Truncated Activin Type I Receptor Alk4 Isoforms Are Dominant Negative Receptors Inhibiting Activin Signaling

Bone Morphogenetic Proteins (BMPs) are potent regulators of embryonic cell fate that are presumed to initiate signal transduction in recipient cells through multimeric, transmembrane, serine/threonine kinase complexes made up of type I and type II receptors. BMPRII was identified previously in mammals as the only type II receptor that binds BMPs, but not activin or TGFbeta, in vitro. We report the cloning and functional analysis in vivo of its Xenopus homolog, XBMPRII. XBMPRII is expressed maternally and zygotically in an initially unrestricted manner. Strikingly, XBMPRII transcripts then become restricted to the mesodermal precursors during gastrulation. Expression is lower in the dorsal organizer region, potentially providing a mechanism to suppress the actions of BMP4 on dorsally fated tissues. Similar to the results seen for a truncated type I BMP receptor (tBR), a dominant-negative form of XBMPRII (tBRII) can dorsalize ventral mesoderm, induce extensive secondary body axes, block mesoderm induction by BMP4 and directly neuralize ectoderm, strongly suggesting that XBMPRII mediates BMP signals in vivo. However, although both tBRII and tBR can induce partial secondary axes, marker analysis shows that tBRII-induced axes are more anteriorly extended. Additionally, coinjection of tBRII and tBR synergistically increases the incidence of secondary axis formation. A truncated activin type II receptor (deltaXAR1) is known to block both activin and BMP signaling in vivo. Here we show that such crossreactivity does not occur for tBRII, in that it does not affect activin signaling. Furthermore, our studies indicate that the full-length activin type II receptor (XAR1) overcomes a block in BMP4 signaling imposed by tBRII, implicating XAR1 as a common component of BMP and activin signaling pathways in vivo. These data implicate XBMPRII as a type II receptor with high selectivity for BMP signaling, and therefore as a critical mediator of the effects of BMPs as mesodermal patterning agents and suppressors of neural fate during embryogenesis.

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Evidence for a dominant‐negative effect of a missense mutation in the SERPING1 gene responsible for hereditary angioedema type I

The Journal of Dermatology ◽

10.1111/1346-8138.15930 ◽

2021 ◽

Author(s):

Shuichiro Yasuno ◽

Osamu Ansai ◽

Ryota Hayashi ◽

Sawako Nakamura ◽

Yutaka Shimomura

Keyword(s):

Missense Mutation ◽

Hereditary Angioedema ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Type I ◽

Negative Effect ◽

Serping1 Gene

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TGF-β and the Smad Signaling Pathway Support Transcriptomic Reprogramming during Epithelial-Mesenchymal Cell Transition

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0658 ◽

2005 ◽

Vol 16 (4) ◽

pp. 1987-2002 ◽

Cited By ~ 366

Author(s):

Ulrich Valcourt ◽

Marcin Kowanetz ◽

Hideki Niimi ◽

Carl-Henrik Heldin ◽

Aristidis Moustakas

Keyword(s):

Target Genes ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Smooth Muscle Actin ◽

Expression Patterns ◽

Ectopic Expression ◽

Dominant Negative ◽

Type I ◽

Mesenchymal Transition ◽

Smad Signaling

Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-β/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-β superfamily establishes that TGF-β but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-β–induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-β target genes with ligand-specific responses. Using a TGF-β type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-β1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, α-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-β and predict functional links to the control of cell proliferation and EMT.

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The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis

Journal of Cell Science ◽

10.1242/jcs.111.23.3451 ◽

1998 ◽

Vol 111 (23) ◽

pp. 3451-3458 ◽

Cited By ~ 3

Author(s):

G. Banting ◽

R. Maile ◽

E.P. Roquemore

Keyword(s):

Steady State ◽

Cell Surface ◽

Dominant Negative ◽

Type I ◽

Steady State Distribution ◽

State Distribution ◽

Trans Golgi Network ◽

Defective Mutant ◽

Dynamin I ◽

Golgi Network

It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

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HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0211 ◽

2021 ◽

Author(s):

Toshihiro Kobayashi ◽

Hitomi Imachi ◽

Kensaku Fukunaga ◽

Jingya Lyu ◽

Seisuke Sato ◽

...

