María L. Magri

Estradiol Increases the Bax/Bcl-2 Ratio and Induces Apoptosis in the Anterior Pituitary Gland

Background: Estrogens are recognized as acting as modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals, thus participating in anterior pituitary homeostasis during the estrous cycle. The balance of pro- and antiapoptotic proteins of the Bcl-2 family is known to regulate cell survival and apoptosis. Aims: In order to understand the mechanisms underlying apoptosis during the estrous cycle, we evaluated the expression of the proapoptotic protein Bax and the antiapoptotic proteins Bcl-2 and Bcl-xL in the anterior pituitary gland in cycling female rats as well as the influence of estradiol on the expression of these proteins in anterior pituitary cells of ovariectomized rats. Methods/Results: As determined by Western blot, the expression of Bax was higher in anterior pituitary glands from rats at proestrus than at diestrus I, Bcl-2 protein levels showed no difference and Bcl-xL expression was lower, thus increasing the Bax/Bcl-2 ratio at proestrus. Assessed by annexin V binding and flow cytometry, the percentage of apoptotic anterior pituitary cells was higher in rats at proestrus than at diestrus I. Chronic estrogen treatment in ovariectomized rats enhanced the Bax/Bcl-2 ratio and induced apoptosis. Moreover, incubation of cultured anterior pituitary cells from ovariectomized rats with 17β-estradiol for 24 h increased the Bax/Bcl-2 ratio, decreased Bcl-xL expression and induced apoptosis. Conclusion: Our results demonstrate that estradiol increases the ratio between proapoptotic and antiapoptotic proteins of the Bcl-2 family. This effect could participate in the sensitizing action of estrogens to proapoptotic stimuli and therefore be involved in the high apoptotic rate observed at proestrus in the anterior pituitary gland.

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG2) showed the highest immobilization capacity (8.2 mg SBP g−1 PANIG2). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the Km was conserved while the specific Vmax dropped from 14.6 to 11.4 µmol min−1 µg−1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.

Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose

Abstract Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca 2+ /Mg 2+ concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and α- d -methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca 2+ /Mg 2+ in the absence of salt, at a flow rate up to 3.2 cm/min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M α- d -methylmannopyranoside and 0.97 M NaCl. Under these conditions, the dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3%. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M α- d -methylmannopyranoside to yield 75% of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively.

Estradiol Increases the Expression of TNF-α and TNF Receptor 1 in Lactotropes

Background: Estrogens are recognized modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that plays an important role in tissue homeostasis modulating cell proliferation, differentiation and death. We previously demonstrated that TNF-α-induced apoptosis of anterior pituitary cells from female rats is estrogen-dependent and predominant in cells from rats at proestrus when estradiol levels are the highest. Aims: Considering that one of the mechanisms involved in the apoptotic action of estrogens can result from increased expression of cytokines and/or their receptors, the aim of the present study was to evaluate the effect of estrogens on the expression of TNF-α and its receptor, TNF receptor 1 (TNFR1), in anterior pituitary cells. Methods/Results: TNFR1 expression, determined by Western blot, was higher in anterior pituitary glands from rats at proestrus than at diestrus. Incubation of anterior pituitary cells from ovariectomized rats with 17β-estradiol enhanced TNFR1 protein expression. As determined by double immunocytochemistry, the expression of TNF-α and TNFR1 was detected in prolactin-, GH-, LH- and ACTH-bearing cells. 17β-estradiol increased the percentage of TNF-α and TNFR1-immunoreactive lactotropes but did not modify the number of GH-bearing cells expressing TNF-α or TNFR1. Conclusion: Our results demonstrate that estradiol increases the expression of TNF-α and TNFR1 in anterior pituitary cells, especially in lactotropes. The sensitizing action of estrogens to proapoptotic stimuli at proestrus in the anterior pituitary gland may involve changes in the expression of the TNF-α/TNFR1 system.

Estrogens induce expression of membrane-associated estrogen receptor α isoforms in lactotropes.

Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2 participates in anterior pituitary cell renewal during the estrous cycle.

Inhibition of nuclear factor-kappa B sensitises anterior pituitary cells to tumour necrosis factor-α- and lipopolysaccharide-induced apoptosis.

Nuclear factor‐kappa B (NF‐κB), an important pro‐inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)‐α activate NF‐κB signalling. Oestrogens were shown to suppress NF‐κB activation. Oestrogens exert a sensitising action to pro‐apoptotic stimuli such as LPS and TNF‐α in anterior pituitary cells. In the present study, we show by western blotting that 17β‐oestradiol (E2) decreases TNF‐α‐induced NF‐κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E2 decreases LPS‐induced NF‐κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF‐κB pathway sensitises anterior pituitary cells to pro‐apoptotic stimuli, we used an inhibitor of NF‐κB activity, BAY 11‐7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF‐α‐induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF‐κB signalling enhances E2‐sensitising effect to TNF‐α‐induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS‐induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS‐induced expression of Bcl‐xL in pituitaries of OVX rats is decreased by E2 administration. Our results show that inhibition of the NF‐κB signalling pathway sensitises anterior pituitary cells to the pro‐apoptotic action of LPS and TNF‐α. Because E2 inhibits LPS‐ and TNF‐α‐activated NF‐κB nuclear translocation, the present study suggests that E2 sensitises anterior pituitary cells to TNF‐α‐ and LPS‐induced apoptosis by inhibiting NF‐κB activity.

Immobilisation of soybean seed coat peroxidase on its natural support for phenol removal from wastewater

Soybean seed coat peroxidase (SBP; EC 1.11.1.7) was immobilised on its natural support, soybean seed coats, anticipating its use in phenol removal. Periodate and glutaraldehyde chemistries were assayed. Periodate failed to immobilise any SBP, whereas glutaraldehyde was effective. The optimum concentration of glutaraldehyde was found to be 1%. Immobilisation shifted the optimum pH for phenol removal from 4.0 to 6.0. Treated seed coat retained its activity over a 4-week period, and reusability assays showed that treated seed coats could be reused once for phenol removal. Polyethylene glycol (PEG) increased the stability of phenol degradation activity. In addition, the phenolic polymer was adsorbed on to seed coats, thus making removal of the polymeric product easier.

Lack of Oestrogenic Inhibition of the Nuclear Factor-κB Pathway in Somatolactotroph Tumour Cells

Activation of nuclear factor (NF)‐κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)‐α by inhibiting NF‐κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF‐κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17β‐oestradiol (E2) (10−9 m) increased the nuclear concentration of NF‐κB/p105, p65 and p50 in GH3 cells. However, E2 did not modify the expression of Bcl‐xL, a NF‐κB target gene. TNF‐α induced apoptosis of GH3 cells incubated in either the presence or absence of E2. Inhibition of the NF‐kB pathway using BAY 11‐7082 (BAY) (5 μm) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive GH3 cells. BAY also increased TNF‐α‐induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c‐Jun N‐terminal protein kinase pathway, SP600125 (10 μm). We also analysed the role of the NF‐κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL‐positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF‐κB pathway and that the pro‐apoptotic effect of TNF‐α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF‐κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF‐κB pathway could interfere with pituitary tumour progression.

Antiapoptotic Factor Humanin Is Expressed in Normal and Tumoral Pituitary Cells and Protects Them from TNF-α-Induced Apoptosis

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.

Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells.

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17β-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.