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PubMed2002

Interference with TGF-beta1 and -beta3 in tumor stroma lowers tumor interstitial fluid pressure independently of growth in experimental carcinoma.

A high tumor interstitial fluid pressure (TIFP) is a pathologic characteristic distinguishing the stroma of carcinomas from normal interstitial loose connective tissues. The role of TGF-beta1 and -beta3 in generating a high TIFP was investigated in xenografted experimental anaplastic thyroid carcinoma (ATC) derived from the human ATC cell line KAT-4. A single intravenous injection of a soluble recombinant TGF-beta receptor type II-murine Fc:IgG(2A) chimeric protein that specifically inhibits TGF-beta1 and -beta3, significantly lowered TIFP in a time and concentration dependent manner but did not change total tissue water content in the tumors. Tumor growth rate was higher in tumors treated with the TGF-beta1 and -beta3 inhibitor compared to control tumors during the first 10 days after administration of the inhibitor. The apoptotic index of carcinoma cells, and expression of the cell cycle inhibitor p27(Kip1), were, however, increased in TGF-beta1 and -beta3 inhibitor-treated tumors. Prolonged treatment periods and administration of a second dose of the inhibitor decreased tumor growth rate. The TGF-beta1 and -beta3 inhibitor did not affect proliferation or expression of phosphorylated Smad2 protein in KAT-4 cells cultured in vitro. Our results indicate that members of the TGF-beta family are potential targets for novel anti-cancer treatment directed to the stroma. First by controlling TIFP and by that potentially the uptake of anticancer drugs into tumors and second by their suggested role in maintaining a supportive tumor stroma.

Animals↗
PubMed1997

Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.

TGF-beta signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-beta receptor induces phosphorylation of two such proteins, Smad2 and Smad3, which form hetero-oligomeric complex(es) with Smad4/DPC4 that translocate to the nucleus, where they then regulate transcriptional responses. However, the mechanisms by which the intracellular signals of TGF-beta are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signalling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses.

Amino Acid Sequence↗
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↗
PubMed2001

Transforming growth factor-beta-induced growth inhibition in a Smad4 mutant colon adenoma cell line.

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

Adenoma↗
PubMed2004

Interferon alpha-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor beta(1).

In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.

Animals↗
PubMed1999

Xenopus Smad4beta is the co-Smad component of developmentally regulated transcription factor complexes responsible for induction of early mesodermal genes.

Smad4 is defined as the common-mediator Smad (co-Smad) required for transducing signals for all TGF-beta superfamily members. This paper describes two Smad4s in Xenopus: XSmad4alpha, which is probably the Xenopus orthologue of human Smad4, and a distinct family member, XSmad4beta, which differs primarily at the extreme N-terminus and in the linker region. Both XSmad4s act as co-Smads, forming ligand-dependent complexes with receptor-regulated Smads 1 and 2 and synergizing with them to activate transcription of mesodermal genes in Xenopus embryos. The two XSmad4 genes have reciprocal temporal expression patterns in Xenopus embryos and are expressed in varying ratios in adult tissues, suggesting distinct functional roles in vivo. XSmad4beta is the predominant maternal co-Smad and we go on to demonstrate its role in the transcriptional regulation of early mesodermal genes. We have identified two distinct nuclear complexes that bind the activin-responsive element of the Xenopus Mix.2 promoter: one formed in response to high levels of activin signaling and the other activated by endogenous signaling pathways. Using specific antisera we demonstrate the presence of endogenous XSmad4beta and also XSmad2 in both of these complexes, and our data indicate that the DNA-binding components of the complexes are different. Furthermore, we show that the presence of these complexes in the nucleus perfectly correlates with the transcriptional activity of the target gene, Mix.2, and we show that one of the XSmad4beta-containing transcription factor complexes undergoes a developmentally regulated nuclear translocation.

Activins↗
PubMed2001

The amino terminus of Smads permits transcriptional specificity.

