231. Mouse oocyte paracrine signalling to cumulus cells by TGF-β superfamily molecules is indispensable for cumulus expansion

2005 ◽  
Vol 17 (9) ◽  
pp. 90
Author(s):  
R. A. Dragovic ◽  
L. J. Ritter ◽  
S. J. Schulz ◽  
D. T. Armstrong ◽  
R. B. Gilchrist
Keyword(s):  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Cumulus Cells ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Cumulus Expansion ◽  
Maximum Expansion ◽  
Paracrine Signalling ◽  
Growth Differentiation Factor 9

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex (COC), which is necessary for ovulation. Members of the transforming growth factor-β (TGF-β) superfamily are prime candidates for the mouse cumulus expansion-enabling factor (CEEF), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. This study was conducted to examine TGF-β superfamily processes regulating cumulus expansion. COCs were collected from eCG-primed mice and the oocyte microsurgically removed to generate oocytectomised (OOX) complexes. An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P < 0.05) induced by either GDF9 (score: mean ± SEM, 3.7 ± 0.1), activin A (2.6 ± 0.1), or co-culture with oocytes (3.2 ± 0.2). The type-I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively. We tested the ability of the ALK4/5/7 kinase inhibitor, SB431542, to neutralise cumulus expansion. SB431542 completely neutralised (P < 0.05) the response of OOX complexes to GDF9, activin and oocyte-induced cumulus expansion. SB431542 also neutralised (P < 0.05) COC expansion in a dose dependent manner. Follistatin, an activin antagonist was effective at neutralising the response of OOX complexes to activin (score: 0), but had no significant effect (P > 0.05) on the expansion of OOX complexes co-cultured with oocytes (score: 2.7 ± 0.2). This study provides evidence that activin is not the sole CEEF, but signalling through the ALK4/5/7 pathway is indispensable for mouse cumulus expansion.

scholarly journals 251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)

2004 ◽  
Vol 16 (9) ◽  
pp. 251
Author(s):  
R. A. Dragovic ◽  
L. J. Ritter ◽  
F. Amato ◽  
S. J. Scott ◽  
M. Cranfield ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Cumulus Expansion ◽  
Maximum Expansion ◽  
Paracrine Signalling ◽  
Differentiation Factor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Hyaluronan Synthase 2 ◽  
Candidate Molecule

Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.

scholarly journals NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I receptor

2005 ◽  
Vol 386 (3) ◽  
pp. 461-470 ◽  
Author(s):  
Go KURATOMI ◽  
Akiyoshi KOMURO ◽  
Kouichiro GOTO ◽  
Masahiko SHINOZAKI ◽  
Keiji MIYAZAWA ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Ubiquitin Ligases ◽  
Transforming Growth Factor Β ◽  
Precursor Cell ◽  
Neural Precursor ◽  
Type I ◽  
Dependent Manner ◽  
E3 Ubiquitin Ligases ◽  
Neural Precursor Cell

Inhibitory Smad, Smad7, is a potent inhibitor of TGF-β (transforming growth factor-β) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-β superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-β type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-β-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-β and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-β superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-β superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.

scholarly journals Inhibition of Pulmonary and Skeletal Metastasis by a Transforming Growth Factor-β Type I Receptor Kinase Inhibitor

2006 ◽  
Vol 66 (13) ◽  
pp. 6714-6721 ◽  
Author(s):  
Abhik Bandyopadhyay ◽  
Joseph K. Agyin ◽  
Long Wang ◽  
Yuping Tang ◽  
Xiufen Lei ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Skeletal Metastasis ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Receptor Kinase

scholarly journals Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

2014 ◽  
Vol 25 (10) ◽  
pp. 1620-1628 ◽  
Author(s):  
Keren E. Shapira ◽  
Tal Hirschhorn ◽  
Lior Barzilay ◽  
Nechama I. Smorodinsky ◽  
Yoav I. Henis ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Lateral Diffusion ◽  
Adaptor Protein ◽  
Transforming Growth Factor Β ◽  
Cholesterol Depletion ◽  
Type I ◽  
Dependent Manner ◽  
A Cell ◽  
Β Receptor ◽  
Signaling Receptors

Transforming growth factor-β (TGF-β) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context–dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-β signaling. However, the molecular mechanism(s) involved in the regulation of TGF-β signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-β receptor (TβRI) with TGF-β phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TβRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA–mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-β–induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-β in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TβRI in the membrane, balancing TGF-β signaling via the Smad and JNK pathways.

scholarly journals Transforming Growth Factor-β Induces Nuclear Import of Smad3 in an Importin-β1 and Ran-dependent Manner

2001 ◽  
Vol 12 (4) ◽  
pp. 1079-1091 ◽  
Author(s):  
Akira Kurisaki ◽  
Shingo Kose ◽  
Yoshihiro Yoneda ◽  
Carl-Henrik Heldin ◽  
Aristidis Moustakas
Keyword(s):  
Growth Factor ◽  
Nuclear Import ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Structural Basis ◽  
Type I ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Terminal Domain

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-β (TGF-β) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-β type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-β1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-β1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-β1. 4) Importin-β1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-β receptor leads to enhanced interaction with importin-β1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

scholarly journals Autocrine TGFβ1 Opposes Exogenous TGFβ1-Induced Cell Migration and Growth Arrest through Sustainment of a Feed-Forward Loop Involving MEK-ERK Signaling

