Caveolin-2 is a negative regulator of anti-proliferative function and signaling of transforming growth factor-β in endothelial cells

Using a combination of wild-type (WT) and caveolin-2 (Cav-2) knockout along with retroviral reexpression approaches, we provide the evidence for the negative role of Cav-2 in regulating anti-proliferative function and signaling of transforming growth factor β (TGF-β) in endothelial cells (ECs). Although, TGF-β had a modest inhibitory effect on WT ECs, it profoundly inhibited proliferation of Cav-2 knockout ECs. To confirm the specificity of the observed difference in response to TGF-β, we have stably reexpressed Cav-2 in Cav-2 knockout ECs using a retroviral approach. Similar to WT ECs, the anti-proliferative effect of TGF-β was dramatically reduced in the Cav-2 reexpressing ECs. The reduced anti-proliferative effect of TGF-β in Cav-2-positive cells was evidenced by three independent proliferation assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell count, and bromodeoxyuridine incorporation and correlated with a loss of TGF-β-mediated upregulation of cell cycle inhibitor p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the retinoblastoma protein in Cav-2 reexpressing ECs. Mechanistically, Cav-2 inhibits anti-proliferative action of TGF-β by suppressing Alk5-Smad2/3 pathway manifested by reduced magnitude and length of TGF-β-induced Smad2/3 phosphorylation as well as activation of activin receptor-like kinase-5 (Alk5)-Smad2/3 target genes plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive ECs. Expression of Cav-2 does not appear to significantly change targeting of TGF-β receptors I and Smad2/3 to caveolar and lipid raft microdomains as determined by sucrose fractionation gradient. Overall, the negative regulation of TGF-β signaling and function by Cav-2 is independent of Cav-1 expression levels and is not because of changing targeting of Cav-1 protein to plasma membrane lipid raft/caveolar domains.

Download Full-text

SnoN facilitates ALK1–Smad1/5 signaling during embryonic angiogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201208113 ◽

2013 ◽

Vol 202 (6) ◽

pp. 937-950 ◽

Cited By ~ 10

Author(s):

Qingwei Zhu ◽

Yong Hwan Kim ◽

Douglas Wang ◽

S. Paul Oh ◽

Kunxin Luo

Keyword(s):

Endothelial Cells ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Endothelial Cell Proliferation ◽

Type I ◽

Positive Role ◽

And Migration ◽

Proliferation And Migration

In endothelial cells, two type I receptors of the transforming growth factor β (TGF-β) family, ALK1 and ALK5, coordinate to regulate embryonic angiogenesis in response to BMP9/10 and TGF-β. Whereas TGF-β binds to and activates ALK5, leading to Smad2/3 phosphorylation and inhibition of endothelial cell proliferation and migration, BMP9/10 and TGF-β also bind to ALK1, resulting in the activation of Smad1/5. SnoN is a negative regulator of ALK5 signaling through the binding and repression of Smad2/3. Here we uncover a positive role of SnoN in enhancing Smad1/5 activation in endothelial cells to promote angiogenesis. Upon ligand binding, SnoN directly bound to ALK1 on the plasma membrane and facilitated the interaction between ALK1 and Smad1/5, enhancing Smad1/5 phosphorylation. Disruption of this SnoN–Smad interaction impaired Smad1/5 activation and up-regulated Smad2/3 activity. This resulted in defective angiogenesis and arteriovenous malformations, leading to embryonic lethality at E12.5. Thus, SnoN is essential for TGF-β/BMP9-dependent biological processes by its ability to both positively and negatively modulate the activities of Smad-dependent pathways.

Download Full-text

Myostatin Signals through a Transforming Growth Factor β-Like Signaling Pathway To Block Adipogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.23.20.7230-7242.2003 ◽

2003 ◽

Vol 23 (20) ◽

pp. 7230-7242 ◽

Cited By ~ 386

Author(s):

A. Rebbapragada ◽

H. Benchabane ◽

J. L. Wrana ◽

A. J. Celeste ◽

L. Attisano

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Signal Transduction Pathway ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Type I ◽

