Biomedical subjects

A B Roberts

Publications and source records attributed to A B Roberts.

At least 19 recordsLinked to original sources

PubMed2001

Meeting report: signaling schemes for TGF-beta.

The transforming growth factor-beta (TGF-beta) superfamily of signaling molecules regulates many developmental processes in a range of organisms from worms to humans. Understanding the mechanisms by which they exert their repertoire of effects has required identification of the components of signaling pathways that they control. Roberts and Derynck focus on this aspect of TGF-beta biology in their review of a recent Federation of American Societies for Experimental Biology (FASEB) meeting on TGF-beta signaling and development and summarize current signaling paradigms and future prospects in TGF-beta signaling from the cell surface to the nucleus.

Animals↗

PubMed2001

Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion.

The Smad family of intracellular signaling intermediates transduce signals downstream from the transforming growth factor beta (TGF-beta) family of receptor serine threonine kinases. The original member of this family, Smad1, has been shown to mediate signals from receptors for the bone morphogenetic proteins (BMPs), a large group of ligands in the TGF-beta superfamily that mediate important developmental events. We have targeted the Smad1 gene in mice and created mutants null at this locus. Smad1 mutant mice die at approximately 9.5 days postcoitum due to defects in allantois formation. In Smad1 mutant mice, the allantois fails to fuse to the chorion, resulting in a lack of placenta and failure to establish a definitive embryonic circulation. Although vasculogenesis is initiated in the mutant allantois, the vessels formed are disorganized, and VCAM-1 protein, a marker for distal allantois development, is not expressed. Smad1 null fibroblasts are still able to respond to BMP2, however, suggesting that the defect observed in the developing extraembryonic tissue is caused by a very specific loss of transcriptional activity regulated by Smad1. Our data further demonstrate that although highly similar structurally, Smad proteins are not functionally homologous.

Allantoin↗

PubMed2001

SNIP1 inhibits NF-kappa B signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators.

SNIP1 is a 396-amino acid nuclear protein shown to be an inhibitor of the TGF-beta signal transduction pathway and to be important in suppressing transcriptional activation dependent on the co-activators CBP and p300. In this report we show that SNIP1 potently inhibits the activity of NF-kappa B, which binds the C/H1 domain of CBP/p300, but does not interfere with the activity of transcription factors such as p53, which bind to other domains of p300, or factors such as VP16, which are independent of these co-activators. Inhibition of NF-kappa B activity is a function of the N-terminal domain of SNIP1 and involves competition of SNIP1 and the NF-kappa B subunit, RelA/p65, for binding to p300, similar to the mechanism of inhibition of Smad signaling by SNIP1. Immunohistochemical staining shows that expression of SNIP1 is strictly regulated in development and that it colocalizes, in certain tissues, with nuclear staining for RelA/p65 and for p300, suggesting that they may regulate NF-kappa B activity in vivo in a spatially and temporally controlled manner. These data led us to suggest that SNIP1 may be an inhibitor of multiple transcriptional pathways that require the C/H1 domain of CBP/p300.

Animals↗

PubMed2001

Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.

Smad proteins transduce signals from TGF-beta receptors and regulate transcription of target genes either directly or in combination with other sequence-specific transcription factors. AP-1 sites and their cognate transcription factors also play important roles in the gene regulatory activities of TGF-beta. In this report, we have investigated the functional interactions of the Smad and AP-1 transcription factors. We demonstrate that Smad and AP-1 complexes specifically bind to their cognate cis-elements and do not interact with each other on-DNA, whereas off-DNA interactions occur between Smad3 and both c-Jun and JunB. Using both artificial constructs specific for either the Smad or AP-1 signaling pathways or natural promoters known to be TGF-beta-responsive, we have determined that Jun family members downregulate Smad3-mediated gene transactivation whereas AP-1-dependent promoters are synergistically activated by Smad3 and Jun proteins. We propose a model where the presence of Smad- and/or AP-1-specific cis-elements within TGF-beta-responsive genes allows dynamic modulation of gene expression, in contrast to the existing model where interactions between Smad and AP-1 proteins are merely an on/off mechanism to regulate TGF-beta/Smad targets.

Activin Receptors, Type I↗

PubMed2001

X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-beta signaling.

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of apoptosis. Here we report that XIAP can also function as a cofactor in the regulation of gene expression by transforming growth factor-beta (TGF-beta). XIAP, but not the related proteins c-IAP1 or c-IAP2, associated with several members of the type I class of the TGF-beta receptor superfamily and potentiated TGF-beta-induced signaling. Although XIAP-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B was found to require the TGF-beta signaling intermediate Smad4, the ability of XIAP to suppress apoptosis was found to be Smad4-independent. These data implicate a role for XIAP in TGF-beta-mediated signaling that is distinct from its anti-apoptotic functions.