Keyword(s):

Gene Expression ◽

Signaling Pathways ◽

Promoter Activity ◽

Active Form ◽

Specific Inhibitor ◽

Density Lipoprotein ◽

Dominant Negative ◽

Activation Mechanism ◽

Dominant Negative Mutant ◽

Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

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Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00279.2019 ◽

2020 ◽

Vol 318 (1) ◽

pp. C215-C224 ◽

Cited By ~ 1

Author(s):

Joaquin M. Muriel ◽

Andrea O’Neill ◽

Jaclyn P. Kerr ◽

Emily Kleinhans-Welte ◽

Richard M. Lovering ◽

...

Keyword(s):

Skeletal Muscle ◽

Isometric Force ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Type I ◽

Mrna Levels ◽

Force Transmission ◽

Keratin 18 ◽

Murine Skeletal Muscle

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout ( Krt18-KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

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Statin-induced Ras Activation Integrates the Phosphatidylinositol 3-Kinase Signal to Akt and MAPK for Bone Morphogenetic Protein-2 Expression in Osteoblast Differentiation

Journal of Biological Chemistry ◽

10.1074/jbc.m606706200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4983-4993 ◽

Cited By ~ 100

Author(s):

Nandini Ghosh-Choudhury ◽

Chandi Charan Mandal ◽

Goutam Ghosh Choudhury

Keyword(s):

Bone Morphogenetic Protein ◽

Osteoblast Differentiation ◽

Dominant Negative ◽

Bone Morphogenetic Protein 2 ◽

Regulatory Subunit ◽

Type I ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

Morphogenetic Protein ◽

Phosphatidylinositol 3

Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation.

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Activin receptor-like kinase-2 inhibits activin signaling by blocking the binding of activin to its type II receptor

Journal of Endocrinology ◽

10.1677/joe-07-0281 ◽

2007 ◽

Vol 195 (1) ◽

pp. 95-103 ◽

Cited By ~ 19

Author(s):

Nina Renlund ◽

Francis H O’Neill ◽

LiHua Zhang ◽

Yisrael Sidis ◽

Jose Teixeira

Keyword(s):

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Underlying Mechanism ◽

Luciferase Reporter ◽

Type I ◽

Type Ii ◽

Activin Receptor ◽

Activin Signaling ◽

Endogenous Expression ◽

Receptor Like Kinase

Activin receptor-like kinase-2 (Alk2) has been shown to be a promiscuous type I receptor for the transforming growth factor β (TGFβ) family of growth and differentiation factors, such as activin, bone morphogenetic proteins, and Müllerian inhibiting substance (MIS). We have studied the putative role of Alk2 in activin signaling using MA-10 cells, a mouse transformed Leydig cell line, in which endogenous expression of cytochrome P450 c17 hydroxylase/C17–20 lyase mRNA is inhibited by both MIS and activin A. Overexpression of Alk2 in MA-10 cells inhibited the activation of the activin-responsive CAGA-luciferase reporter and, conversely, transfection of siRNA for Alk2 increased the response. In contrast, overexpression of the MIS type II receptor in MA-10 cells increased the activin-mediated induction of CAGA-luciferase approximately fivefold, which we hypothesized occurs by MIS type II receptor sequestering endogenous Alk2. Binding experiments with 125I-labeled activin show that the underlying mechanism of Alk2-mediated inhibition of activin signaling involves Alk2 blocking the access of activin to its type II receptor, which we show can bind Alk2 in the absence of ligand. These results show that the complement of other type I receptors in addition to the ligand-specific type I receptor can provide an important mechanism for modulating cell-specific responses to members of the TGFβ family.