Type Ii ◽

Transduction Pathway

ABSTRACT Myostatin, a transforming growth factor β (TGF-β) family member, is a potent negative regulator of skeletal muscle growth. In this study we characterized the myostatin signal transduction pathway and examined its effect on bone morphogenetic protein (BMP)-induced adipogenesis. While both BMP7 and BMP2 activated transcription from the BMP-responsive I-BRE-Lux reporter and induced adipogenic differentiation, myostatin inhibited BMP7- but not BMP2-mediated responses. To dissect the molecular mechanism of this antagonism, we characterized the myostatin signal transduction pathway. We showed that myostatin binds the type II Ser/Thr kinase receptor. ActRIIB, and then partners with a type I receptor, either activin receptor-like kinase 4 (ALK4 or ActRIB) or ALK5 (TβRI), to induce phosphorylation of Smad2/Smad3 and activate a TGF-β-like signaling pathway. We demonstrated that myostatin prevents BMP7 but not BMP2 binding to its receptors and that BMP7-induced heteromeric receptor complex formation is blocked by competition for the common type II receptor, ActRIIB. Thus, our results reveal a strikingly specific antagonism of BMP7-mediated processes by myostatin and suggest that myostatin is an important regulator of adipogenesis.

Download Full-text

RLP, a novel Ras-like protein, is an immediate-early transforming growth factor-β (TGF-β) target gene that negatively regulates transcriptional activity induced by TGF-β

Biochemical Journal ◽

10.1042/bj20040774 ◽

2004 ◽

Vol 383 (1) ◽

pp. 187-199 ◽

Cited By ~ 11

Author(s):

Ester PIEK ◽

Maarten van DINTHER ◽

W. Tony PARKS ◽

John M. SALLEE ◽

Erwin P. BÖTTINGER ◽

...

Keyword(s):

Growth Factor ◽

Target Gene ◽

Target Genes ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Small Gtpases ◽

Negative Regulator ◽

Immediate Early ◽

Ras Superfamily ◽

Β Receptor

We have described previously the use of microarray technology to identify novel target genes of TGF-β (transforming growth factor-β) signalling in mouse embryo fibroblasts deficient in Smad2 or Smad3 [Yang, Piek, Zavadil, Liang, Xie, Heyer, Pavlidis, Kucherlapati, Roberts and Böttinger (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 10269–10274]. Among the TGF-β target genes identified, a novel gene with sequence homology to members of the Ras superfamily was identified, which we have designated as RLP (Ras-like protein). RLP is a Smad3-dependent immediate-early TGF-β target gene, its expression being induced within 45 min. Bone morphogenetic proteins also induce expression of RLP, whereas epidermal growth factor and phorbol ester PMA suppress TGF-β-induced expression of RLP. Northern-blot analysis revealed that RLP was strongly expressed in heart, brain and kidney, and below the detection level in spleen and skeletal muscles. At the protein level, RLP is approx. 30% homologous with members of the Ras superfamily, particularly in domains characteristic for small GTPases. However, compared with prototypic Ras, RLP contains a modified P-loop, lacks the consensus G2 loop and the C-terminal prenylation site and harbours amino acid substitutions at positions that render prototypic Ras oncogenic. However, RLP does not have transforming activity, does not affect phosphorylation of mitogen-activated protein kinase and is unable to bind GTP or GDP. RLP was found to associate with certain subtypes of the TGF-β receptor family, raising the possibility that RLP plays a role in TGF-β signal transduction. Although RLP did not interact with Smads and did not affect TGF-β receptor-induced Smad2 phosphorylation, it inhibited TGF-β-induced transcriptional reporter activation, suggesting that it is a novel negative regulator of TGF-β signalling.

Download Full-text

Endothelin‐1 increases CHSY ‐1 expression in aortic endothelial cells via transactivation of transforming growth factor β type I receptor induced by type B receptor endothelin‐1

Journal of Pharmacy and Pharmacology ◽

10.1111/jphp.13081 ◽

2019 ◽

Vol 71 (6) ◽

pp. 988-995 ◽

Cited By ~ 2

Author(s):

Faezeh Seif ◽

Peter J. Little ◽

Reyhaneh Niayesh‐Mehr ◽

Masoumeh Zamanpour ◽

Hossein Babaahmadi‐Rezaei

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Type I ◽

Type B ◽

Aortic Endothelial Cells ◽

Endothelin 1 ◽

Aortic Endothelial

Download Full-text

Axin Facilitates Smad3 Activation in the Transforming Growth Factor β Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5132-5141.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5132-5141 ◽

Cited By ~ 100

Author(s):

Masao Furuhashi ◽

Ken Yagi ◽

Hideki Yamamoto ◽

Yoichi Furukawa ◽

Shinji Shimada ◽

...

