Effect of Three NR3C1 mutations in Pathogenesis of Pituitary ACTH Adenoma

Endocrinology ◽  
2021 ◽  
Author(s):  
Hui Miao ◽  
Yang Liu ◽  
Lin Lu ◽  
Fengying Gong ◽  
Linjie Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Nuclear Translocation ◽  
Treatment Approach ◽  
Wild Type ◽  
Acth Secretion ◽  
Novel Treatment ◽  
Generation Sequencing ◽  
Nuclear Receptor Subfamily ◽  
Corticotropic Adenoma

Abstract Objective Glucocorticoids act through the glucocorticoid receptor (GR) encoded by the Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) gene. This study aimed to examine the function of NR3C1 variants and their possible pathogenic role in Cushing’s disease (CD). Methods Next Generation Sequencing was conducted in 49 CD patients. Corticotroph tumor GR protein expression was examined by immunohistochemistry (IHC). Constructs harboring the three NR3C1-mutants and wild-type (WT) GR were transfected into the murine corticotropic adenoma cell line (AtT-20) and GR protein expression was quantified by western blot. Translocation activity was assessed by immunofluorescence and effects of the GR mutants on corticotroph tumor proliferation, pro-opiomelanocortin (POMC) transcription and ACTH secretion were tested. Results Clinical features were similar in patients harboring the NR3C1 mutations and WT GR. Recurrent adenomas showed higher GR IHC score than non-recurrent tumors. In vitro studies demonstrated that the p.R469X mutant generated a truncated GR protein, and the p.D590G and p.Y693D GR mutants resulted in lower GR expression. Dexamethasone (DEX) treatment of AtT-20 cells demonstrated decreased DEX-induced nuclear translocation, increased cell proliferation and attenuated suppression of POMC transcription of 3 GR mutants. Interestingly, the p.R469X GR mutant resulted in increased murine corticotroph tumor ACTH secretion compared to WT GR. Conclusion Our findings identify 3/49 (6.1%) consecutive human corticotroph tumors harboring GR mutations. Further findings demonstrate the role NR3C1 plays in CD pathogenesis and offer insights into a novel treatment approach in this patient subset.

scholarly journals Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 878
Author(s):  
Oskar Gustafsson ◽  
Julia Rädler ◽  
Samantha Roudi ◽  
Tõnis Lehto ◽  
Mattias Hällbrink ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Cell Penetrating Peptide ◽  
Wild Type ◽  
Efficient Manner ◽  
Editing Efficiency ◽  
Freeze Thaw ◽  
Enzyme Digest ◽  
Clinical Potential ◽  
Generation Sequencing

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

Octreotide exerts different effects in vivo and in vitro in Cushing's disease

1994 ◽  
Vol 130 (2) ◽  
pp. 125-131 ◽  
Author(s):  
Günter K Stalla ◽  
Steffi J Brockmeier ◽  
Ulrich Renner ◽  
Chris Newton ◽  
Michael Buchfelder ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Cushing’S Disease ◽  
Cushing's Disease ◽  
Dependent Manner ◽  
Acth Secretion ◽  
Adenoma Cell ◽  
Cortisol Levels ◽  
Corticotropic Adenoma

