NORMAL GENOMIC

• Lei X, Yang J, Nichols RW, Sun LZ
• Abrogation of TGFbeta signaling induces apoptosis through the modulationof MAP kinase pathways in breast cancer cells.
• Exp Cell Res. 2007; 313: 1687-95
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• Transforming growth factor beta (TGFbeta) can modulate the activity ofvarious MAP kinases. However, how this pathway may mediate TGFbeta-inducedmalignant phenotypes remains elusive. We investigated the role ofautocrine TGFbeta signaling through MAP kinases in the regulation of cellsurvival in breast carcinoma MCF-7 cells and untransformed human mammaryepithelial cells (HMECs). Our results show that abrogation of autocrineTGFbeta signaling with the expression of a dominant negative type IITGFbeta receptor (DNRII) or the treatment with a TGFbeta type I receptorinhibitor significantly increased apoptosis in MCF-7 cell, but not inHMEC. The expression of DNRII markedly decreased activated/phosphorylatedErk, whereas increased activated/phosphorylated p38 in MCF-7 cells. Incontrast, there was no or little change of phosphorylated Erk and p38 inHMECs after the expression of DNRII. Inhibition of Erk activity in MCF-7control cell induced apoptosis whereas restoration of Erk activity inMCF-7 DNRII cell reduced apoptosis. Similarly, inhibition of p38 activityalso inhibited apoptosis in MCF-7 DNRII cell. Thus, autocrine TGFbetasignaling can enhance the survival of MCF-7 cells by maintaining the levelof active Erk high and the level of active p38 low. Furthermore, thesurvival properties of TGFbeta pathway appear related to transformationsupporting the notion that it may be a potential target for cancertherapy.

• Lebrin F et al.
• Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signaltransduction.
• EMBO J. 2004; 23: 4018-28
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• Endoglin is a transmembrane accessory receptor for transforming growthfactor-beta (TGF-beta) that is predominantly expressed on proliferatingendothelial cells in culture and on angiogenic blood vessels in vivo.Endoglin, as well as other TGF-beta signalling components, is essentialduring angiogenesis. Mutations in endoglin and activin receptor-likekinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, havebeen linked to the vascular disorder, hereditary haemorrhagictelangiectasia. However, the function of endoglin in TGF-beta/ALKsignalling has remained unclear. Here we report that endoglin is requiredfor efficient TGF-beta/ALK1 signalling, which indirectly inhibitsTGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not growbecause TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signallingis increased. Surviving cells adapt to this imbalance by downregulatingALK5 expression in order to proliferate. The ability of endoglin topromote ALK1 signalling also explains why ectopic endoglin expression inendothelial cells promotes proliferation and blocks TGF-beta-inducedgrowth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our resultsindicate a pivotal role for endoglin in the balance of ALK1 and ALK5signalling to regulate endothelial cell proliferation.

• Chaturvedi K, Sarkar DK
• Involvement of protein kinase C-dependent mitogen-activated protein kinasep44/42 signaling pathway for cross-talk between estradiol and transforminggrowth factor-beta3 in increasing basic fibroblast growth factor infolliculostellate cells.
• Endocrinology. 2004; 145: 706-15
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• We have recently shown that TGF-beta3, in the presence of estradiol,increases the release of basic fibroblast growth factor (bFGF) fromfolliculostellate (FS) cells in the pituitary. We determined theinteractive effects of TGF-beta3 and estradiol on bFGF production andrelease from FS cells, and the role of the MAPK pathway in TGF-beta3 andestradiol interaction. We found that TGF-beta3 and estradiol alonemoderately increased cell content and release of bFGF from FS cells; buttogether, they markedly increased the peptide. Estradiol and TGF-beta3alone moderately activated MAPK p44/42; together they produced markedactivation of MAPK p44/42. Pretreatment of FS cells with an MAPK kinase1/2 inhibitor or with protein kinase C inhibitors suppressed theactivation of MAPK p44/42, bFGF release, and protein level increases, allof which were induced by TGF-beta3 and estradiol. Estradiol and TGF-beta3,either alone or in combination, increased the levels of active Ras.Furthermore, bFGF induction by TGF-beta3 and estradiol was blocked byoverexpression of Ras N17, a dominant negative mutant of Ras p21. Estrogenreceptor blocker ICI 182,780 failed to prevent estrogen's and TGF-beta3'seffects on bFGF. These data suggest that an estradiol receptor-independentprotein kinase C- activated Ras-dependent MAPK pathway is involved in thecross-talk between TGF-beta3 and estradiol to increase bFGF productionand/or release from FS cells.