Smad proteins transduce transforming growth factor-beta signals from the cell surface to the nucleus, regulating a variety of physiologic processes. In the nucleus, Smads control gene expression by binding to both DNA and transcription factors. Individual Smads regulate distinct subsets of target genes. The key residues important for this specificity are thought to reside in the carboxyl-terminal MH2 domain. To further examine Smad specificity in vivo, we undertook structure-function studies in Xenopus laevis embryos and found that truncated Smads containing the MH2 domain activate gene transcription. A striking finding revealed by the in vivo analyses was that the functional truncated Smads all behaved identically and had lost wild-type specificity. For most Smads, wild-type activity required the presence of an MH1 domain, either in cis or in trans. Of note, even heterologous MH1 domains could restore wild-type signaling specificity to effector MH2 domains. We found a possible mechanism to account for these observations, as Smad MH1 domains altered the binding of pathway-specific transcription factors to the MH2 domain. Thus, Smad MH1 domains are important to the regulation of transcriptional specificity.

Animals↗
PubMed2001

Expression of SMAD signal transduction molecules in the pancreas.

Members of the TGF-beta superfamily of cytokines have been implicated in pancreatic cancer, pancreatitis and in regulation and differentiation of pancreatic endocrine and exocrine cells. Different TGF-beta members signal through phosphorylation of different signal transduction proteins, which eventually form oligomers with SMAD 4 and translocate to the nucleus. Reverse transcriptase-polymerase chain reaction showed that SMADs 1, 2 and 4 are expressed in pancreatic islets. Immunostaining revealed that SMAD 1 and 4 predominantly were expressed by islet insulin and glucagon cells. Since SMAD 1 is known to transduce signals from receptors binding bone morphogenetic protein (BMP) these results indicate a previously unknown role of BMP-like ligands in islet function.

Animals↗
PubMed2004

Cirrhotic hepatocytes exhibit decreased TGFbeta growth inhibition associated with downregulated Smad protein expression.

TGFbeta controls hepatocyte growth through cell cycle arrest and apoptosis, and resistance to TGFbeta is a mechanism of malignant transformation. The aim of this study was to assess differences in TGFbeta-mediated growth inhibition in normal and cirrhotic hepatocytes. Cirrhosis was induced in mice and normal and cirrhotic hepatocytes were isolated by collagenase perfusion and treated with or without TGFbeta (5 ng/ml). DNA synthesis, Smad protein expression, and DNA binding activity were determined. TGFbeta reduced DNA synthesis to a greater degree in normal hepatocytes than in cirrhotic hepatocytes (87% vs. 68%; p<0.05). Smad protein expression was decreased in cirrhotic hepatocytes and Smad 2/3/4 complex formation was suppressed. Furthermore, cirrhotic hepatocytes had decreased DNA binding activity at 120 min following TGFbeta treatment. In conclusion, decreased Smad protein expression may impair TGFbeta-mediated growth inhibition in cirrhotic hepatocytes.

Active Transport, Cell Nucleus↗
PubMed2004

New insights into TGF-beta-Smad signalling.

Transforming growth factor beta (TGF-beta) initiates its diverse cellular responses by binding to and activating specific cell surface receptors that have intrinsic serine/threonine kinase activity. These activated TGF-beta receptors stimulate the phosphorylation of receptor-regulated Smad proteins, which in turn form complexes with Smad4 that accumulate in the nucleus and regulate the transcription of target genes. TGF-beta responses can be cell-type specific and are dependent on both the concentration of TGF-beta signalling components and the activity of other signal transduction pathways, which can either synergize with or antagonize the TGF-beta pathway. Recent research has provided insights into the specificity determinants of TGF-beta-Smad signalling, including combinatorial ligand-receptor associations, selective interactions between the Smads and other pathway components that are mediated through defined binding motifs, and the differential regulation of duration and intensity of signalling.

Active Transport, Cell Nucleus↗
PubMed2006

p38 mitogen-activated protein kinase and alkaline phosphatase in human dental pulp cells.

OBJECTIVE: The objective of this study was to investigate the implication of p38 mitogen-activated protein kinase (MAPK) in mediating alkaline phosphatase (ALPase) activity in human dental pulp cells (HPCs). STUDY DESIGN: Nuclear translocation of p38 was observed by immunofluorescence in isolated HPCs treated with transforming growth factor beta1 (TGF-beta1). TGF-beta1 was used to examine the interaction between p38 MAPK and Smad pathway. Role of p38 kinase in mediating ALPase activity was determined with SB203580, a specific inhibitor for the p38 pathway. Lipopolysaccharide (LPS) or TGF-beta1 was added to inhibit or increase ALPase activity. Statistical analysis was performed by unpaired t test. RESULTS: TGF-beta1 induced nuclear translocalization of p38. Blockage of p38 pathway with SB203580 inhibited translocation of Smad2/3 to nuclei. ALPase activity decreased in cells treated with SB203580, in contrast to its vehicle (P < .05). Inhibition on enzyme activity by LPS was exacerbated by SB203580 (P < .05). Treatment with SB203580 before addition of TGF-beta1 also made a significant decrease in ALPase activity (P < .05). CONCLUSIONS: These results suggest that p38 MAPK is implicated in regulating ALPase activity in HPCs and may interact with Smad pathway.