Stalla GK, Brockmeier SJ, Renner U, Newton C, Buchfelder M, Stalla J, Müller OA. Octreotide exerts different effects in vivo and in vitro in Cushing's disease. Eur J Endocrinol 1994;130:125–31. ISSN 0804–4643 The effect of the long-acting somatostatin analog octreotide (SMS 201-995) on adrenocorticotropin (ACTH) secretion was studied in five patients with untreated Cushing's disease in vivo and in six human corticotropic adenoma cell cultures in vitro. For the in vivo study, 100 μg of octreotide sc was given 30 and 180 min after cannulation of the cubital vein and 100 μg of corticotropin-releasing hormone (CRH) was injected iv at 210 min. Serum ACTH and cortisol levels were measured for 390 min. In vivo, octreotide had no significant effect either on basal or CRH-stimulated ACTH levels and did not influence cortisol levels. The in vitro studies were conducted with corticotropic adenoma cell cultures derived from adenoma tissue obtained from six patients with Cushing's disease. In four of six cell cultures, octreotide (1 nmol/l–1 μmol/l) inhibited basal ACTH secretion in a dose-dependent manner. The inhibition ranged from 70 to 92% for 1 nmol/l octreotide to 14–46% for 1 μmol/l octreotide as compared to controls (100%). In three of three octreotide-responsive adenoma cell cultures investigated, CRH-stimulated ACTH secretion was suppressed by octreotide. Hydrocortisone pretreatment in vitro abolished the inhibitory effect of octreotide on ACTH secretion in one octreotide-responsive corticotropic adenoma cell culture. In conclusion, we showed that octreotide in most cases could inhibit the ACTH release from human corticotropic adenoma cells in vitro but had no suppressive effect on ACTH levels of patients with Cushing's disease in vivo. This discrepancy could be due to a somatostatin receptor down-regulation by cortisol at the hypercortisolemic state in vivo. Günter K Stalla, Max-Planck-Institute of Psychiatry, Clinical Institute, Kraepelinstr. 10, D-80804 Munich, Germany

scholarly journals Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

2015 ◽  
Vol 26 (13) ◽  
pp. 2475-2490 ◽  
Author(s):  
Galina Schevzov ◽  
Anthony J. Kee ◽  
Bin Wang ◽  
Vanessa B. Sequeira ◽  
Jeff Hook ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Actin Filaments ◽  
Nuclear Translocation ◽  
Genetic Manipulation ◽  
Wild Type ◽  
Proximity Ligation ◽  
Mouse Embryo Fibroblasts ◽  
Wild Type Mefs

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

scholarly journals Wnt/β-Catenin–Promoted Macrophage Alternative Activation Contributes to Kidney Fibrosis

2017 ◽  
Vol 29 (1) ◽  
pp. 182-193 ◽  
Author(s):  
Ye Feng ◽  
Jiafa Ren ◽  
Yuan Gui ◽  
Wei Wei ◽  
Bingyan Shu ◽  
...  
Keyword(s):  
Normal Development ◽  
Nuclear Translocation ◽  
Alternative Activation ◽  
Wild Type ◽  
Kidney Fibrosis ◽  
Ureter Obstruction ◽  
M2 Polarization ◽  
Macrophage Accumulation

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4– or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro. Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4– or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

scholarly journals Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

1987 ◽  
Vol 7 (12) ◽  
pp. 4255-4265 ◽  
Author(s):  
W Markland ◽  
A E Smith ◽  
B L Roberts
Keyword(s):  
Rat Liver ◽  
Nuclear Translocation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.

Lipid-nanopotentiated combinatorial delivery of tamoxifen and sulforaphane: ex vivo, in vivo and toxicity studies

Nanomedicine ◽  
2020 ◽  
Vol 15 (26) ◽  
pp. 2563-2583 ◽  
Author(s):  
Bharti Mangla ◽  
Yub R Neupane ◽  
Archu Singh ◽  
Pankaj Kumar ◽  
Sadat Shafi ◽  
...  
Keyword(s):  
Intestinal Permeability ◽  
Oral Delivery ◽  
Chemotherapeutic Agent ◽  
Treatment Approach ◽  
Ex Vivo ◽  
Toxicity Assessment ◽  
Drug Release Profile ◽  
Novel Treatment

Aim: This study aims to load tamoxifen (TAM) and sulforaphane (SFN) into nanostructured lipid carriers (NLCs) to enhance their oral delivery. Materials & methods: TAM-SFN-NLCs were prepared using Precirol® ATO5 and Transcutol® HP, characterized and evaluated in vitro and ex vivo to assess the drug release profile and intestinal permeability, respectively. In vivo pharmacokinetic and acute toxicity assessment was performed in Wistar rats. Results: Optimized TAM-SFN-NLCs exhibited a particle size of 121.9 ± 6.42 nm and zeta potential of -21.2 ± 2.91 mV. The NLCs enhanced intestinal permeability of TAM and SFN and augmented oral bioavailability of TAM and SFN 5.2-fold and 4.8-fold, respectively. SFN significantly reduced TAM-associated toxicity in vivo. Conclusion: This coencapsulation of a chemotherapeutic agent with a herbal bioactive in NLCs could pave a novel treatment approach against cancer.

