Biomedical subjects

Peter Ten Dijke

Publications and source records attributed to Peter Ten Dijke.

15 recordsLinked to original sources

PubMed2007

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Methylation of Smad6 by protein arginine N-methyltransferase 1.

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.

Alanine↗

PubMed2006

Smad7-induced beta-catenin degradation alters epidermal appendage development.

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.

Animals↗

PubMed2005

The deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta signalling.

Disruption of components in the transforming growth factor-beta (TGF-beta) signalling cascade is a common occurrence in human cancers. TGF-beta pathway activation is accomplished via serine/threonine kinase receptors and intracellular Smad transcription factors. A key regulatory step involves specific ubiquitination by Smurfs that mediate the proteasomal degradation of Smads and/or receptors. Here, we report a novel interaction between Smads and ubiquitin C-terminal hydrolase UCH37, a deubiquitinating enzyme that could potentially reverse Smurf-mediated ubiquitination. In GST pull down experiments, UCH37 bound weakly to Smad2 and Smad3, and bound very strongly to Smad7 in a region that is distinct from the -PY- motif in Smad7 that interacts with Smurf ubiquitin ligases. Endogenous Smad7 and UCH37 formed a stable complex in U4A/JAK1 cells, and FLAG-Smad7 co-immunoprecipitated with HA-UCH37 in transfected HEK-293 cells. In addition, we show that UCH37 can deubiquitinate and stabilize the type I TGF-beta receptor. Furthermore, overexpression of UCH37 upregulates TGF-beta-dependent transcription, and this effect is reversed in cells subject to RNAi-mediated knockdown of endogenous UCH37. These findings support a new role for deubiquitinating enzymes in the control of the TGF-beta signalling pathway, and provide a novel molecular target for the design of inhibitors with therapeutic potential in cancer.

Activin Receptors, Type I↗

PubMed2005

TGF-beta receptor function in the endothelium.

Genetic studies in mice and humans have revealed the pivotal role of transforming growth factor-beta (TGF-beta) signaling during angiogenesis. Mice deficient for various TGF-beta signaling components present an embryonic lethality due to vascular defects. In patients, mutations in the TGF-beta type I receptor ALK1 or in the accessory TGF-beta receptor endoglin are linked to an autosomal dominant disorder of vascular dysplasia termed Hereditary Haemorrhagic Telangiectasia (HHT). It has puzzled researchers for years to explain the effects of TGF-beta being a stimulator and an inhibitor of angiogenesis in vitro and in vivo. Recently, a model has been proposed in which TGF-beta by binding to the TGF-beta type II receptor can activate two distinct type I receptors in endothelial cells (ECs), i.e., the EC-restricted ALK1 and the broadly expressed ALK-5, which have opposite effects on ECs behavior. ALK1 via Smad1/5 transcription factors stimulates EC proliferation and migration, whereas ALK5 via Smad2/3 inhibits EC proliferation and migration. Here, the new findings are presented concerning the molecular mechanisms that take place in ECs to precisely regulate and even switch between TGF-beta-induced biological responses. In particular, the role of the accessory TGF-beta receptor endoglin in the regulation of EC behavior is addressed and new insights are discussed concerning the possible mechanisms that are implicated in the development of HHT.

Animals↗

PubMed2005

New mechanisms of skin innate immunity: ASK1-mediated keratinocyte differentiation regulates the expression of beta-defensins, LL37, and TLR2.

