Juan Pablo Petiti

Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.

Activation of PKC epsilon induces lactotroph proliferation through ERK1/2 in response to phorbol ester.

The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.

Bromocriptine induces parapoptosis as the main type of cell death responsible for experimental pituitary tumor shrinkage.

Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.

17β-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.

Heterogeneous vancomycin-intermediate susceptibility in a community-associated methicillin-resistant Staphylococcus aureus epidemic clone, in a case of Infective Endocarditis in Argentina

BackgroundCommunity-Associated Methicillin Resistant Staphylococcus aureus (CA-MRSA) has traditionally been related to skin and soft tissue infections in healthy young patients. However, it has now emerged as responsible for severe infections worldwide, for which vancomycin is one of the mainstays of treatment. Infective endocarditis (IE) due to CA-MRSA with heterogeneous vancomycin-intermediate susceptibility-(h-VISA) has been recently reported, associated to an epidemic USA 300 CA-MRSA clone.Case PresentationWe describe the occurrence of h-VISA phenotype in a case of IE caused by a strain belonging to an epidemic CA-MRSA clone, distinct from USA300, for the first time in Argentina. The isolate h-VISA (SaB2) was recovered from a patient with persistent bacteraemia after a 7-day therapy with vancomycin, which evolved to fatal case of IE complicated with brain abscesses. The initial isolate-(SaB1) was fully vancomycin susceptible (VSSA). Although MRSA SaB2 was vancomycin susceptible (≤2 μg/ml) by MIC (agar and broth dilution, E-test and VITEK 2), a slight increase of MIC values between SaB1 and SaB2 isolates was detected by the four MIC methods, particularly for teicoplanin. Moreover, Sab2 was classified as h-VISA by three different screening methods [MHA5T-screening agar, Macromethod-E-test-(MET) and by GRD E-test] and confirmed by population analysis profile-(PAP). In addition, a significant increase in cell-wall thickness was revealed for SaB2 by electron microscopy. Molecular typing showed that both strains, SaB1 and SaB2, belonged to ST5 lineage, carried SCCmec IV, lacked Panton-Valentine leukocidin-(PVL) genes and had indistinguishable PFGE patterns (subtype I2), thereby confirming their isogenic nature. In addition, they were clonally related to the epidemic CA-MRSA clone (pulsotype I) detected in our country.ConclusionsThis report demonstrates the ability of this epidemic CA-MRSA clone, disseminated in some regions of Argentina, to produce severe and rapidly fatal infections such as IE, in addition to its ability to acquire low-level vancomycin resistance; for these reasons, it constitutes a new challenge for the Healthcare System of this country.

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes

Electron Microscopy is an indispensable tool to investigate the intricate structures of the cell and organelles, and also to study the cellular biological processes implicated in the responses to changes in the microenvironment. However, several cellular events may be missed if conventional ultrastructural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins which can become rearranged as part of their own dynamic processes. Thus, immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology, and ultrastructural studies, by placing macromolecular functions within a cellular context.

17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells.

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.

Testosterone abrogates TLR4 activation in prostate smooth muscle cells contributing to the preservation of a differentiated phenotype.

Prostate smooth muscle cells (pSMCs) are capable of responding to inflammatory stimuli by secreting proinflammatory products, which causes pSMCs to undergo dedifferentiation. Although it has been proposed that androgens decrease proinflammatory molecules in many cells and under various conditions, the role of testosterone in the prostate inflammatory microenvironment is still unclear. Therefore, our aim was to evaluate if testosterone was able to modulate the pSMCs response to bacterial LPS by stimulating primary pSMC cultures, containing testosterone or vehicle, with LPS (1 or 10 µg/ml) for 24–48 h. The LPS challenge induced pSMCs dedifferentiation as evidenced by a decrease of calponin and alpha smooth muscle actin along with an increase of vimentin in a dose‐dependent manner, whereas testosterone abrogated these alterations. Additionally, an ultrastructural analysis showed that pSMCs acquired a secretory profile after LPS and developed proteinopoietic organelles, while pSMCs preincubated with testosterone maintained a more differentiated phenotype. Testosterone downregulated the expression of surface TLR4 in control cells and inhibited any increase after LPS treatment. Moreover, testosterone prevented IκB‐α degradation and the LPS‐induced NF‐κB nuclear translocation. Testosterone also decreased TNF‐α and IL6 production by pSMCs after LPS as quantified by ELISA. Finally, we observed that testosterone inhibited the induction of pSMCs proliferation incited by LPS. Taken together, these results indicate that testosterone reduced the proinflammatory pSMCs response to LPS, with these cells being less reactive in the presence of androgens. In this context, testosterone might have a homeostatic role by contributing to preserve a contractile phenotype on pSMCs under inflammatory conditions. J. Cell. Physiol. 228: 1551–1560, 2013.

Specific subcellular targeting of PKCα and PKCε in normal and tumoral lactotroph cells by PMA-mitogenic stimulus

The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCα and PKCε in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCα to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCε in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCα could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.

Involvement of MEK/ERK1/2 and PI3K/Akt Pathways in the Refractory Behavior of GH3B6 Pituitary Tumor Cells to the Inhibitory Effect of TGFβ1

Pituitary tumor cells have a poor response to the growth inhibitory effect of TGFβ1, possibly resulting from the cross talk of TGFβ/Smads signal with other signaling pathways, an undescribed mechanism in these tumoral cells. To address this hypothesis, we investigated whether the mitogen-activated extracellular signal-regulated kinase (MEK)/ERK1/2 and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathways were able to regulate the antimitogenic effect of TGFβ1 on GH3B6 cells. TGFβ1 treatment decreased the cell proliferation and induced an activation of mothers against decapentaplegic homolog 2/3 (Smad2/3), effects that were potentiated by MEK and PI3K inhibitors, thus indicating the existence of a cross talk between TGFβ1/Smad with the MEK/ERK1/2 or PI3K/Akt pathways. In addition, through immunoprecipitation assays, a direct interaction was observed between Smad2/3-ERK1/2 and Smad2/3-Akt, which decreased when the GH3B6 cells were incubated with TGFβ1 in the presence of MEK or PI3K inhibitors, thereby suggesting that the ERK1/2- and Akt-activated states were involved. These Smad2/3-ERK1/2 and Smad2/3-Akt associations were also confirmed by confocal and transmission electron microscopy. These findings indicate that the TGFβ1-antimitogenic effect in GH3B6 cells was attenuated by the MEK/ERK1/2 and PI3K/Akt pathways via modulating Smad2/3 phosphorylation. This molecular mechanism could explain in part the refractory behavior of pituitary tumor cells to the inhibitory effect of TGFβ1.