• Rotzer D, Roth M, Lutz M, Lindemann D, Sebald W, Knaus P
• Type III TGF-beta receptor-independent signalling of TGF-beta2 viaTbetaRII-B, an alternatively spliced TGF-beta type II receptor.
• EMBO J. 2001; 20: 480-90
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• Transforming growth factor-beta (TGF-beta) signals through membrane-boundserine/threonine kinase receptors, which upon stimulation phosphorylateSmad proteins and thereby trigger their nuclear translocation andtranscriptional activity. Although the three mammalian isoforms ofTGF-beta are highly homologous at the level of sequence, analysis of theirin vivo function by gene knockouts revealed striking differences,suggesting no significant functional redundancy between TGF-beta1, -2 and-3. While signal transduction by TGF-beta1 has been well characterized,receptor binding and activation by the TGF-beta2 isoform is less wellunderstood. Here, we show that TbetaRII-B, an alternatively splicedvariant of the TGF-beta type II receptor, is a TGF-beta2 binding receptor,which mediates signalling via the Smad pathway in the absence of anyTGF-beta type III receptor (TbetaRIII). L6 cells lacking endogenousTbetaRIII as well as TbetaRII-B do not respond to TGF-beta2. Transfectionof these cells with TbetaRII-B restores TGF-beta2 sensitivity. Theexpression of TbetaRII-B is restricted to cells originating from tissuessuch as bone where the isoform TGF-beta2 has a predominant role. Thisreflects the importance of this receptor in TGF-beta isoform-specificsignalling.

• Prunier C, Ferrand N, Frottier B, Pessah M, Atfi A
• Mechanism for mutational inactivation of the tumor suppressor Smad2.
• Mol Cell Biol. 2001; 21: 3302-13
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• Transforming growth factor beta (TGF-beta) is a potent naturalantiproliferative agent that plays an important role in suppressingtumorigenicity. In numerous tumors, loss of TGF-beta responsiveness isassociated with inactivating mutations that can occur in components ofthis signaling pathway, such as the tumor suppressor Smad2. Although ageneral framework for how Smads transduce TGF-beta signals has beenproposed, the physiological relevance of alterations of Smad2 functions inpromoting tumorigenesis is still unknown. Here, we show that expression ofSmad2.P445H, a tumor-derived mutation of Smad2 found in human cancer,suppresses the ability of the Smads to mediate TGF-beta-induced growtharrest and transcriptional responses. Smad2.P445H is phosphorylated by theactivated TGF-beta receptor at the carboxy-terminal serine residues andassociates with Smad3 and Smad4 but is unable to dissociate from thereceptor. Upon ligand-induced phosphorylation, Smad2.P445H interactsstably with wild-type Smad2, thereby blocking TGF-beta-induced nuclearaccumulation of wild-type Smad2 and Smad2-dependent transcription. Theability of the Smad2.P445H to block the nuclear accumulation of wild-typeSmad2 protein reveals a new mechanism for loss of sensitivity to thegrowth-inhibitory functions of TGF-beta in tumor development.

