A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1689-1698 ◽  
Author(s):  
M.E. Horb ◽  
G.H. Thomsen
Keyword(s):  
Critical Role ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Cell Fates ◽  
Full Spectrum ◽  
T Box ◽  
Spemann Organizer ◽  
Embryonic Cleavage ◽  
Mesoderm Formation ◽  
Body Patterning

Pattern formation in early embryogenesis is guided by maternal, localized determinants and by inductive interactions between cells. In Xenopus eggs, localized molecules have been identified and some, such as Vg1 and Xwnt-11, can specify cell fates by functioning as inducers or patterning agents. We have used differential screening to identify new Xenopus genes that regulate mesodermal patterning, and we have isolated a new member of the T-box family of transcription factors. This gene, named Brat, is expressed maternally and its transcripts are localized to the vegetal hemisphere of the egg. During early embryonic cleavage, Brat mRNA becomes partitioned primarily within vegetal cells that are fated to form the endoderm. Zygotic expression of Brat begins at the onset of gastrulation within the presumptive mesoderm of the marginal zone. Consistent with its zygotic expression pattern, Brat induces, in a dose-dependent manner, a full spectrum of mesodermal genes that mark tissues across the dorsal-ventral axis, from the blood through the Spemann organizer. Brat also induces endoderm, consistent with its vegetal localization, making Brat a good candidate for a maternal determinant of the endoderm. We tested whether endogenous Brat is required for mesoderm formation by expressing a dominant-negative, transcriptional repressor form of Brat in embryos. This treatment inhibited mesoderm formation and severely disrupted normal development, thereby establishing that Brat plays a critical role in embryonic mesoderm formation and body patterning.

scholarly journals α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation

2017 ◽  
Vol 114 (5) ◽  
pp. 1183-1188 ◽  
Author(s):  
Seong Su Kang ◽  
Zhentao Zhang ◽  
Xia Liu ◽  
Fredric P. Manfredsson ◽  
Li He ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Lewy Bodies ◽  
Cell Loss ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Ph Domain ◽  
Dopaminergic Cell ◽  
Neuroprotective Actions

The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson’s disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP+), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L’s neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.

scholarly journals Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

2005 ◽  
Vol 169 (2) ◽  
pp. 331-339 ◽  
Author(s):  
Wanli W. Smith ◽  
Darrell D. Norton ◽  
Myriam Gorospe ◽  
Haibing Jiang ◽  
Shino Nemoto ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Redox Regulation ◽  
Critical Role ◽  
Ectopic Expression ◽  
Amyloid Β ◽  
Dominant Negative ◽  
Neuronal Cultures ◽  
Amyloid Β Peptide ◽  
Dependent Manner ◽  
Primary Neuronal Cultures

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

Abstract 266: Screening and Validation of Small Molecule Inhibitors of Serum and Glucocorticoid Regulated Kinase-1 as Novel Therapy for Cardiac Arrhythmia Disorders

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Vassilios J Bezzerides ◽  
Bridget Simonson ◽  
Kumaran Shanmugasundaram ◽  
Ottaviano Phyllis ◽  
Stacey Lynch ◽  
...  
Keyword(s):  
Small Molecule ◽  
Cardiac Arrhythmias ◽  
Small Molecule Inhibitors ◽  
Critical Role ◽  
Active Form ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Av Block ◽  
Lead Compound ◽  
Dose Dependent

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. Recent data demonstrates a critical role for the serum and glucocorticoid regulated kinase-1 (SGK1) by modulation of INa in the heart, by regulating the voltage-gated sodium channel NaV1.5. To better understand and pharmacologically probe the significance of SGK1 in cardiac dysrhythmias, we have used computer aided drug discovery (CADD) to identify small molecule inhibitors of SGK1. Expression of a constitutively active form of SGK1 (SGK1-CA) increased INa (1.7 fold, p <0.005) in a stable line of HEK cells expressing NaV1.5. Conversely, expression of a dominant negative form (SGK-DN) decreased NaV1.5 channel activity (2.8 fold, p <0.005). We examined the effects of SGK1 inhibition in a LQT model, by quantifying the ability of SGK1 inhibition to rescue the 2:1 AV block phenotype of the potassium channel zebrafish mutant, breakdance (bkd). Morpholino injection or expression of SGK1-DN significantly rescued the 2:1 AV block phenotype as compared to controls (p < 0.05). Using CADD partnered with iterative empirical screens we identified several hit chemical scaffolds. Our lead compound inhibits the phosphorylation of the SGK1 target gene, GSK3-β in a dose dependent manner in cardiomyocytes (CMs) expressing SGK1-CA at the lowest effective concentration of 0.5μM. There was no significant inhibition of AKT dependent phosphorylation of GSK3-β up to a concentration of 50μM, demonstrating specificity of the inhibitor for SGK1. Incubation of bkd zebrafish mutants with the inhibitor rescued the 2:1 AV block in a dose dependent manner (60% rescue with 45μM, p < 0.05). Acute application of the inhibitor dramatically inhibited INa with either expression of SGK1-CA (90.8% reduction, p <0.05 ) or with RFP only (77.5% reduction p < 0.005). The half-time of inhibition was 200s with resulting current densities that were not statistically different than those observed with genetic inhibition by expression of SGK1-DN. We conclude SGK1 activity regulates INa and speculate that structure activity relationship (SAR) derivatives of our lead compound might have a role in treatment of human cardiac arrhythmias.