Active Transport, Cell Nucleus↗
PubMed2005

Modulation of the TGFbeta/Smad signaling pathway in mesangial cells by CTGF/CCN2.

Transforming growth factor-beta (TGFbeta) drives fibrosis in diseases such as diabetic nephropathy (DN). Connective tissue growth factor (CTGF; CCN2) has also been implicated in this, but the molecular mechanism is unknown. We show that CTGF enhances the TGFbeta/Smad signaling pathway by transcriptional suppression of Smad 7 following rapid and sustained induction of the transcription factor TIEG-1. Smad 7 is a known antagonist of TGFbeta signaling and TIEG-1 is a known repressor of Smad 7 transcription. CTGF enhanced TGFbeta-induced phosphorylation and nuclear translocation of Smad 2 and Smad 3 in mesangial cells. Antisense oligonucleotides directed against TIEG-1 prevented CTGF-induced downregulation of Smad 7. CTGF enhanced TGFbeta-stimulated transcription of the SBE4-Luc reporter gene and this was markedly reduced by TIEG-1 antisense oligonucleotides. Expression of the TGFbeta-responsive genes PAI-1 and Col III over 48 h was maximally stimulated by TGFbeta+CTGF compared to TGFbeta alone, while CTGF alone had no significant effect. TGFbeta-stimulated expression of these genes was markedly reduced by both CTGF and TIEG-1 antisense oligonucleotides, consistent with the endogenous induction of CTGF by TGFbeta. We propose that under pathological conditions, where CTGF expression is elevated, CTGF blocks the negative feedback loop provided by Smad 7, allowing continued activation of the TGFbeta signaling pathway.

Cell Cycle Proteins↗
PubMed2004

RNA degradation in human breast tissue after surgical removal: a time-course study.

There is much interest in the study of human malignancy using gene expression profiling techniques. Expression profiles obtained from microarrays utilize RNA extracted from the tissue in question. Currently, cell cultures or fresh tissue processed "quickly" are used in these studies. To our knowledge, there are no published reports of a time-course of RNA degradation in surgically removed breast tissue. Such a time-course study is critically needed. We obtained normal breast tissue from breast reduction surgery. Portions of breast tissue kept at room temperature were sampled and placed into RNAlater to preserve RNA at different time-points from 10 min to 3 h after the surgical removal. We evaluated total RNA integrity from each specimen using agarose gel electrophoresis and real-time quantitative RT-PCR analysis of four genes. Electrophoresis showed good-quality, intact RNA at all time points up to 3 h. Quantitative RT-PCR showed no difference in amplified products among all samples. Our study showed that there was no loss of RNA integrity in normal breast tissue for up to 3 h after surgical removal.

Actins↗
PubMed1998

Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion.

Growth factors of the transforming growth factor-beta (TGF-beta) family inhibit the proliferation of epithelial, endothelial, and hematopoietic cells, and stimulate the synthesis of extracellular matrix components. TGF-beta s are secreted from cells in high-molecular-mass protein complexes that are composed of three proteins, the mature TGF-beta-dimer, the TGF-beta propeptide dimer, or latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Mature TGF-beta is cleaved from its propeptide during secretion, but the proteins remain associated by noncovalent interactions. LTBP is required for efficient secretion and processing of latent TGF-beta and it binds to LAP via disulfide bond(s). LTBP is a component of extracellular matrix microfibrils, and it targets the latent TGF-beta complex to the extracellular matrix. TGF-beta signaling is initiated by proteolytic cleavage of LTBP that results in the release of the latent TGF-beta complex from the extracellular matrix. TGF-beta is activated by dissociation of LAP from the mature TGF-beta. Subsequent signaling involves binding of active TGF-beta to its type II cell surface receptors, which phosphorylate and activate type I TGF-beta receptors. Type I receptors, in turn, phosphorylate cytoplasmic transcriptional activator proteins Smad2 and Smad3, inducing their translocation to the nucleus. Recent evidence suggests that acquisition of resistance to TGF-beta growth inhibition plays a major role in the progression of epithelial and hematopoietic cell malignancies. The role of secretion of TGF-beta in tumorigenesis is more complex. The secretion of TGF-beta s by tumor cells may contribute to autocrine growth inhibition, but on the other hand, it may also promote invasion, metastasis, angiogenesis, and even immunosuppression. Tumor cells may also fail to deposit LTBP:TGF-beta complexes to the extracellular matrix. The elucidation of the mechanisms of the release of TGF-beta from the matrix and its subsequent activation aids the understanding of the pathophysiologic roles of TGF-beta in malignant growth, and allows the development of therapeutic agents that regulate the activity of TGF-beta.