Epidermal keratinocytes differentiate and form a multilayered epidermis, which is the primary barrier between the body and the outer environment. As the epidermis is constantly exposed to a variety of microbial pathogens, its function of resisting microbial pathogens is vital. This characteristic feature is formed during differentiation. Immunohistochemical analysis revealed that the upper epidermis of normal human skin expresses beta-defensins 1-3 and LL37. We hypothesized that epidermal keratinocytes develop an innate immune barrier based on human beta-defensins (hBD) and LL37 during differentiation. To prove this, we introduced an active form of the apoptosis signal-regulating kinase-1 (ASK1), an intracellular regulator of keratinocyte differentiation, into cultured normal human keratinocytes. Transfection of this active form, ASK1-DeltaN, significantly enhanced the expression of hBD1-3 and LL37. In addition, a p38 inhibitor abolished this induction, indicating that the ASK1-p38 cascade regulates the expression of hBD1-3 and LL37. Furthermore, the ASK1-p38 pathway also regulated the expression of Toll-like receptor (TLR)2 in keratinocytes. Contact between S. aureus and keratinocytes resulted in the phosphorylation of p38 and induced the expression of hBD2 and hBD3. Moreover, the p38 inhibitor reduced this induction. In conclusion, the ASK1-p38 cascade regulates the innate immunity of the skin by forming an immune barrier consisting of hBD, LL37, and TLR2 during epidermal differentiation.

Antimicrobial Cationic Peptides↗

PubMed2004

Defective paracrine signalling by TGFbeta in yolk sac vasculature of endoglin mutant mice: a paradigm for hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder in humans that is characterised by multisystemic vascular dyplasia and recurrent haemorrhage. Germline mutations in one of two different genes, endoglin or ALK1 can cause HHT. Both are members of the transforming growth factor (TGF) beta receptor family of proteins, and are expressed primarily on the surface of endothelial cells (ECs). Mice that lack endoglin or activin receptor like kinase (ALK) 1 die at mid-gestation as a result of defects in the yolk sac vasculature. Here, we have analyzed TGFbeta signalling in yolk sacs from endoglin knockout mice and from mice with endothelial-specific deletion of the TGFbeta type II receptor (TbetaRII) or ALK5. We show that TGFbeta/ALK5 signalling from endothelial cells to adjacent mesothelial cells is defective in these mice, as evidenced by reduced phosphorylation of Smad2. This results in the failure of vascular smooth muscle cells to differentiate and associate with endothelial cells so that blood vessels remain fragile and become dilated. Phosphorylation of Smad2 and differentiation of smooth muscle can be rescued by culture of the yolk sac with exogenous TGFbeta1. Our data show that disruption of TGFbeta signalling in vascular endothelial cells results in reduced availability of TGFbeta1 protein to promote recruitment and differentiation of smooth muscle cells, and provide a possible explanation for weak vessel walls associated with HHT.

Activin Receptors, Type I↗

PubMed2004

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

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

Amino Acid Sequence↗

PubMed2003

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats.

BACKGROUND & AIMS: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS: Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.

Actins↗

PubMed2003

Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation.

Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.

Actins↗

PubMed2003

Smad protein and TGF-beta signaling in vascular smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) plays a role in vascular remodeling by stimulating vascular smooth muscle cell (SMC) growth and matrix-protein synthesis at sites of vascular injury. Smad proteins have been shown to mediate intracellular signaling of this growth factor. We investigated the expression and phosphorylation of Smads in cultured rat aortic smooth muscle cells. In addition, we evaluated the effects of overexpression of Smad proteins on TGF-beta signal transduction by adenovirus-mediated gene transfer. In rat SMC, Smad1, Smad2, Smad3, Smad4 and Smad5 were detected by immunoprecipitation. Using antisera against phosphorylated Smad2, we showed that TGF-beta1-induced Smad2 phosphorylation in a concentration- and time-dependent manner. Using adenovirus-mediated transfection method, we demonstrated that overexpression of Smad2 or Smad4 was associated with an increased production of TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1). However, the most prominent expression of PAI-1 was observed upon cotransfection of both Smad2 and Smad4. Both the proliferative effect of TGF-beta1 under serum-free conditions and its anti-proliferative effect under serum-rich conditions were suppressed by the adenovirus-mediated overexpression of Smad7. These results indicated that Smads proteins were expressed in vascular SMC and that they mediated TGF-beta signaling in those cells.

Adenoviridae↗

PubMed2002

Overexpression of Smad7 results in severe pathological alterations in multiple epithelial tissues.