• Wells RG, Gilboa L, Sun Y, Liu X, Henis YI, Lodish HF
• Transforming growth factor-beta induces formation of adithiothreitol-resistant type I/Type II receptor complex in live cells.
• J Biol Chem. 1999; 274: 5716-22
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• Transforming growth factor-beta (TGF-beta) binds to and signals via twoserine-threonine kinase receptors, the type I (TbetaRI) and type II(TbetaRII) receptors. We have used different and complementary techniquesto study the physical nature and ligand dependence of the complex formedby TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptorssuggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complexthat is most likely a heterotetramer. Antibody-mediated immunofluorescenceco-patching of epitope-tagged receptors provides the first evidence inlive cells that TbetaRI. TbetaRII complex formation occurs at a low butmeasurable degree in the absence of ligand, increasing significantly afterTGF-beta binding. In addition, we demonstrate that pretreatment of cellswith dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI,does not prevent formation of the TbetaRI.TbetaRII complex, but increasesits sensitivity to detergent and prevents TGF-beta-activated TbetaRI fromphosphorylating Smad3 in vitro. This indicates that either a specificconformation of the TbetaRI. TbetaRII complex, disrupted bydithiothreitol, or direct binding of TGF-beta to TbetaRI is required forsignaling.

• Kang SH, Bang YJ, Jong HS, Seo JY, Kim NK, Kim SJ
• Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in theTGF-beta-induced cell cycle arrest of gastric carcinoma cells.
• Br J Cancer. 1999; 80: 1144-9
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• Transforming growth factor-beta (TGF-beta) is a multifunctionalpolypeptide that inhibits cellular proliferation in most epithelial cells.cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15INK4B,p21WAF1/Cip1 and p27Kip1) have been implicated in the TGF-beta-inducedcell cycle arrest. More recently, down-regulation of Cdc25A, a cdkactivator, was additionally suggested as a mechanism underlying growthinhibition by TGF-beta. The existence of diverse cellular mediators ofTGF-beta, however, raises the question of whether their involvement mightoccur in a redundant manner or coordinately in a certain cell type. Usingtwo TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), weaddressed the contributory roles of several cdk inhibitors, and of cdk4and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing theirtemporal expression pattern in response to TGF-beta. Among the cdkinhibitors examined, p21 mRNA was most rapidly (in less than 1 h) andprominently induced by TGF-beta. In contrast, p15 mRNA was more slowlyinduced than p21 in SNU-620 cells, and not expressed in SNU-16 cellsharbouring homozygous deletion of p15. Western blotting results confirmedthe rapid increase of p21, while opposite patterns of p27 expression wereobserved in the two cell lines. The down-regulation of Cdc25A mRNAoccurred, but was more delayed than that of p15 or p21. Until G1 arrestwas established, changes in the protein levels of both Cdc25A and cdk4were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed thatinduced p21 associates with cdk4 and that its kinase activity is reducedby TGF-beta, which kinetically correlates closely with G1 arrest followingTGF-beta treatment of both cell lines. These results suggest that incertain human epithelial cells, p21 may play an early role inTGF-beta-induced cell cycle arrest, and its cooperation with other cdkinhibitors is different depending on cell type. Delayed down-regulation ofCdc25A and cdk4 may contribute to cell adaptation to the quiescent statein the two gastric carcinoma cell lines studied.

• Kretzschmar M, Doody J, Timokhina I, Massague J
• A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras.
• Genes Dev. 1999; 13: 804-16
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• TGFbeta can override the proliferative effects of EGF and otherRas-activating mitogens in normal epithelial cells. However, epithelialcells harboring oncogenic Ras mutations often show a loss of TGFbetaantimitogenic responses. Here we report that oncogenic Ras inhibitsTGFbeta signaling in mammary and lung epithelial cells by negativelyregulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activatedRas inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3and Smad-dependent transcription. Ras acting via Erk MAP kinases causesphosphorylation of Smad2 and Smad3 at specific sites in the region linkingthe DNA-binding domain and the transcriptional activation domain. Thesesites are separate from the TGFbeta receptor phosphorylation sites thatactivate Smad nuclear translocation. Mutation of these MAP kinase sites inSmad3 yields a Ras-resistant form that can rescue the growth inhibitoryresponse to TGFbeta in Ras-transformed cells. EGF, which is weaker thanoncogenic mutations at activating Ras, induces a less extensivephosphorylation and cytoplasmic retention of Smad2 and Smad3. Our resultssuggest a mechanism for the counterbalanced regulation of Smad2/Smad3 byTGFbeta and Ras signals in normal cells, and for the silencing ofantimitogenic TGFbeta functions by hyperactive Ras in cancer cells.