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1357
Author(s):  
Hendrik Ungefroren ◽  
Jessica Christl ◽  
Caroline Eiden ◽  
Ulrich F. Wellner ◽  
Hendrik Lehnert ◽  
...  
Keyword(s):  
Cell Motility ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Growth Arrest ◽  
Transforming Growth Factor Β ◽  
Erk Signaling ◽  
Ductal Adenocarcinoma ◽  
Type I ◽  
Cyclin Dependent Kinase Inhibitor

Autocrine transforming growth factor β (aTGFβ) has been implicated in the regulation of cell invasion and growth of several malignant cancers such as pancreatic ductal adenocarcinoma (PDAC) or triple-negative breast cancer (TNBC). Recently, we observed that endogenous TGFB1 can inhibit rather than stimulate cell motility in cell lines with high aTGFβ production and mutant KRAS, i.e., Panc1 (PDAC) and MDA-MB-231 (TNBC). The unexpected anti-migratory role prompted us to evaluate if aTGFβ1 may be able to antagonize the action of exogenous (recombinant human) TGFβ (rhTGFβ), a well-known promoter of cell motility and growth arrest in these cells. Surprisingly, RNA interference-mediated knockdown of the endogenous TGFB1 sensitized genes involved in EMT and cell motility (i.e., SNAI1) to up-regulation by rhTGFβ1, which was associated with a more pronounced migratory response following rhTGFβ1 treatment. Ectopic expression of TGFB1 decreased both basal and rhTGFβ1-induced migratory activities in MDA-MB-231 cells but had the opposite effect in Panc1 cells. Moreover, silencing TGFB1 reduced basal proliferation and enhanced growth inhibition by rhTGFβ1 and induction of cyclin-dependent kinase inhibitor, p21WAF1. Finally, we show that aTGFβ1 promotes MEK-ERK signaling and vice versa to form a self-perpetuating feedforward loop that is sensitive to SB431542, an inhibitor of the TGFβ type I receptor, ALK5. Together, these data suggest that in transformed cells an ALK5-MEK-ERK-aTGFβ1 pathway opposes the promigratory and growth-arresting function of rhTGFβ1. This observation has profound translational implications for TGFβ signaling in cancer.

scholarly journals Transforming Growth Factor β Facilitates β-TrCP-Mediated Degradation of Cdc25A in a Smad3-Dependent Manner

2005 ◽  
Vol 25 (8) ◽  
pp. 3338-3347 ◽  
Author(s):  
Dipankar Ray ◽  
Yasuhisa Terao ◽  
Dipali Nimbalkar ◽  
Li-Hao Chu ◽  
Maddalena Donzelli ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Anaphase Promoting Complex ◽  
Mutant Proteins ◽  
Major Mechanism ◽  
Β Receptor ◽  
Interfering Rna

ABSTRACT Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-β-TrCP (SCFβ-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor β (TGF-β)-Smad3 pathway promotes SCFβ-TrCP-mediated Cdc25A ubiquitination. Cells treated with TGF-β, as well as cells transfected with Smad3 or a constitutively active type I TGF-β receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-β-induced ubiquitination is associated with Cdc25A phosphorylation at the β-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and β-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-β-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits β-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-β. β-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFβ-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-β-Smad3 pathway.

scholarly journals FKBP12 functions as an adaptor of the Smad7–Smurf1 complex on activin type I receptor

2006 ◽  
Vol 36 (3) ◽  
pp. 569-579 ◽  
Author(s):  
T Yamaguchi ◽  
A Kurisaki ◽  
N Yamakawa ◽  
K Minakuchi ◽  
H Sugino
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Regulatory Factor ◽  
Fk506 Binding Protein ◽  
Adaptor Molecule ◽  
Inhibitory Function ◽  
Inhibitory Molecule

The cytoplasmic immunophilin FKBP12, a 12 kDa FK506-binding protein, has been shown to act as an inhibitor for transforming growth factor-β (TGF-β) signaling. FKBP12 binds to the glycine- and serine-rich motif (GS motif) of the TGF-β type I receptor, and functions as a secure switch to prevent the leaky signal. Upon stimulation with ligand, FKBP12 is released from the receptor to fully propagate the signal. We found that activin, a member of TGF-β superfamily, also induced the dissociation of FKBP12 from the activin type I receptor (ALK4). However, we observed that the released FKBP12 associates again with the receptor a few hours later. FKBP12 also interacted with another inhibitory molecule of activin signal, Smad7, in an activin-dependent manner, and formed a complex with Smad7 on the type I receptor. FK506, a chemical ligand for FKBP12, which dissociates FKBP12 from the receptor, decreased the interaction between Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1). FK506 also inhibited the ubiquitination of the type I receptor by Smurf1. These findings indicate a new inhibitory function of FKBP12 as an adaptor molecule for the Smad7–Smurf1 complex to regulate the duration of the activin signal.

scholarly journals Screening and Identification of a Novel Class of TGF-β Type 1 Receptor Kinase Inhibitor

2011 ◽  
Vol 16 (7) ◽  
pp. 724-733 ◽  
Author(s):  
Q. Khai Huynh ◽  
Sarah J. Wise ◽  
Keith A. Koch ◽  
Laurie A. Castonguay ◽  
Brian G. Reid ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Promising Target ◽  
Ic50 Value ◽  
Screening And Identification ◽  
Response Testing

Transforming growth factor β (TGF-β) type I receptor (activin receptor–like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420 000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration–response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC50 value of 0.7 µM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC50 value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.

Sign in / Sign up

Export Citation Format

Share Document