Keyword(s):

Growth Factor ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Glycogen Synthase ◽

Transforming Growth Factor Β ◽

Receptor Activation ◽

Negative Regulator ◽

Type I ◽

Ligand Stimulation

ABSTRACT Axin acts as a negative regulator in Wnt signaling through interaction with various molecules involved in this pathway, including β-catenin, adenomatous polyposis coli, and glycogen synthase kinase 3β. We show here that Axin also regulates the effects of Smad3 on the transforming growth factor β (TGF-β) signaling pathway. In the absence of activated TGF-β receptors. Axin physically interacted with Smad3 through its C-terminal region located between the β-catenin binding site and Dishevelled-homologous domain. An Axin homologue, Axil (also called conductin), also interacted with Smad3. In the absence of ligand stimulation, Axin was colocalized with Smad3 in the cytoplasm in vivo. Upon receptor activation, Smad3 was strongly phosphorylated by TGF-β type I receptor (TβR-I) in the presence of Axin, and dissociated from TβR-I and Axin. Moreover, the transcriptional activity of TGF-β was enhanced by Axin and repressed by an Axin mutant which is able to bind to Smad3. Axin may thus function as an adapter of Smad3, facilitating its activation by TGF-β receptors for efficient TGF-β signaling.

Download Full-text

The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling

Oncogene ◽

10.1038/s41388-021-01803-8 ◽

2021 ◽

Author(s):

Panagiotis Papoutsoglou ◽

Dorival Mendes Rodrigues-Junior ◽

Anita Morén ◽

Andrew Bergman ◽

Fredrik Pontén ◽

...

Keyword(s):

Growth Factor ◽

Target Genes ◽

Rna Binding ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Tgfβ Signaling ◽

Ribonucleoprotein Complexes ◽

Expression Of Genes ◽

Downstream Effectors ◽

Human Carcinomas

AbstractActivation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2–3p, miR-125b-5p, and miR-125b-1–3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2–3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.

Download Full-text

Intracisternal administration of transforming growth factor-β evokes fever through the induction of cyclooxygenase-2 in brain endothelial cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00181.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. R266-R275 ◽

Cited By ~ 3

Author(s):

Shigenobu Matsumura ◽

Tetsuro Shibakusa ◽

Teppei Fujikawa ◽

Hiroyuki Yamada ◽

Kiyoshi Matsumura ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Proinflammatory Cytokines ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Cyclooxygenase 2 ◽

Dependent Manner ◽

Cox 2 ◽

Β Receptor

Transforming growth factor-β (TGF-β), a pleiotropic cytokine, regulates cell proliferation, differentiation, and apoptosis, and plays a key role in development and tissue homeostasis. TGF-β functions as an anti-inflammatory cytokine because it suppresses microglia and B-lymphocyte functions, as well as the production of proinflammatory cytokines. However, we previously demonstrated that the intracisternal administration of TGF-β induces fever like that produced by proinflammatory cytokines. In this study, we investigated the mechanism of TGF-β-induced fever. The intracisternal administration of TGF-β increased body temperature in a dose-dependent manner. Pretreatment with cyclooxygenase-2 (COX-2)-selective inhibitor significantly suppressed TGF-β-induced fever. COX-2 is known as one of the rate-limiting enzymes of the PGE2 synthesis pathway, suggesting that fever induced by TGF-β is COX-2 and PGE2 dependent. TGF-β increased PGE2 levels in cerebrospinal fluid and increased the expression of COX-2 in the brain. Double immunostaining of COX-2 and von Willebrand factor (vWF, an endothelial cell marker) revealed that COX-2-expressing cells were mainly endothelial cells. Although not all COX-2-immunoreactive cells express TGF-β receptor, some COX-2-immunoreactive cells express activin receptor-like kinase-1 (ALK-1, an endothelial cell-specific TGF-β receptor), suggesting that TGF-β directly or indirectly acts on endothelial cells to induce COX-2 expression. These findings suggest a novel function of TGF-β as a proinflammatory cytokine in the central nervous system.

Download Full-text