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Genetic, Phenotypic, and Interferon Biomarker Status in ADAR1-Related Neurological Disease

Neuropediatrics ◽

10.1055/s-0037-1601449 ◽

2017 ◽

Vol 48 (03) ◽

pp. 166-184 ◽

Cited By ~ 25

Author(s):

Gillian Rice ◽

Naoki Kitabayashi ◽

Magalie Barth ◽

Tracy Briggs ◽

Annabel Burton ◽

...

Keyword(s):

Neurological Disease ◽

De Novo ◽

Phenotypic Variability ◽

Spastic Paraparesis ◽

Dominant Negative ◽

Compound Heterozygous ◽

Motor Disorder ◽

Type I ◽

Missense Mutations ◽

Dominant Negative Mutation

AbstractWe investigated the genetic, phenotypic, and interferon status of 46 patients from 37 families with neurological disease due to mutations in ADAR1. The clinicoradiological phenotype encompassed a spectrum of Aicardi–Goutières syndrome, isolated bilateral striatal necrosis, spastic paraparesis with normal neuroimaging, a progressive spastic dystonic motor disorder, and adult-onset psychological difficulties with intracranial calcification. Homozygous missense mutations were recorded in five families. We observed a p.Pro193Ala variant in the heterozygous state in 22 of 23 families with compound heterozygous mutations. We also ascertained 11 cases from nine families with a p.Gly1007Arg dominant-negative mutation, which occurred de novo in four patients, and was inherited in three families in association with marked phenotypic variability. In 50 of 52 samples from 34 patients, we identified a marked upregulation of type I interferon-stimulated gene transcripts in peripheral blood, with a median interferon score of 16.99 (interquartile range [IQR]: 10.64–25.71) compared with controls (median: 0.93, IQR: 0.57–1.30). Thus, mutations in ADAR1 are associated with a variety of clinically distinct neurological phenotypes presenting from early infancy to adulthood, inherited either as an autosomal recessive or dominant trait. Testing for an interferon signature in blood represents a useful biomarker in this context.

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Integrin β1Signaling Is Necessary for Transforming Growth Factor-β Activation of p38MAPK and Epithelial Plasticity

Journal of Biological Chemistry ◽

10.1074/jbc.m106176200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 46707-46713 ◽

Cited By ~ 264

Author(s):

Neil A. Bhowmick ◽

Roy Zent ◽

Mayshan Ghiassi ◽

Maureen McDonnell ◽

Harold L. Moses

Keyword(s):

Extracellular Matrix ◽

Growth Factor ◽

Transcriptional Activation ◽

Intracellular Signaling ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Dominant Negative ◽

Receptor Expression ◽

Type I ◽

Type Ii

Transforming growth factor-β (TGF-β) can induce epithelial to mesenchymal transdifferentiation (EMT) in mammary epithelial cells. TGF-β-meditated EMT involves the stimulation of a number of signaling pathways by the sequential binding of the type II and type I serine/threonine kinase receptors, respectively. Integrins comprise a family of heterodimeric extracellular matrix receptors that mediate cell adhesion and intracellular signaling, hence making them crucial for EMT progression. In light of substantial evidence indicating TGF-β regulation of various β1integrins and their extracellular matrix ligands, we examined the cross-talk between the TGF-β and integrin signal transduction pathways. Using an inducible system for the expression of a cytoplasmically truncated dominant negative TGF-β type II receptor, we blocked TGF-β-mediated growth inhibition, transcriptional activation, and EMT progression. Dominant negative TGF-β type II receptor expression inhibited TGF-β signaling to the SMAD and AKT pathways, but did not block TGF-β-mediated p38MAPK activation. Interestingly, blocking integrin β1function inhibited TGF-β-mediated p38MAPK activation and EMT progression. Limiting p38MAPK activity through the expression of a dominant negative-p38MAPK also blocked TGF-β-mediated EMT. In summary, TGF-β-mediated p38MAPK activation is dependent on functional integrin β1, and p38MAPK activity is required but is not sufficient to induce EMT.

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