Apoptosis↗

PubMed2001

Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.

A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.

Animals↗

PubMed2001

Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases.

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

Amino Acid Sequence↗

PubMed2001

Transforming growth factor-beta receptor-associated protein 1 is a Smad4 chaperone.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-beta receptors and suggested to play a role in TGF-beta signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-beta and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-beta signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-beta signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

Activins↗

PubMed2001

The hepatitis B virus encoded oncoprotein pX amplifies TGF-beta family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis.

Hepatitis B, one of the most common infectious diseases in the world, is closely associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Many clinical investigations have revealed that hepatic fibrosis is an important component of these liver diseases caused by chronic hepatitis B. TGF-beta signaling plays an important role in the pathogenesis of fibrosis in chronic hepatitis and cirrhosis. As these diseases are associated with hepatitis B virus (HBV) infection, we examined the possibility that the HBV-encoded pX oncoprotein regulates TGF-beta signaling. We show that pX enhances transcriptional activity in response to TGF-beta, BMP-2, and activin by stabilizing the complex of Smad4 with components of the basic transcriptional machinery. Additionally, confocal microscopic studies suggest that pX facilitates and potentiates the nuclear translocation of Smads, further enhancing TGF-beta signaling. Our studies suggest a new paradigm for amplification of Smad-mediated signaling by an oncoprotein and suggest that enhanced Smad-mediated signaling may contribute to HBV-associated liver fibrosis.

3T3 Cells↗

PubMed2001

Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas.

The eight mammalian Smad proteins mediate cellular signaling from members of the transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and activin families. Smads 1, 5, and 8 transmit signals from BMPs, while Smads 2 and 3 transmit signals from TGF-betas and activin. Smad 4 is a common mediator of both pathways, while Smads 6 and 7 inhibit signaling. Signal transduction involves translocation of Smad complexes to the nucleus and subsequent gene activation. Little is known about the expression of endogenous Smad proteins during development. We identified commercially available Smad antibodies that specifically recognize a unique Smad protein and are suitable for immunohistochemistry. Here we compare the localization of Smads 1, 2, 3, 4, 5, and 6 in tissues of the 15-day gestation mouse embryo. Immunoreactive Smad proteins are seen in many tissues with differences in the localization being dependent upon the cell type. All tissues express Smad 4 and at least one each of the BMP-specific and TGF-beta-specific Smads, while expression of Smad 6 is more restricted. Differences are observed in the nuclear versus cytoplasmic localization among the Smads in different cell types or tissues, suggesting selective activation of Smads during this stage of development.

Animals↗

PubMed2001

Suppressor and oncogenic roles of transforming growth factor-beta and its signaling pathways in tumorigenesis.

Transforming growth factor-beta (TGF-beta) has been implicated in oncogenesis since the time of its discovery almost 20 years ago. The complex, multifunctional activities of TGF-beta endow it with both tumor suppressor and tumor promoting activities, depending on the stage of carcinogenesis and the responsivity of the tumor cell. Dysregulation or alteration of TGF-beta signaling in tumorigenesis can occur at many different levels, including activation of the ligand, mutation or transcriptional suppression of the receptors, or alteration of downstream signal transduction pathways resulting from mutation or changes in expression patterns of signaling intermediates or from changes in expression of other proteins which modulate signaling. New insights into signaling from the TGF-beta receptors, including the identification of Smad signaling pathways and their interaction with mitogen-activated protein (MAP) kinase pathways, are providing an understanding of the changes involved in the change from tumor suppressor to tumor promoting activities of TGF-beta. It is now appreciated that loss of sensitivity to inhibition of growth by TGF-beta by most tumor cells is not synonymous with complete loss of TGF-beta signaling but rather suggests that tumor cells gain advantage by selective inactivation of the tumor suppressor activities of TGF-beta with retention of its tumor promoting activities, especially those dependent on cross talk with MAP kinase pathways and AP-1.

Animals↗

PubMed2001

Ultrasonographic surveillance in red blood cell alloimmunization.

