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Aristidis Moustakas

Publications and source records attributed to Aristidis Moustakas.

At least 19 recordsLinked to original sources
PubMed2006

Transforming growth factor-beta employs HMGA2 to elicit epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) occurs during embryogenesis, carcinoma invasiveness, and metastasis and can be elicited by transforming growth factor-beta (TGF-beta) signaling via intracellular Smad transducers. The molecular mechanisms that control the onset of EMT remain largely unexplored. Transcriptomic analysis revealed that the high mobility group A2 (HMGA2) gene is induced by the Smad pathway during EMT. Endogenous HMGA2 mediates EMT by TGF-beta, whereas ectopic HMGA2 causes irreversible EMT characterized by severe E-cadherin suppression. HMGA2 provides transcriptional input for the expression control of four known regulators of EMT, the zinc-finger proteins Snail and Slug, the basic helix-loop-helix protein Twist, and inhibitor of differentiation 2. We delineate a pathway that links TGF-beta signaling to the control of epithelial differentiation via HMGA2 and a cohort of major regulators of tumor invasiveness and metastasis. This network of signaling/transcription factors that work sequentially to establish EMT suggests that combinatorial detection of these proteins could serve as a new tool for EMT analysis in cancer patients.

Animals↗
PubMed2006

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer.

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.

Animals↗
PubMed2006

A new twist in Smad signaling.

Signaling by members of the TGFbeta family is much dependent on the common-mediator Smad4, which forms transcriptionally active complexes with all receptor-activated Smads (R-Smads). New findings demonstrate that transcriptional intermediary factor 1gamma (TIF1gamma) also can bind to R-Smads, as an alternative to Smad4, and mediate different transcriptional effects.

Models, Biological↗
PubMed2006

The mechanism of nuclear export of Smad3 involves exportin 4 and Ran.

Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.

Active Transport, Cell Nucleus↗
PubMed2005

Non-Smad TGF-beta signals.

During the past 10 years, it has been firmly established that Smad pathways are central mediators of signals from the receptors for transforming growth factor beta (TGF-beta) superfamily members to the nucleus. However, growing biochemical and developmental evidence supports the notion that alternative, non-Smad pathways also participate in TGF-beta signalling. Non-Smad signalling proteins have three general mechanisms by which they contribute to physiological responses to TGF-beta: (1) non-Smad signalling pathways directly modify (e.g. phosphorylate) the Smads and thus modulate the activity of the central effectors; (2) Smads directly interact and modulate the activity of other signalling proteins (e.g. kinases), thus transmitting signals to other pathways; and (3) the TGF-beta receptors directly interact with or phosphorylate non-Smad proteins, thus initiating parallel signalling that cooperates with the Smad pathway in eliciting physiological responses. Thus, non-Smad signal transducers under the control of TGF-beta provide quantitative regulation of the signalling pathway, and serve as nodes for crosstalk with other major signalling pathways, such as tyrosine kinase, G-protein-coupled or cytokine receptors.

Animals↗
PubMed2005

Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis.

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.

Apoptosis↗
PubMed2005

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner.

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.

Animals↗
PubMed2005

Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases.

Smad4 mediates signaling by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-beta receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-beta signaling may be critical for proper physiological response to this pathway.

Adenoviridae↗
PubMed2005

TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition.

Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-beta/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-beta superfamily establishes that TGF-beta 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-beta-induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-beta target genes with ligand-specific responses. Using a TGF-beta 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-beta1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, alpha-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-beta and predict functional links to the control of cell proliferation and EMT.

Animals↗
PubMed2005

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta.

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.

Actin Depolymerizing Factors↗
PubMed2005

Smad pathway-specific transcriptional regulation of the cell cycle inhibitor p21(WAF1/Cip1).

Transforming growth factor-beta (TGF-beta) inhibits epithelial cell growth, in part via transcriptional induction of the cell cycle inhibitor p21(WAF1/Cip1) (p21). We show that bone morphogenetic protein (BMP)-7 induces higher p21 expression than TGF-beta1 in various epithelial cells. Despite this, BMP-7 only weakly suppresses epithelial cell proliferation, as Id2, a cell cycle-promoting factor, becomes concomitantly induced by BMP-7. Signaling studies with all type I receptors of the TGF-beta superfamily show that BMP receptors induce higher p21 expression than TGF-beta/activin receptors. Smad4 is essential for p21 regulation by all receptor pathways. Based on the previously known ability of c-Myc to block p21 expression and epithelial growth arrest in response to TGF-beta1, we demonstrate that ectopic c-Myc expression can abrogate Smad-mediated p21 induction by all TGF-beta and BMP receptors. Furthermore, p21 induction by all receptor pathways can be blocked by the natural inhibitors of the TGF-beta superfamily. Smad7 inhibits all pathways whereas Smad6 selectively inhibits the BMP pathways. The observed pathway specificity reflects the efficiency by which BMP Smads, compared to TGF-beta Smads, transactivate the p21 promoter. In addition, BMP-specific Smads, Smad1, Smad5, and especially Smad8, induce endogenous p21 mRNA and protein levels, while they fail to induce epithelial growth inhibition when compared to TGF-beta receptor-phosphorylated Smads (R-Smads), Smad2 and Smad3. Thus, p21 is a common target of all TGF-beta superfamily pathways. However, the ability of TGF-beta superfamily members to induce cell growth arrest depends on the regulation of additional gene targets.

Bone Morphogenetic Protein 7↗
PubMed2004

Tgf-beta3-induced palatal fusion is mediated by Alk-5/Smad pathway.

Cleft palate is among the most common birth defects in humans, caused by a failure in the complex multistep developmental process of palatogenesis. It has been recently shown that transforming growth factor beta3 (Tgf-beta3) is an absolute requirement for successful palatal fusion, both in mice and humans. However, very little is known about the mechanisms of Tgf-beta3 signaling during this process. Here we show that putative Tgf-beta type I receptors, Alk-1, Alk-2, and Alk-5, are all endogenously expressed in the palatal epithelium. Activation of Alk-5 in the Tgf-beta3 (-/-) palatal epithelium is able to rescue palatal fusion, whereas inactivation of Alk-5 in the wild-type palatal epithelium prevents palatal fusion. The effect of Alk-2 is similar, but less pronounced. The induction of fusion by activation of Alk-5 or Alk-2 is stronger in the posterior parts of the palates at the embryonic day 14 (E14), while their activation at E13.5 also restores anterior fusion, reflecting the natural anterior-posterior direction of palate maturation in vivo. We also show that Smad2 is endogenously activated in the palatal midline epithelial seam (MES) during the fusion process. By using a mutant Alk-5 receptor that is an active kinase but is unable to activate Smads, we show that activation of Smad-independent Tgf-beta responses is not sufficient to induce fusion of shelves deficient in Tgf-beta3. Based on these observations, we conclude that the Smad2-dependent Alk-5 signaling pathway is dominant in palatal fusion driven by Tgf-beta3.

Activin Receptors, Type I↗
PubMed2004

Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor beta signaling.

BACKGROUND: Transforming growth factor-betas (TGF-betas), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors. RESULTS: There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (associated molecule with the SH3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in Jun kinase activation domain binding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-beta-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-beta signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-beta. CONCLUSIONS: This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-beta signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-beta signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein.

Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.

Animals↗
PubMed2003

Differential ubiquitination defines the functional status of the tumor suppressor Smad4.

Smad4 is an essential signal transducer of all transforming growth factor-beta (TGF-beta) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.

Amino Acid Sequence↗
PubMed2003

Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.

We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads.

Animals↗