The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling [correction of controling] dorsoventral patterning of Xenopus mesoderm

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3045-3053 ◽  
Author(s):  
D. Onichtchouk ◽  
V. Gawantka ◽  
R. Dosch ◽  
H. Delius ◽  
K. Hirschfeld ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Homeobox Gene ◽  
Dominant Negative ◽  
Signalling Pathway ◽  
Dependent Manner ◽  
Dorsoventral Patterning ◽  
Spemann Organizer ◽  
Dorsal Mesoderm ◽  
Dose Dependent

We describe a novel Xenopus homeobox gene, Xvent-2, which together with the previously identified homeobox gene Xvent-1, defines a novel class of homeobox genes. vent genes are related by sequence homology, expression pattern and gain-of-function phenotype. Evidence is presented for a role of Xvent-2 in the BMP-4 pathway involved in dorsoventral patterning of mesoderm. (1) Xvent-2 is expressed in regions that also express BMP-4. (2) Xvent-2 and BMP-4 interact in a positive feedback loop. (3) Xvent-2 ventralizes dorsal mesoderm in a dose-dependent manner resulting in phenoytpes ranging from microcephaly to Bauchstuck pieces, as does BMP-4. (4) Like BMP-4 and gsc, Xvent-2 and gsc are able to interact in a crossregulatory loop to suppress each other. (5) Microinjection of Xvent-2 mRNA can rescue dorsalization by a dominant-negative BMP-4 receptor. The results suggest that Xvent-2 functions in the BMP-4 signalling pathway that antagonizes the Spemann organizer.

scholarly journals Characterization of NF-κΒ/IκΒ Proteins in Zebra Fish and Their Involvement in Notochord Development

2004 ◽  
Vol 24 (12) ◽  
pp. 5257-5268 ◽  
Author(s):  
Ricardo G. Correa ◽  
Vinay Tergaonkar ◽  
Jennifer K. Ng ◽  
Ilir Dubova ◽  
Juan Carlos Izpisua-Belmonte ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Dependent Manner ◽  
Vertebrate Development ◽  
Zebra Fish ◽  
Promoter Regions ◽  
T Box ◽  
Biochemical Assays ◽  
Dominant Negative Form ◽  
Fish Cells

ABSTRACT Although largely involved in innate and adaptive immunity, NF-κB plays an important role in vertebrate development. In chicks, the inactivation of the NF-κΒ pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-κB activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-κΒ/IκB proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-κΒ/IκB members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-κΒ proteins are able to bind consensus DNA-binding (κB) sites and inhibitory IκBα proteins from mammals. We show that zebra fish IκBαs are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-κΒ activity in IκBα−/− MEFs. Expression of a dominant-negative form of the murine IκBα (mIκBαM), which is able to block NF-κΒ in zebra fish cells, interferes with the notochord differentiation, generating no tail (ntl)-like embryos. This phenotype can be rescued by coinjection of the T-box gene ntl (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that ntl lies downstream of NF-κΒ. We further show that ntl and Brachyury promoter regions contain functional κB sites and NF-κΒ can directly modulate ntl expression. Our study illustrates the conservation and compatibility of NF-κΒ/IκB proteins among vertebrates and the importance of NF-κΒ pathway in mesoderm formation during early embryogenesis.

scholarly journals Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian Gonadotropin-Releasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the Carboxyl-Terminal Tail Are Critical

Endocrinology ◽  
2003 ◽  
Vol 144 (9) ◽  
pp. 3860-3871 ◽  
Author(s):  
Adam J. Pawson ◽  
Stuart R. Maudsley ◽  
John Lopes ◽  
Arieh A. Katz ◽  
Yuh-Man Sun ◽  
...  
Keyword(s):  
Critical Role ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Coated Vesicle ◽  
Dependent Manner ◽  
Structural Determinants ◽  
Hek 293 ◽  
Carboxyl Terminal ◽  
Gonadotropin Releasing Hormone Receptor