scholarly journals Tenascin-C deficiency attenuates TGF-β-mediated fibrosis following murine lung injury

2010 ◽  
Vol 299 (6) ◽  
pp. L785-L793 ◽  
Author(s):  
William A. Carey ◽  
Glen D. Taylor ◽  
Willow B. Dean ◽  
James D. Bristow
Keyword(s):  
Lung Injury ◽  
Nuclear Translocation ◽  
Interstitial Fibrosis ◽  
Smooth Muscle Actin ◽  
Transcript Abundance ◽  
Lung Fibroblasts ◽  
Wild Type ◽  
Tenascin C ◽  
Smad 3

Tenascin-C (TNC) is an extracellular matrix glycoprotein of unknown function that is highly expressed in adult lung parenchyma following acute lung injury (ALI). Here we report that mice lacking TNC are protected from interstitial fibrosis in the bleomycin model of ALI. Three weeks after exposure to bleomycin, TNC-null mice had accumulated 85% less lung collagen than wild-type mice. The lung interstitium of TNC-null mice also appeared to contain fewer myofibroblasts and fewer cells with intranuclear Smad-2/3 staining, suggesting impaired TGF-β activation or signaling. In vitro, TNC-null lung fibroblasts exposed to constitutively active TGF-β expressed less α-smooth muscle actin and deposited less collagen I into the matrix than wild-type cells. Impaired TGF-β responsiveness was correlated with dramatically reduced Smad-3 protein levels and diminished nuclear translocation of Smad-2 and Smad-3 in TGF-β-exposed TNC-null cells. Reduced Smad-3 in TNC-null cells reflects both decreased transcript abundance and enhanced ubiquitin-proteasome-mediated protein degradation. Together, these studies suggest that TNC is essential for maximal TGF-β action after ALI. The clearance of TNC that normally follows ALI may restrain TGF-β action during lung healing, whereas prolonged or exaggerated TNC expression may facilitate TGF-β action and fibrosis after ALI.

scholarly journals Functional Potentiation of Leptin-Signal Transducer and Activator of Transcription 3 Signaling by the Androgen Receptor

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 6028-6036 ◽  
Author(s):  
WuQiang Fan ◽  
Toshihiko Yanase ◽  
Yoshihiro Nishi ◽  
Seiichi Chiba ◽  
Taijiro Okabe ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Nuclear Translocation ◽  
Steroid Hormone Receptors ◽  
Signal Transducer ◽  
Male Mice ◽  
Wild Type ◽  
Stat3 Signaling ◽  
Transcription 3 ◽  
Arcuate Neurons

Hypogonadism is associated with increased fat mass and dysregulation of metabolic homeostasis in men. Our previous study revealed that androgen receptor (AR)-null male mice (ARL-/Y) develop late-onset obesity and are leptin-resistant. The present study evaluated how hypothalamic AR contributes to central leptin-signal transducer and activator of transcription 3 (STAT3) signaling. We evaluated leptin action in wild-type and ARL-/Y mice, the anatomic co-relationship between AR and leptin signaling in the hypothalamus, and the effects of AR on leptin-mediated STAT3 transactivation and nuclear translocation. AR deletion in male mice results in a weaker leptin-induced suppression of food intake and body weight drop even before the onset of overt obesity. In wild-type male but not female mice, AR was highly expressed in various hypothalamic nuclei that also expressed the long-form leptin receptor (OBRB) and co-resided with OBRB directly in the arcuate neurons. In vitro, AR significantly enhanced STAT3-mediated transcription of leptin target genes including POMC and SOCS3. This effect relied on the AR N-terminal activation function-1 (AF-1) domain and was specific to AR in that none of the other sex steroid hormone receptors tested showed similar effects. AR enhanced the low concentrations of leptin-induced STAT3 nuclear translocation in vitro, and ARL-/Y mice receiving leptin had impaired STAT3 nuclear localization in the arcuate neurons. These findings indicate that AR in the hypothalamus functions as a regulator of central leptin-OBRB-STAT3 signaling and has a physiological role in energy homeostasis and metabolic regulation in male mice.