• Kretzschmar M, Doody J, Massague J
• Opposing BMP and EGF signalling pathways converge on the TGF-beta familymediator Smad1.
• Nature. 1997; 389: 618-22
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• The growth factor TGF-beta, bone morphogenetic proteins (BMPs) and relatedfactors regulate cell proliferation, differentiation and apoptosis,controlling the development and maintenance of most tissues. Their signalsare transmitted through the phosphorylation of the tumour-suppressor SMADproteins by receptor protein serine/threonine kinases (RS/TKs), leading tothe nuclear accumulation and transcriptional activity of SMAD proteins.Here we report that Smadl, which mediates BMP signals, is also a target ofmitogenic growth-factor signalling through epidermal growth factor andhepatocyte growth factor receptor protein tyrosine kinases (RTKs).Phosphorylation occurs at specific serines within the region linking theinhibitory and effector domains of Smad1, and is catalysed by the Erkfamily of mitogen-activated protein kinases. In contrast to theBMP-stimulated phosphorylation of Smad1, which affects carboxy-terminalserines and induces nuclear accumulation of Smad1, Erk-mediatedphosphorylation specifically inhibits the nuclear accumulation of Smad1.Thus, Smadl receives opposing regulatory inputs through RTKs and RS/TKs,and it is this balance that determines the level of Smad1 activity in thenucleus, and so possibly the role of Smad1 in the control of cell fate.

• Savage C et al.
• Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conservedfamily of transforming growth factor beta pathway components.
• Proc Natl Acad Sci U S A. 1996; 93: 790-4
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• Although transforming growth factor beta (TGF-beta) superfamily ligandsplay critical roles in diverse developmental processes, how cellstransduce signals from these ligands is still poorly understood. Cellsurface receptors for these ligands have been identified, but theircytoplasmic targets are unknown. We have identified three Caenorhabditiselegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypessimilar to those of the TGF-beta-like receptor gene daf-4, indicating thatthey are required for daf-4-mediated developmental processes. We show thatsma-2 functions in the same cells as daf-4, consistent with a role intransducing signals from the receptor. These three genes define a proteinfamily, the dwarfins, that includes the Mad gene product, whichparticipates in the decapentaplegic TGF-beta-like pathway in Drosophila[Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. &Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification ofhomologous components of these pathways in distantly related organismssuggests that dwarfins may be universally required for TGF-beta-likesignal transduction. In fact, we have isolated highly conserved dwarfinsfrom vertebrates, indicating that these components are not idiosyncraticto invertebrates. These analyses suggest that dwarfins are conservedcytoplasmic signal transducers.

• Feng XH, Derynck R
• Ligand-independent activation of transforming growth factor (TGF) betasignaling pathways by heteromeric cytoplasmic domains of TGF-betareceptors.
• J Biol Chem. 1996; 271: 13123-9
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• Transforming growth factor beta (TGF-beta) transduces signals through tworelated serine/threonine kinase receptors, the type I and type IIreceptors, which have the ability to interact with each other. In theheteromeric complex, the type II receptor is the primary determinant ofligand binding and phosphorylates the cytoplasmic domain of the type Ireceptor. Using a chimeric receptor strategy, we and others have shownpreviously that a functional TGF-beta receptor complex requiresheteromerization of both extracellular and intracellular domains of type Iand type II receptors. In the current study, we show that overexpressionof two receptors carrying a heteromeric combination of cytoplasmic domainsresulted in ligand-independent responses, further supporting thefunctional requirement of the two heterologous cytoplasmic domains inTGF-beta signaling. Furthermore, coexpression of only the cytoplasmicdomains of both the type I and II receptors or tethering the type II tothe type I cytoplasmic domain activated TGF-beta responses in aligand-independent manner. In cotransfected COS-1 cells, both cytoplasmicdomains are associated with each other. Our results indicate that thecytoplasmic domains of the type I and type II TGF-beta receptorsphysically and functionally interact with each other in the heteromericcomplex.