Abstract The chicken GnRH receptor (cGnRH-R) differs from all mammalian GnRH-Rs in possessing a cytoplasmic carboxyl-terminal tail. We have previously demonstrated that the cGnRH-R undergoes more rapid agonist-induced internalization than the mammalian GnRH-Rs and requires the carboxyl-terminal tail for this process. To investigate the structural determinants mediating this rapid internalization, a series of mutant receptors was generated, including progressive truncations of the tail and substitution of serine and threonine residues with alanine. Truncation of the carboxyl-terminal tail to position 366 and then to position 356 resulted in a progressive attenuation of the rate and total extent of receptor internalization. However, truncation between positions 356 and 346 did not alter the kinetics of internalization further, whereas a further truncation to position 337 resulted in an additional marked reduction of internalization. We show that the membrane-proximal Cys328 and the Thr369Thr370 doublet located in the distal carboxyl terminus play a critical role in mediating rapid internalization. We demonstrate that the cGnRH-R, when expressed in both COS-7 and HEK 293 cells, preferentially undergoes rapid agonist-induced internalization in a caveolae-like, dynamin-dependent manner. These conclusions are based on our observation that pretreatments with filipin and methyl-β-cyclodextrin, agents that disrupt lipid rafts such as caveolae, and coexpression of dominant-negative dynamin-1 (K44A) and caveolin-1 (Δ1–81) mutants, effectively inhibited rapid agonist-induced internalization. Furthermore, cGnRH-Rs appeared to be mobilized to the β-arrestin- and clathrin-coated, vesicle-mediated endocytic pathway upon β-arrestin overexpression.

scholarly journals Exogenous C2-ceramide activates c-fos serum response element via Rac-dependent signalling pathway

1998 ◽  
Vol 330 (2) ◽  
pp. 1009-1014 ◽  
Author(s):  
Byung-Chul KIM ◽  
Jae-Hong KIM
Keyword(s):  
Phospholipase A2 ◽  
Reporter Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Response Element ◽  
Serum Response ◽  
Negative Mutant

Ceramide is an important regulatory molecule implicated in a variety of biological processes in response to stress and cytokines. To understand the signal transduction pathway of ceramide to the nucleus, in the present study, we examined whether C2-ceramide, a cell permeable ceramide, activates c-fos serum response element (SRE). Treatment of Rat-2 fibroblast cells with C2-ceramide caused the stimulation of c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner by transient transfection analysis. Next, we examined the role of Rho family GTPases in the ceramide-induced signalling to SRE activation. By reporter gene analysis following transient transfections with various plasmids expressing a dominant negative mutant form of Cdc42, Rac1 or RhoA, C2-ceramide-induced SRE activation was shown to be selectively repressed by pEXV-RacN17 encoding a dominant negative mutant of Rac1, suggesting that Rac activity is essential for the signalling cascade of ceramide to the nucleus. In a further study to analyse the downstream mediator of Rac in the ceramide-signalling pathway, we observed that either pretreatment with mepacrine, a potent and specific inhibitor of phospholipase A2, or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide repressed the C2-ceramide-induced SRE activation selectively, implying a critical role of cPLA2 in C2-ceramide-induced signalling to nucleus. Consistent with these results, the translocation of cPLA2 protein as well as the release of arachidonic acid, a principal product of phospholipase A2, was rapidly induced by the addition of C2-ceramide in a Rac-dependent manner. Together, our findings suggest the critical role of ‘Rac and subsequent activation of phospholipase A2’ in ceramide-signalling to nucleus.

scholarly journals Gonadotropin-Releasing Hormone Induction of Extracellular-Signal Regulated Kinase Is Blocked by Inhibition of Calmodulin

2005 ◽  
Vol 19 (9) ◽  
pp. 2412-2423 ◽  
Author(s):  
Mark S. Roberson ◽  
Stuart P. Bliss ◽  
Jianjun Xie ◽  
Amy M. Navratil ◽  
Todd A. Farmerie ◽  
...  
Keyword(s):  
Critical Role ◽  
Dominant Negative ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Erk Activation ◽  
Raf Kinase ◽  
Rat Pituitary Cells

Abstract Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in αT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone α-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.

scholarly journals STAT3 modulates reprogramming efficiency of human somatic cells; insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations

Biology Open ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. bio052662
Author(s):  
Zhen Yu ◽  
Natalia I. Dmitrieva ◽  
Avram D. Walts ◽  
Hui Jin ◽  
Yangtengyu Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Molecular Mechanisms ◽  
Autosomal Dominant ◽  
Critical Role ◽  
Somatic Cells ◽  
Dominant Negative ◽  
Skin Fibroblasts ◽  
Dependent Manner ◽  
Cell Based Therapy ◽  
Reprogramming Efficiency

ABSTRACTHuman induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.

Protein kinase Cδ-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

2007 ◽  
Vol 292 (6) ◽  
pp. C2150-C2160 ◽  
Author(s):  
Carlos Clavijo ◽  
Jo-Lin Chen ◽  
Kwang-Jin Kim ◽  
Mary E. Reyland ◽  
David K. Ann
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Dominant Negative ◽  
Fine Tuning ◽  
Time Dependent ◽  
Response To Treatment ◽  
Dependent Manner

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKCδ is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKCδ by RNAi (sh-PKCδ) or expression of the kinase-dead (Lys376Arg) mutant of PKCδ (PKCδKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (γ-H2AX), a hallmark for DNA damage, was altered by sh-PKCδ, and sh-PKCδ completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKCδ-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKCδ and caspase-3, suggesting that PKCδKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKCδ-dependent and -independent manners. In summary, these findings suggest that the activation of a PKCδ-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment.

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