Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice

2012 ◽  
Vol 303 (10) ◽  
pp. L852-L860 ◽  
Author(s):  
S. Yoshida ◽  
N. Minematsu ◽  
S. Chubachi ◽  
H. Nakamura ◽  
M. Miyazaki ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Apoptosis ◽  
Pulmonary Emphysema ◽  
Keratinocyte Growth Factor ◽  
Annexin V ◽  
Phenotypic Change ◽  
Chronic Obstructive ◽  
Mrna Levels

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

scholarly journals MACC1 Suppresses Cell Apoptosis in Hepatocellular Carcinoma by Targeting the HGF/c-MET/AKT Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 983-996 ◽  
Author(s):  
Yingmin Yao ◽  
Chanwei Dou ◽  
Zhongtang Lu ◽  
Xin Zheng ◽  
Qingguang Liu
Keyword(s):  
Cell Apoptosis ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Annexin V ◽  
Akt Pathway ◽  
Caspase 9 ◽  
Dapi Staining ◽  
In Vivo Experiments

Background & Aims: To investigate the expression and prognostic value of MACC1 in patients with HCC and identify the mechanism by which MACC1 inhibits HCC cell apoptosis. Methods: MACC1 and p-AKT expression was studied using immunohistochemistry of both HCC tissues and adjacent liver tissues. qRT-PCR and western immunoblotting were used to examine the expression of target genes at the mRNA and protein levels, respectively. The MTT assay was used to assess cell viability, and cell apoptosis was determined by DAPI staining, Annexin V/PI staining and Caspase 3/7 assay. Nude mice were used to perform in vivo experiments. Results: The overexpression of MACC1 was found in HCC tissues and was correlated with poor postsurgical prognosis. There was a positive relationship between MACC1 and p-AKT expression in HCC tissues. In vitro experiments showed that MACC1 repressed HCC cell apoptosis and promoted cell growth. Knockdown of c-MET abolished the anti-apoptotic function of MACC1. Next, MACC1 was verified to activate PI3K/AKT signaling by sensitizing HGF/c-MET signaling in HCC. MACC1 overexpression enhanced the HGF-driven phosphorylation of BAD, Caspase 9 and FKHRL1 and inhibited their pro-apoptotic functions in HCC cells. Finally, MACC1 was shown to inhibit cell apoptosis and promote HCC growth in vivo. Conclusions: This investigation revealed that MACC1 overexpression predicted worse prognosis after liver resection, which was attributed to the repression of HCC cell apoptosis via a molecular mechanism in which MACC1 accelerated the activation of the HGF/c-MET/PI3K/AKT pathway and phosphorylated BAD, Caspase 9 and FKHRL1, ultimately preventing their nuclear translocation and their pro-apoptotic function.

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
J.-S. Lee ◽  
E.-B. Jeung
Keyword(s):  
Mrna Expression ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Oestrogen Receptors ◽  
In Vivo Models ◽  
Ru 486 ◽  
Final Injection ◽  
M 10

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

scholarly journals The Ashwell-Morell Receptor Regulates Hepatic Thrombopoietin Production Via JAK2-STAT3 Signaling in Vivo and in Vitro

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2-2
Author(s):  
Renata Grozovsky ◽  
Antonija Jurak Begonja ◽  
John H. Hartwig ◽  
Herve Falet ◽  
Karin M Hoffmeister
Keyword(s):  
Bone Marrow ◽  
Sialic Acid ◽  
Mrna Expression ◽  
Hepg2 Cells ◽  
Mrna Levels ◽  
Platelet Production ◽  
Platelet Survival ◽  
Post Infusion

Abstract The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of platelet precursors, bone marrow megakaryocytes. Hepatocytes are a major source of production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. Here, we provide experimental evidence that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor), thereby regulating platelet survival and hepatic TPO levels. These conclusions are based on the following evidence: 1) Mice lacking the AMR Asgr2 subunit had increased platelet survival, compared to wild type (WT) mice. Platelets from Asgr2-null mice showed increased loss of sialic acid, as evidenced by flow cytometry using the galactose specific lectins RCAI and ECL, showing that removal of desialylated platelets by the AMR regulates in vivo platelet survival. 2) Livers isolated from Asgr2-null mice had TPO mRNA levels decreased by 40%, compared to WT mice. In contrast, liver TPO mRNA levels were increased by 30% in St3gal4-null mice lacking the sialyltransferase ST3GalIV, where desialylated platelet clearance is increased and specifically mediated by the AMR. Both plasma TPO levels and platelet TPO contents were similarly altered in both mutant mice. Thus, desialylated platelet uptake by the AMR regulated liver TPO levels. 3) Desialylated platelets isolated from St3gal4-null or Asgr2-null mice infused into WT mice increased hepatic TPO mRNA levels as early as 12h post-infusion. Plasma TPO concentrations and bone marrow megakaryocyte numbers increased in parallel with TPO mRNA levels, peaking by day 2 post-infusion, followed by new platelet release at day 10 post-infusion. In contrast, desialylated platelets infused into Asgr2-null mice had no effect on TPO mRNA synthesis, TPO plasma levels and bone marrow megakaryocyte numbers. 4) Incubation of human hepatoma cell line, HepG2 cells, with human desialylated platelets by sialidase treatment resulted in TPO mRNA expression increase by 2.2 and 2.9-fold after 4 and 6h, respectively, followed by significant increase in TPO secretion. 5) The signaling pathways activated by uptake of desialylated platelets by the AMR to induce TPO mRNA transcription were investigated in vivo and in vitro. Major polypeptides of 60-70 and 125 kDa were highly tyrosine phosphorylated in WT liver cells, as evidenced by SDS-PAGE. Using a specific antibody directed against JAK2, we identified the 125-kDa phosphoprotein as the tyrosine kinase JAK2 in mouse liver cells and human HepG2 cells. Analysis of liver samples revealed a marked reduction in JAK2 phosphorylation in Asgr2-null mice and significant increase in St3gal4-null mice. 6) The JAK1/2 inhibitor AZD1480 significantly decreased phosphorylation of JAK2, phosphorylation and translocation to the nucleus of the acute phase response transcription factor STAT3, TPO mRNA expression and TPO secretion in HepG2 cells incubated with desialylated platelets. In vivo treatment of WT mice with AZD1480 blocked TPO mRNA increase promoted by injection of endogenously desialylated platelets. Therefore we conclude that platelets desialylate as they circulate, thereby becoming the primary AMR ligand and providing a novel physiological feedback mechanism to regulate plasma TPO levels and platelet production in vivo and in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo

2001 ◽  
Vol 26 (3) ◽  
pp. 175-184 ◽  
Author(s):  
D Marcantonio ◽  
LE Chalifour ◽  
MA Alaoui-Jamali And H T Huynh ◽  
MA Alaoui-Jamali ◽  
MA Alaoui-Jamali ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Muscle Cells ◽  
Ventral Prostate ◽  
Mrna Levels ◽  
Rat Ventral Prostate ◽  
Steroid Sensitive

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles

2013 ◽  
Vol 25 (8) ◽  
pp. 1194 ◽  
Author(s):  
G. L. Vasconcelos ◽  
M. V. A. Saraiva ◽  
J. J. N. Costa ◽  
M. J. Passos ◽  
A. W. B. Silva ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Related Factors ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Before And After ◽  
Vitro Development

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e. hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL–1 from Days 0 to 6 and 500 ng mL–1 from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P < 0.05). Alone, 200 ng mL–1 GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL–1 bovine serum albumin, 10 µg mL–1 insulin, 5.5 µg mL–1 transferrin, 5 ng mL–1 selenium, 2 mM glutamine, 2 mM hypoxanthine and 50 μg mL–1 ascorbic acid (α-MEM+). Comparisons of uncultured (0.2 mm) and α-MEM+ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P < 0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice

2007 ◽  
Vol 293 (5) ◽  
pp. L1230-L1239 ◽  
Author(s):  
Laurent Plantier ◽  
Sylvain Marchand-Adam ◽  
Valeria G. Antico ◽  
Laurent Boyer ◽  
Cécile De Coster ◽  
...  
Keyword(s):  
Dna Damage ◽  
Growth Factor ◽  
Pulmonary Emphysema ◽  
Alveolar Epithelial Cell ◽  
Keratinocyte Growth Factor ◽  
Epithelial Cell Line ◽  
Alveolar Cells ◽  
Monocyte Chemoattractant Protein 1

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg·kg−1·day−1 KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2α) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro

2007 ◽  
Vol 292 (4) ◽  
pp. G1019-G1028 ◽  
Author(s):  
Stephan Brand ◽  
Julia Dambacher ◽  
Florian Beigel ◽  
Kathrin Zitzmann ◽  
Malte H. J. Heeg ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Expression ◽  
Liver Cell ◽  
Cytokine Signaling ◽  
Cell Regeneration ◽  
Mrna Levels ◽  
Hepatic Cells ◽  
Hepatocyte Regeneration

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [3H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-α. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.

scholarly journals Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

2014 ◽  
Vol 28 (1) ◽  
pp. 40-52 ◽  
Author(s):  
Dimiter Avtanski ◽  
Horacio J. Novaira ◽  
Sheng Wu ◽  
Christopher J. Romero ◽  
Rhonda Kineman ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Mrna Expression ◽  
Transcriptional Control ◽  
Estrogen Receptor Α ◽  
In Vitro Models ◽  
Mrna Levels ◽  
Ovariectomized Mice ◽  
Serum Gh

Abstract Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α– and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

scholarly journals MicroRNA-132 controls water homeostasis through regulating MECP2-mediated vasopressin synthesis

2018 ◽  
Vol 315 (4) ◽  
pp. F1129-F1138 ◽  
Author(s):  
Roel Bijkerk ◽  
Christiane Trimpert ◽  
Coen van Solingen ◽  
Ruben G. de Bruin ◽  
Barend W. Florijn ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Plasma Osmolality ◽  
Fine Tuning ◽  
Mrna Levels ◽  
Membrane Expression ◽  
Aquaporin 2 ◽  
Osmotic Balance ◽  
Avp Gene

Fine-tuning of the body’s water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.

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