scholarly journals Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-β autocrine regulatory loop

2004 ◽  
Vol 381 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Séverine ALLEGRA ◽  
Jacques Yuan LI ◽  
José Maria SAEZ ◽  
Dominique LANGLOIS
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Terminal Differentiation ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Time Interval ◽  
Tgfβ Signalling ◽  
Regulatory Loop ◽  
Myod Expression

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the βMHC (β-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFβ (transforming growth factor β) on βMHC expression in Sol 8 cells. TGFβ1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited βMHC expression in Sol 8 cells. This inhibition of βMHC expression was progressively lost when TGFβ1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFβ1 did not inhibit βMHC accumulation any longer. Two independent approaches showed that a TGFβ autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFβs released by the cells into DM increased βMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFβ type II receptor was overexpressed in Sol 8 cells under the control of the βMHC promoter. Both the dominant-negative receptor and the βMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFβ signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote βMHC expression. To conclude, TGFβ inhibits Sol 8 cell terminal differentiation within a narrow time interval (24–34 h) that coincides with the onset of βMHC expression.

Expression of mutant human epidermal receptor 3 attenuates lung fibrosis and improves survival in mice

2005 ◽  
Vol 99 (1) ◽  
pp. 298-307 ◽  
Author(s):  
David E. Nethery ◽  
Bethany B. Moore ◽  
George Minowada ◽  
James Carroll ◽  
Jihane A. Faress ◽  
...  
Keyword(s):  
Growth Factor ◽  
Lung Injury ◽  
Transgenic Mice ◽  
Bronchoalveolar Lavage ◽  
Bronchoalveolar Lavage Fluid ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Lavage Fluid ◽  
Dominant Negative ◽  
Nontransgenic Littermate

Neuregulin-1 (NRG-1), binding to the human epidermal growth factor receptor HER2/HER3, plays a role in pulmonary epithelial cell proliferation and recovery from injury in vitro. We hypothesized that activation of HER2/HER3 by NRG-1 would also play a role in recovery from in vivo lung injury. We tested this hypothesis using bleomycin lung injury of transgenic mice incapable of signaling through HER2/HER3 due to lung-specific dominant-negative HER3 (DNHER3) expression. In animals expressing DNHER3, protein leak, cell infiltration, and NRG-1 levels in bronchoalveolar lavage fluid increased after injury, similar to that in nontransgenic littermate control animals. However, HER2/HER3 was not activated, and DNHER3 animals displayed fewer lung morphological changes at 10 and 21 days after injury ( P = 0.01). In addition, they contained 51% less collagen in injured lungs ( P = 0.04). Transforming growth factor-β1 did not increase in bronchoalveolar lavage fluid from DNHER3 mice compared with nontransgenic littermate mice ( P = 0.001), suggesting that a mechanism for the decreased fibrosis was lack of transforming growth factor-β1 induction in DNHER3 mice. Severe lung injury (0.08 units bleomycin) resulted in 80% mortality of nontransgenic mice, but only 35% mortality of DNHER3 transgenic mice ( P = 0.04). Thus inhibition of HER2/HER3 signaling protects against pulmonary fibrosis and improves survival.

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

2001 ◽  
Vol 276 (50) ◽  
pp. 46707-46713 ◽  
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.

scholarly journals Arkadia Activates Smad3/Smad4-Dependent Transcription by Triggering Signal-Induced SnoN Degradation

2007 ◽  
Vol 27 (17) ◽  
pp. 6068-6083 ◽  
Author(s):  
Laurence Levy ◽  
Michael Howell ◽  
Debipriya Das ◽  
Sean Harkin ◽  
Vasso Episkopou ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Transforming Growth Factor ◽  
E3 Ubiquitin Ligase ◽  
Cancer Cell Line ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Esophageal Cancer Cell ◽  
Esophageal Cancer Cell Line

ABSTRACT E3 ubiquitin ligases play important roles in regulating transforming growth factor β (TGF-β)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-β induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-β response. Knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-β-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-β/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-β-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer.

scholarly journals Transforming Growth Factor β/Smad3 Signaling Regulates IRF-7 Function and Transcriptional Activation of the Beta Interferon Promoter

2004 ◽  
Vol 24 (3) ◽  
pp. 1411-1425 ◽  
Author(s):  
Jing Qing ◽  
Cheng Liu ◽  
Lisa Choy ◽  
Rui-Yun Wu ◽  
Joseph S. Pagano ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transcriptional Activation ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Negative Interference ◽  
Rapid Induction ◽  
Dominant Negative Interference ◽  
Smad3 Expression

ABSTRACT The rapid induction of alpha interferon (IFN-α) and IFN-β expression plays a critical role in the innate immune response against viral infection. We studied the effects of transforming growth factor β (TGF-β) and its intracellular effectors, the Smads, on the function of IRF-7, an essential transcription factor for IFN-α and -β induction. IRF-7 interacted with Smads, and IRF-7, but not IRF-3, cooperated with Smad3 to activate IFN-β transcription. This transcriptional cooperation occurred at the IRF-binding sequences in the IFN-β promoter, and dominant-negative interference with TGF-β receptor signaling and Smad3 function decreased IRF-7-mediated transcription. Furthermore, elimination of Smad3 expression in Smad3−/− fibroblasts delayed and decreased double-stranded RNA-induced expression of endogenous IFN-β, whereas restoration of Smad3 expression enhanced IFN-β induction. The IRF-7-Smad3 cooperativity resulted from the regulation of the transactivation activity of IRF-7 by Smad3, and dominant-negative interference with Smad3 function decreased IRF-7 activity. Consistent with the regulation by Smad3, the transcriptional activity of IRF-7 depended on and was regulated by TGF-β signaling. Our studies underscore a role of TGF-β/Smad3 signaling in IRF-7-mediated induction of IFN-β expression.

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