scholarly journals Retinoblastoma and the Related Pocket Protein p107 Act as Coactivators of NeuroD1 to Enhance Gene Transcription

2005 ◽  
Vol 280 (16) ◽  
pp. 16088-16095 ◽  
Author(s):  
Eric Batsché ◽  
Pandelis Moschopoulos ◽  
Julien Desroches ◽  
Steve Bilodeau ◽  
Jacques Drouin
Keyword(s):  
Gene Transcription ◽  
Protein Interactions ◽  
Mrna Levels ◽  
Orphan Nuclear Receptor ◽  
Knock Out ◽  
Helix Loop Helix ◽  
Knock Out Mice ◽  
Interfering Rna

Gene inactivation studies have suggested that the product of the retinoblastoma gene,Rb, is particularly limiting in pituitary pro-opiomelanocortin (POMC)-expressing cell lineages. Indeed, in Rb knock-out mice, these cells develop tumors with high frequency. To understand the implication of limiting Rb expression in these cells, we investigated the action of Rb and its related pocket proteins, p107 and p130, onPOMCgene transcription. This led to the identification of the neurogenic basic helix-loop-helix transcription factor, NeuroD1, as a target of Rb action. Rb and to a lesser extent p107, but not p130, enhance NeuroD1-dependent transcription, and this activity appears to depend on direct protein interactions between the Rb pocket and the helix-loop-helix domain of NeuroD1.In vivo, NeuroD is found in a complex that includes Rb and also the orphan nuclear receptor NGFI-B, which mediates corticotropin-releasing hormone activation of POMC transcription. The formation of a similar complexin vitrorequires the presence of Rb as a bridge between NeuroD and NGFI-B. In POMC-expressing AtT-20 cells, Rb and p107 are present on the POMC promoter and inhibition of their expression through small interfering RNA decreases POMC mRNA levels. The action of Rb and its related proteins on POMC transcription may contribute to the establishment and/or maintenance of the differentiation phenotype.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

Abstract In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

scholarly journals Orphan Nuclear Receptor ERRγ Is a Novel Transcriptional Regulator of IL-6 Mediated Hepatic BMP6 Gene Expression in Mice

2020 ◽  
Vol 21 (19) ◽  
pp. 7148
Author(s):  
Kamalakannan Radhakrishnan ◽  
Yong-Hoon Kim ◽  
Yoon Seok Jung ◽  
Jina Kim ◽  
Don-Kyu Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Molecular Mechanism ◽  
Nuclear Receptor ◽  
Iron Homeostasis ◽  
Luciferase Reporter ◽  
Orphan Nuclear Receptor ◽  
Knock Out

Bone morphogenetic protein 6 (BMP6) is a multifunctional growth factor involved in organ development and homeostasis. BMP6 controls expression of the liver hormone, hepcidin, and thereby plays a crucial role in regulating iron homeostasis. BMP6 gene transcriptional regulation in liver is largely unknown, but would be of great help to externally modulate iron load in pathologic conditions. Here, we describe a detailed molecular mechanism of hepatic BMP6 gene expression by an orphan nuclear receptor, estrogen-related receptor γ (ERRγ), in response to the pro-inflammatory cytokine interleukin 6 (IL-6). Recombinant IL-6 treatment increases hepatic ERRγ and BMP6 expression. Overexpression of ERRγ is sufficient to increase BMP6 gene expression in hepatocytes, suggesting that IL-6 is upstream of ERRγ. In line, knock-down of ERRγ in cell lines or a hepatocyte specific knock-out of ERRγ in mice significantly decreases IL-6 mediated BMP6 expression. Promoter studies show that ERRγ directly binds to the ERR response element (ERRE) in the mouse BMP6 gene promoter and positively regulates BMP6 gene transcription in IL-6 treatment conditions, which is further confirmed by ERRE mutated mBMP6-luciferase reporter assays. Finally, an inverse agonist of ERRγ, GSK5182, markedly inhibits IL-6 induced hepatic BMP6 expression in vitro and in vivo. Taken together, these results reveal a novel molecular mechanism on ERRγ mediated transcriptional regulation of hepatic BMP6 gene expression in response to IL-6.

scholarly journals Regulation of gonadotropin-releasing hormone-1 gene transcription by members of the purine-rich element-binding protein family

2010 ◽  
Vol 298 (3) ◽  
pp. E524-E533 ◽  
Author(s):  
Sheng Zhao ◽  
Robert J. Kelm ◽  
Russell D. Fernald
Keyword(s):  
Gene Transcription ◽  
Peptide Mapping ◽  
Cichlid Fish ◽  
Gonadotropin Releasing Hormone ◽  
Protein Family ◽  
Upstream Region ◽  
Mrna Levels ◽  
Releasing Hormone