OBJECTIVE: This study was undertaken to audit ultrasonographic measurements of fetal liver length and middle cerebral artery peak velocity in cases of red blood cell alloimmunization between 1986 and 1999. STUDY DESIGN: A total of 200 fetuses at risk for anemia because of red blood cell alloimmunization underwent ultrasonographic measurement of the length of the right lobe of the liver, 45 underwent Doppler recording of middle cerebral artery peak velocity, and 119 underwent fetal blood sampling. RESULTS: The overall survival was 188 of 200 (94%). Among 69 fetuses found to have anemia, liver length values in 64 (93%) were at the 95th percentile or greater, and the other 5 were in the upper part of the normal range. The middle cerebral artery peak velocity was > or =95th percentile in 15 of the 19 cases of anemia in which this value was measured (79%). Among those measured within 1 week of birth, all liver lengths were at least in the upper part of the normal range, with most >95th percentile, including 1 case with a cord blood hemoglobin concentration <90 g/L. CONCLUSIONS: All fetuses with anemia identified at fetal blood sampling had enlarged livers with 93% at > or =95th percentile. The peak velocity in the middle cerebral artery was abnormal in most fetuses with anemia.

Blood Flow Velocity↗

PubMed2001

Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?

Transforming growth factor (TGF)-beta plays a central role in fibrosis, contributing both to the influx and activation of inflammatory cells, as well as to activation of fibroblasts to elaborate extracellular matrix. In the past few years, new insight has been gained into signal transduction pathways downstream of the TGF-beta receptor serine-threonine kinases with the identification of a family of evolutionarily conserved Smad proteins. Two receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated by the activated TGF-beta type I receptor kinase, after which they partner with the common mediator, Smad4, and are translocated to the nucleus to where they participate in transcriptional complexes to control expression of target genes. We have shown in wound healing studies of mice null for Smad3, that loss of this key signaling intermediate interferes with the chemotaxis of inflammatory cells to TGF-beta as well as with their ability to autoinduce TGF-beta. Moreover, studies with mouse embryo fibroblasts null for Smad3 show that TGF-beta-dependent induction of c-Jun and c-Fos, important in induction of collagen as well as in autoinduction of TGF-beta, is mediated by Smad3. Based on these observations, we hypothesize that loss of Smad3 will confer resistance to fibrosis and result in reduced inflammatory cell infiltrates, reduced autoinduction of TGF-beta, important to sustain the process, and reduced elaboration of collagen. Preliminary observations in a model of radiation-induced fibrosis confirm this hypothesis and suggest that inhibitors of Smad3 might have clinical application both to improve wound healing and to reduce fibrosis.

DNA-Binding Proteins↗

PubMed2000

Characterization of the mouse Smad1 gene and its expression pattern in adult mouse tissues.

Smad1 belongs to a family of receptor-activated proteins which mediate signals from TGF-beta superfamily ligands, including TGF-beta and BMPs. Although much is known about the biochemistry of Smad1 signal transduction, the role of Smad1 in vivo is still unclear. Here we present the first description of the genomic structure of the mouse Smad1 gene and the characterization of its expression pattern in adult mouse tissues by immunohistochemistry. The Smad1 gene contains 7 exons and spans >42 kb of genomic DNA. Its coding region is contained within 6 exons and all introns, except intron 1, follow the GT/AG rule. Immunohistochemical analysis shows that Smad1 is widely expressed in adult mouse tissues, with a varying degree of nuclear localization in different cell types, suggesting a regulated function for this protein. This study assigns all of the exon-intron boundaries of the mouse Smad1 gene and provides the basis for assessing the functional significance of this gene using targeted gene manipulation in the mouse.

5' Untranslated Regions↗

PubMed2000

Role of transforming growth factor-beta signaling in cancer.

Signaling from transforming growth factor-beta (TGF-beta) through its unique transmembrane receptor serine-threonine kinases plays a complex role in carcinogenesis, having both tumor suppressor and oncogenic activities. Tumor cells often escape from the antiproliferative effects of TGF-beta by mutational inactivation or dysregulated expression of components in its signaling pathway. Decreased receptor function and altered ratios of the TGF-beta type I and type II receptors found in many tumor cells compromise the tumor suppressor activities of TGF-beta and enable its oncogenic functions. Recent identification of a family of intracellular mediators, the Smads, has provided new paradigms for understanding mechanisms of subversion of TGF-beta signaling by tumor cells. In addition, several proteins recently have been identified that can modulate the Smad-signaling pathway and may also be targets for mutation in cancer. Other pathways such as various mitogen-activated protein kinase cascades also contribute substantially to TGF-beta signaling. Understanding the interplay between these signaling cascades as well as the complex patterns of cross-talk with other signaling pathways is an important area of investigation that will ultimately contribute to understanding of the bifunctional tumor suppressor/oncogene role of TGF-beta in carcinogenesis.

Animals↗

PubMed2000

A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.

Members of the transforming growth factor-beta superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-beta family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, we sought to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-beta and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-beta family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-beta-induced transcriptional activation involving CBP/p300.

Amino Acid Sequence↗

PubMed2000

The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.

The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.

Amino Acid Sequence↗

PubMed2000

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

Amino Acid Sequence↗