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

Blood ◽  
2011 ◽  
Vol 117 (16) ◽  
pp. 4338-4348 ◽  
Author(s):  
Ewa Surdziel ◽  
Maciej Cabanski ◽  
Iris Dallmann ◽  
Marcin Lyszkiewicz ◽  
Andreas Krueger ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Target Prediction ◽  
Mouse Bone Marrow ◽  
Granulocytic Differentiation ◽  
Over Expression ◽  
Small Noncoding Rnas

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF–induced granulocytic differentiation and enables G-CSF–dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF–dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b–mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

scholarly journals Epac ameliorates tubulointerstitial inflammation in diabetic nephropathy via C/EBPβ/SOCS3/STAT3 pathway

2020 ◽  
Author(s):  
Wenxia Yang ◽  
Shumin Zhang ◽  
Huafen Wang ◽  
Yifei Liu ◽  
Jialu Liu ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Glucose ◽  
Nuclear Translocation ◽  
Morphological Changes ◽  
Stat3 Pathway ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Gene And Protein Expression ◽  
Tubulointerstitial Inflammation

Abstract Background: Tubulointerstitial inflammation plays a pivotal role in the progression of diabetic nephrology (DN), and tubular cells act as the driving force in the inflammation cascade. The aim of the study is to investigate whether activation of Epac alleviates tubulointerstitial inflammation through SOCS3/JAK/STAT3 pathway under DN. Methods: The kidney morphological changes and the level of oxidative stress were observed in db/db mice treated with Epac agonist (8-pCPT-2’-O-Me-cAMP). The expression of inflammatory cytokines, fibrosis markers, C/EBPβ, SOCS3 and p-STAT3 in renal tissues were assessed. In in vitro study, the changes of above genes and proteins were also detected in human proximal tubular cell line (HK-2) incubated with high glucose (HG) with and without Epac agonist. Overexpression of SOCS3 plasmid, SOCS3 siRNA and C/EBP-β siRNA were employed and the translocation of C/EBP-β and p-STAT3 were observed by immunofluorescence. Macrophage migration assay were detected in Transwell system. Results: In db/db mice, we observed that the expression of MCP-1 was up-regulated in renal tubular cells, which concurrent with increased influx of macrophages, cytokines production (MCP-1, TNF-α, and IL-6), and tubulointerstitial fibrosis. Moreover, we also found that these changes were accompanied by reduced C/EBP-β expression and increased SOCS3 and phosphorylated STAT3 (p-STAT3) expression. However, after treated db/db mice with 8-O-cAMP, an Epac activator, the expression of SOCS3 further up-regulated while other protein expression profile was partially reversed. In vitro , high glucose (30mM, HG) treatment down-regulated C/EBP-β expression while increased SOCS3, p-STAT3 and MCP-1 expression in HK-2 cells; Activation of Epac by 8-O-cAMP further increased the gene and protein expression of SOCS3 while reversed the changes of the other proteins. Mechanistically, under HG ambience, knockdown of C/EBP-β or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on MCP-1 expression. Furthermore, 8-O-cAMP treatment enhanced the nuclear-translocation of C/EBP-β and further increased the transcription of SOCS3 gene, which decreased MCP-1 expression through inhibiting the phosphorylation of STAT3 in HK-2 cells. Conclusions: These data indicate that Epac ameliorates tubulointerstitial inflammation in DN at least partially through C/EBPβ/SOCS3/STAT3 pathway. Therefore, Activation of Epac by 8-O-cAMP may be a novel therapeutic strategy for DN.

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