Gonadotropin-releasing hormone-1 (GnRH1) controls reproduction by stimulating the release of gonadotropins from the pituitary. To characterize regulatory factors governing GnRH1 gene expression, we employed biochemical and bioinformatics techniques to identify novel GnRH1 promoter-binding proteins from the brain of the cichlid fish, Astatotilapia burtoni ( A. burtoni ). Using an in vitro DNA-binding assay followed by mass spectrometric peptide mapping, we identified two members of the purine-rich element-binding (Pur) protein family, Purα and Purβ, as candidates for GnRH1 promoter binding and regulation. We found that transcripts for both Purα and Purβ colocalize in GnRH1-expressing neurons in the preoptic area of the hypothalamus in A. burtoni brain. Furthermore, we confirmed in vivo binding of endogenous Purα and Purβ to the upstream region of the GnRH1 gene in A. burtoni brain and mouse neuronal GT1–7 cells. Consistent with the relative promoter occupancy exhibited by endogenous Pur proteins, overexpression of Purβ, but not Purα, significantly downregulated GnRH1 mRNA levels in transiently transfected GT1–7 cells, suggesting that Purβ acts as a repressor of GnRH1 gene transcription.

scholarly journals Changes in Liver Gene Expression of Azin1 Knock-out Mice

2010 ◽  
Vol 65 (7-8) ◽  
pp. 519-527 ◽  
Author(s):  
Tao Wan ◽  
Yuan Hu ◽  
Ailong Huang ◽  
Ken-ichi Yamamura ◽  
Hua Tang
Keyword(s):  
Ornithine Decarboxylase ◽  
Regulatory Protein ◽  
Fold Change ◽  
Oligonucleotide Array ◽  
Wild Type ◽  
Polyamine Biosynthesis ◽  
Knock Out ◽  
Knock Out Mice

The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change ≥ 2) and 1466 downregulated genes (fold change ≤ 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype

2013 ◽  
Vol 23 (4) ◽  
pp. 357-369 ◽  
Author(s):  
C. Gineste ◽  
J.M. De Winter ◽  
C. Kohl ◽  
C.C. Witt ◽  
B. Giannesini ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Knock Out ◽  
Knock Out Mice

scholarly journals Adrenocortical function and regulation of the steroid 21-hydroxylase gene in NGFI-B-deficient mice.

1995 ◽  
Vol 15 (8) ◽  
pp. 4331-4316 ◽  
Author(s):  
P A Crawford ◽  
Y Sadovsky ◽  
K Woodson ◽  
S L Lee ◽  
J Milbrandt
Keyword(s):  
Early Gene ◽  
Adrenocortical Function ◽  
Mrna Levels ◽  
Orphan Nuclear Receptor ◽  
Wild Type ◽  
Gene Encoding ◽  
Serum Corticosterone ◽  
B Mice

The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response element (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI-B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21-hydroxylase (P450c21) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450c21 promoter. To examine the influence of these factors on P450c21 expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (-/-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450c21 mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450c21 mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a close homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450c21 mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (-/-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450c21 expression in mice and maintain normal steroidogenesis.

Abstract 341: The P2Y2 Nucleotide Receptor Regulates Vascular Calcification

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Cheikh Seye ◽  
Maxwell Sheldon ◽  
Shaomin Qian
Keyword(s):  
Smooth Muscle ◽  
Vascular Calcification ◽  
Extracellular Nucleotides ◽  
Chow Diet ◽  
Knock Out ◽  
Retroviral Transduction ◽  
High Concentrations ◽  
Knock Out Mice

Objective: Vascular calcification is widespread in individuals with atherosclerosis, and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes. Recent studies identified extracellular nucleotides and P2Y receptor cascade as important regulators of bone remodeling. We investigated the potential role of the P2Y2 receptor (P2Y2R) in vascular calcification. Methods and Results: P2Y2R-null mice were crossed with ApoE-null mice to generate P2Y2R/ApoE double knock-out mice. When fed a standard mouse chow diet for 16 weeks, P2Y2R–/–/ApoE–/– mice showed significant higher intimal calcification as compared to their APOE–/– counterparts. Smooth muscle cells (SMCs) isolated from aortas of P2Y2R +/+ and P2Y2R -/- mice were identical in morphology and stained positively for SM lineage proteins including, desmin, smooth muscle and SM22alpha. When cultured in medium containing high concentrations of inorganic phosphate, an inducer of vascular calcification, a remarkably higher calcification was observed in P2Y2R -/- SMCs compared to P2Y2R +/+ SMCs. Furthermore, retroviral transduction of mouse P2Y2RcDNA into P2Y2R -/- SMCs rescued the calcification phenotype of the cells. Conclusion: These results demonstrate that inactivation of the P2Y2R gene regulates vascular calcification both in vivo and in vitro, suggesting that drugs targeting this receptor could prevent complications associated with vascular calcification

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