• Sankar S, Mahooti-Brooks N, Bensen L, McCarthy TL, Centrella M, Madri JA
• Modulation of transforming growth factor beta receptor levels onmicrovascular endothelial cells during in vitro angiogenesis.
• J Clin Invest. 1996; 97: 1436-46
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• Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D)cultures proliferate rapidly and exhibit an undifferentiated phenotype.Addition of transforming growth factor beta1 (TGFbeta1) increasesfibronectin expression and inhibits proliferation. RFCs cultured inthree-dimensional (3D) type I collagen gels proliferate slowly and arerefractory to the anti-proliferative effects of TGF beta1. TGF beta1promotes tube formation in 3D cultures. TGF beta1 increases fibronectinexpression and urokinase plasminogen activator (uPA) activity andplasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since theTGF beta type I and II receptors have been reported to regulate differentactivities induced by TGF beta1, we compared the TGF beta receptorprofiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited asignificant loss of cell surface type II receptor compared with cells in2D cultures. The inhibitory effect of TGF beta1 on proliferation issuppressed in transfected 2D cultures expressing a truncated form of thetype II receptor, while its stimulatory effect on fibronectin productionis reduced in both 2D and 3D transfected cultures expressing a truncatedform of the type I receptor. These data suggest that the type II receptormediates the antiproliferative effect of TGF beta1 while the type Ireceptor mediates the matrix response of RFCs to TGF beta1 and demonstratethat changes in the matrix environment can modulate the surface expressionof TGF beta receptors, altering the responsiveness of RFCs to TGF beta1.

• Vivien D, Attisano L, Wrana JL, Massague J
• Signaling activity of homologous and heterologous transforming growthfactor-beta receptor kinase complexes.
• J Biol Chem. 1995; 270: 7134-41
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• Transforming growth factor-beta (TGF-beta) signaling in Mv1Lu lungepithelial cells requires coexpression of TGF-beta receptors I (T betaR-I) and II (T beta R-II), two distantly related transmembraneserine/threonine kinases that form a heteromeric complex upon ligandbinding. Here, we examine the formation of TGF-beta receptorhomo-oligomers and their possible contribution to signaling. T beta R-Ican contact ligand bound to T beta R-II, but not ligand free in themedium, and thus cannot form ligand-induced homo-oligomers. T beta R-II,which binds ligand on its own, formed oligomeric complexes whenoverexpressed in transfected COS cells. However, these complexes werelargely ligand-independent and involved immature receptor protein. Sinceligand-induced homo-oligomers could not be obtained with the wild-typeTGF-beta receptors, we studied receptor cytoplasmic domainhomo-oligomerization by using receptor chimeras. The extracellular domainof T beta R-II was fused to the transmembrane and cytoplasmic domains of Tbeta R-I, yielding T beta R-II/I, and the extracellular domain of T betaR-I was fused to the transmembrane and cytoplasmic domains of T beta R-II,yielding T beta R-I/II. When contransfected with wild-type receptors andexposed to ligand, T beta R-II/I formed a complex with T beta R-I, and Tbeta R-I/II formed a complex with T beta R-II, thus yielding complexeswith homologous cytoplasmic domains. T beta R-II/I transfected alone orwith T beta R-I did not restore TGF-beta responsiveness in T betaR-II-defective cell mutants. Furthermore, T beta R-II/I acted in adominant negative fashion, inhibiting restoration of TGF-betaresponsiveness by a cotransfected T beta R-II in T beta R-II-defectivecells and by a cotransfected T beta R-I in T beta R-I-defective cells.Similarly, T beta R-I/II transfected alone or with T beta R-II did notrestore TGF-beta responsiveness and acted in a dominant negative fashionagainst T beta R-I. Together with previous genetic and biochemicalevidence, these results suggest that TGF-beta mediates transcriptional andantiproliferative responses through the heteromeric T beta R-I.T beta R-IIcomplex and not through homo-oligomeric T beta R-I or T beta R-IIcomplexes.