Biochemical studies have shown that Smad7 blocks signal transduction of transforming growth factor beta (TGFbeta); however, its in vivo functions are largely unknown. To determine the functions of Smad7, we have expressed Smad7 in transgenic mice, utilizing a keratin K5 promoter (K5.Smad7). K5.Smad7 mice exhibited pathological changes in multiple tissues and died within 10 days after birth. These mice were born with open eyelids and corneal defects, significantly delayed and aberrant hair follicle morphogenesis, and hyperproliferation in the epidermis and other stratified epithelia. Furthermore, K5.Smad7 mice developed severe thymic atrophy and massive thymocyte death, suggesting that Smad signaling in thymic epithelia is essential for thymocyte survival. Interestingly, in addition to a reduction in Smad phosphorylation, the protein levels of the receptors for TGFbeta, activin and bone morphogenetic protein were significantly decreased in the affected tissues of K5.Smad7 mice. Our study provides evidence that Smad7 is a potent in vivo inhibitor for signal transduction of the TGFbeta superfamily during development and maintenance of homeostasis of multiple epithelial tissues.

Animals↗

PubMed2002

Regulation of cell proliferation by Smad proteins.

Transforming growth factor-beta (TGF-beta) family members which include TGF-betas, activins, and bone morphogenetic proteins (BMPs) regulate a broad spectrum of biological responses on a large variety of cell types. TGF-beta family members initiate their cellular responses by binding to distinct receptors with intrinsic serine/threonine kinase activity and activation of specific downstream intracellular effectors termed Smad proteins. Smads relay the signal from the cell membrane to the nucleus, where they affect the transcription of target genes. Smad activation, subcellular distribution, and stability have been found to be intricately regulated and a broad array of transcription factors have been identified as Smad partners. Important activities of TGF-beta are its potent anti-mitogenic and pro-apoptotic effects that, at least in part, are mediated via Smad proteins. Escape from TGF-beta/Smad-induced growth inhibition and apoptosis is frequently observed in tumors. Certain Smads have been found to be mutated in specific types of cancer and gene ablation of particular Smads in mice has revealed increased rate of tumorigenesis. In late stage tumors, TGF-beta has been shown to function as a tumor promoter. TGF-beta can stimulate the de-differentiation of epithelial cells to malignant invasive and metastatic fibroblastic cells. Interestingly, TGF-beta may mediate these effects directly on tumor cells via subverted Smad-dependent and/or Smad-independent pathways.

Animals↗

PubMed2002

Activation of bone morphogenetic protein/Smad signaling in bronchial epithelial cells during airway inflammation.

Bone morphogenetic proteins (BMPs) are pleiotropic secreted proteins, structurally related to transforming growth factor (TGF)-beta and activins. BMPs play pivotal roles in the regulation of embryonic lung development and branching of airways and have recently been considered to influence inflammatory processes in adults due to their chemotactic activity on fibroblasts, myocytes, and inflammatory cells. In this study, we have investigated the possible involvement of BMPs in a model of experimental allergic-airway inflammation in situ using antibodies that detect activated Smad proteins, and have monitored the modulation of BMP ligands during the inflammatory response. Inflamed bronchial epithelial cells and a few scattered alveolar cells expressed levels of phosphorylated Smad1 (pSmad1/5), indicative of active BMP/Smad signaling. This was in contrast to healthy epithelium, which was devoid of immunoreactivity. A mechanistic explanation for increased pSmad1/5 staining during inflammation was provided by the upregulated expression of all the BMP type I receptors, i.e., activin receptor-like kinase (ALK)2, ALK3, and ALK6, in the inflamed bronchial epithelial cells. Furthermore, the mRNA and protein profiles for BMP ligands were significantly altered during airway inflammation with induction of BMP2, BMP4, and BMP6, and downregulation of BMP5 and BMP7. Collectively, our data demonstrate for the first time active BMP/Smad signaling during airway inflammation in bronchial epithelial cells and thus raise the possibility that BMPs could play a determining role in respiratory pathophysiology.

Animals↗

PubMed2002

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit articular cartilage.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.

Activin Receptors, Type I↗