): e29402. doi:10.1371/journal.pone.0029402 Editor: Kaustubh Datta, University of Nebraska Medical Center, United States of America Received August 16, 2011; Accepted November 28, 2011; Published December 28, 2011 Copyright: ß 2011 Cabilla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Agencia Nacional de Promocio ´ n Cientı ´fica y Tecnolo ´ gica (PICT 32311), Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas (PIP 5536) and Universidad de Buenos Aires (M025 and B052). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jcabilla@ffyb.uba.ar Introduction Nitric oxide sensitive- or soluble guanylyl cyclase (sGC), the main intracellular receptor of nitric oxide is comprised of two subunits, a and b, of which several isoforms (a1, a2, a2i, b1 and b2) have been described. a1b1 is the most abundant and widely expressed heterodimer, showing the greater activity [1]. The major female hormone, 17b-estradiol (E2), is a key regulator of pituitary physiology involved in hormonal release as well as proliferation and cell death in anterior pituitary gland [2– 4]. Previous studies from our laboratory show that acute E2 treatment exerts an inhibitory effect on sGC activity by down- regulating sGC b1 subunit in anterior pituitary gland. However, this treatment increases sGC a1 expression from both, immature and adult rats [5,6]. The E2 effects on anterior pituitary sGC were observed not only after in vivo and in vitro treatment but also during estrous cycle. These observations support a direct effect of E2 on sGC regulation and a differential and independent regulation on both subunits. Previous evidence [7–9] further sustains that under certain conditions, a1 and b1 can be independently expressed. E2 signaling pathways comprise classical and non-classical actions. Classical actions are mediated by nuclear E2 receptor (ER) and include both de novo transcriptional and translational events. E2 non-classical actions are mediated by non-nuclear ER and include the activation of signaling pathways that finally can also trigger transcription of certain genes [10,11]. In many cells, around 5–10% of total ER is found at the plasma membrane, including both a and b ER subtypes depending on cell type [11]. E2 can also regulate many genes post-transcriptionally by affecting mRNA stability. Certain mRNAs have highly conserved sequences, adenine-uracil rich elements (AREs), present on untranslated 39 end. AREs are involved in rapid mRNA degradation and are binding targets of several proteins. They constitute an important regulatory element involved in the control of genetic expression in vertebrates. Human antigen-R RNA binding protein (HuR) is ubiquitously expressed and belongs to embryonic lethal abnormal vision (ELAV) family proteins. HuR PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29402