Cell Division↗
PubMed2000

OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways.

We have identified the 30-zinc finger protein OAZ as a DNA-binding factor that associates with Smads in response to BMP2, forming a complex that transcriptionally activates the homeobox regulator of Xenopus mesoderm and neural development, Xvent-2. OAZ contains a BMP signaling module formed by two clusters of fingers that bind Smads and the Xvent-2 BMP response element, respectively. Previously implicated as a transcriptional partner of Olf-1/EBF in olfactory epithelium and lymphocyte development in the rat, OAZ fulfills this role through clusters of fingers that are separate from the BMP signaling module. The mutually exclusive use of OAZ by the BMP-Smad and Olf pathways illustrates the dual role of a multi-zinc finger protein in signal transduction during development.

Amino Acid Sequence↗
PubMed2000

Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling.

To begin to understand the molecular basis of communication between the parasite Schistosoma mansoni and its mammalian host, we are studying the signaling pathway downstream of S. mansoni receptor kinase-1 (SmRK1), a divergent type I transforming growth factor-beta (TGF-beta) receptor found on the tegumental surface of the parasite. In this study, we have used a homology based PCR approach to clone two S. mansoni Smad (SmSmad) genes; Smads play a pivotal role in the most well understood signaling pathways initiated by the TGF-beta family of ligands in other organisms. Comparison of the amino acid sequences with those of other Smads reveals that the conserved MH1 and MH2 domains of SmSmads show a high degree of identity to homologues in Drosophila. Transcripts for both SmSmads are detected in the same developmental stages as SmRK1, and both are capable of interacting with the intracellular domain of the receptor in vitro. Functional characterization using the human type I TGF-beta receptor further confirms the highly conserved nature of these proteins, as both SmSmads show TGF-beta dependent enhancement of luciferase activity and nuclear translocation in mammalian cells. These data are the first to show a TGF-beta-like receptor/Smad signaling pathway in parasitic helminths and by analogy with other systems, is likely important in regulating schistosome development.

Activin Receptors, Type I↗
PubMed2001

The transcriptional role of Smads and FAST (FoxH1) in TGFbeta and activin signalling.

The Smad family of proteins are critical components of the TGFbeta superfamily signalling pathway. Ligand addition induces phosphorylation of specific receptor-regulated Smads, which then form heteromeric complexes with the common mediator Smad, Smad4. This complex then translocates from the cytoplasm into the nucleus. Once there, the R-Smad/Smad4 complex interacts with a variety of DNA binding proteins and is thereby targetted to a diverse array of gene promoters. The Smad-containing DNA binding complex can then positively or negatively regulate gene expression through the recruitment of co-activators and co-repressors. Xenopus FAST (now known as FoxH1) was the first Smad DNA binding partner identified and the FoxH1 family now includes related proteins from mouse, human and Zebrafish. In all organisms examined, FoxH1 is expressed primarily during the earliest stages of development and thus FoxH1 is thought to play a critical role in mediating TGFbeta superfamily signals during these early developmental stages. Other Smad partners range from those that are ubiquitously expressed to others that are present only in specific cell types or developmental stages. Thus, it is the interaction of Smads with a wide range of specific transcriptional partners that is important for the generation of diverse biological responses to TGFbeta superfamily members.

Activins↗
PubMed2000

XSIP1, a Xenopus zinc finger/homeodomain encoding gene highly expressed during early neural development.

We have isolated a Xenopus homologue of the zinc finger/homeodomain-containing transcriptional repressor Smad-interacting protein-1 (SIP1) from mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs.

Amino Acid Sequence↗