Signal transduction. RIPping tyrosine kinase receptors apart.
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Active Transport, Cell Nucleus↗
Biomedical subjects
C H Heldin
Publications and source records attributed to C H Heldin.
Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads.
Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.
Activin Receptors↗
Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis.
Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.
Adult↗
Ligand stimulation reduces platelet-derived growth factor beta-receptor susceptibility to tyrosine dephosphorylation.
Ligand binding to the platelet-derived growth factor (PDGF) beta-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerization-induced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligand-stimulated PDGF beta-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and non-stimulated forms of tyrosine-phosphorylated PDGF beta-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligand-stimulated PDGF beta-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligand-stimulated forms of the PDGF beta-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF beta-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation.
Agglutinins↗
Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors.
Most solid malignancies display interstitial hypertension and a poor uptake of anticancer drugs. Platelet-derived growth factor (PDGF) and the cognate tyrosine kinase receptors are expressed in many tumors. Signaling through PDGFbeta receptors was shown recently to increase interstitial fluid pressure (IFP) in dermis after anaphylaxis-induced lowering of IFP. In this study, we show that treatment with the selective PDGF receptor kinase inhibitor, STI571, formerly known as CGP57148B, decreased the interstitial hypertension and increased capillary-to-interstitium transport of 51Cr-EDTA in s.c. growing rat PROb colonic carcinomas. Furthermore, treatment with an antagonistic PDGF-B oligonucleotide aptamer decreased interstitial hypertension in these tumors. PDGFbeta receptors were expressed in blood vessels and stromal cells but not in the tumor cells of PROb colonic carcinomas. Our study indicates a previously unrecognized role of PDGF receptors in tumor biology, although similar effects of PDGF on IFP have been demonstrated previously in the dermis. The data suggest interference with PDGF receptors, or their ligands, as a novel strategy to increase drug uptake and therapeutic effectiveness of cancer chemotherapy.
Animals↗
Mechanisms of platelet-derived growth factor-induced chemotaxis.
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Animals↗
Phosphorylation of Smad7 at Ser-249 does not interfere with its inhibitory role in transforming growth factor-beta-dependent signaling but affects Smad7-dependent transcriptional activation.
Smad proteins are major components in the intracellular signaling pathway of transforming growth factor-beta (TGF-beta), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF-beta-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-beta1. Abrogation of the phosphorylation by substitution of Ser-249 with alanine or aspartic acid residues did not affect the ability of Smad7 to inhibit TGF-beta1 and BMP7 signaling. No differences were found in the stability or in the intracellular distribution of Smad7 mutants compared with the wild-type molecule. However, Smad7 fused to the DNA-binding domain of GAL4 induced transcription from a reporter with mutated TATA minimal promoter in a Ser-249-dependent manner. Moreover, a reporter with the SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was also dependent on Ser-249 phosphorylation. The amplitude of effects on transcriptional regulation was dependent on cell type. Our results suggest that phosphorylation of Smad7, unlike phosphorylation of the receptor-regulated Smads, does not regulate TGF-beta signaling but rather affects TGF-beta-independent effects of Smad7 on transcriptional regulation.
3T3 Cells↗
Regulation of Smad signaling by protein kinase C.
Cross talk between transforming growth factor b(TGF-b) serine/threonine kinase receptor signaling and tyrosine kinase receptor signaling modulates cell responsiveness to polypeptide growth factors regulating cell proliferation, differentiation, and apoptosis. Here we provide a mechanism through which Smad-dependent TGF-b signaling is modulated by protein kinase C (PKC). PKC, for example, is activated downstream of tyrosine kinase receptors. We show that PKC directly phosphorylates receptor-regulated Smad proteins. This phosphorylation abrogates the ability of Smad3 to bind directly to DNA, which leads to subsequent inability to mediate transcriptional responses dependent on the direct binding of Smad3 to DNA. Interference with PKC regulation of Smad functions increased cell sensitivity to transformation by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PKC-dependent phosphorylation of Smad3 was found also to be a key event in the PMA-dependent inactivation of TGF-b-stimulated cell death. Thus, PKC-dependent phosphorylation of Smad3 leads to down-regulation of the growth inhibitory and apoptotic action of TGF-b.
Cell Line↗
Growth factor regulation of kinases.
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Animals↗
Involvement of platelet-derived growth factor in disease: development of specific antagonists.
Platelet-derived growth factor (PDGF) is a family of dimeric isoforms that stimulates, e.g., growth, chemotaxis and cell shape changes of various connective tissue cell types and certain other cells. The cellular effects of PDGF isoforms are exerted through binding to two structurally related tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation. This enables a number of SH2 domain containing signal transduction molecules to bind to the receptors, thereby initiating various signaling pathways. PDGF isoforms have important roles during the embryonic development, particularly in the formation of connective tissue in various organs. In the adult, PDGF stimulates wound healing. Overactivity of PDGF has been implicated in certain disorders, including fibrotic conditions, atherosclerosis, and malignancies. Different kinds of PDGF antagonists are currently being developed and evaluated in different animal disease models, as well as in clinical trials.
Animals↗
PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor.
The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.
Amino Acid Sequence↗
Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner.
Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.
Active Transport, Cell Nucleus↗
Activation of the TGF-beta/activin-Smad2 pathway during allergic airway inflammation.
Changes in the levels of transforming growth factor (TGF)-beta cytokines or receptors observed during the progression of several inflammatory and fibrotic disorders have been used to implicate these cytokines in the pathophysiology of these diseases. Although correlative, these studies were inconclusive because they were unable to demonstrate actual continuous TGF-beta-mediated signaling in the involved tissues. We reasoned that the phosphorylation state and subcellular localization of Smad2, the intracellular effector of TGF-beta/activin-mediated signaling, could be used as a marker of active signaling mediated by these cytokines in situ. We therefore used an experimental model of ovalbumin-induced allergic airway inflammation and were able to demonstrate a dramatic increase in the numbers of bronchial epithelial, alveolar, and infiltrating inflammatory cells expressing nuclear phosphorylated Smad2 within the allergen-challenged lungs. This was accompanied by strong upregulation of the activin receptor ALK-4/ActR-IB and redistribution of the TGF-beta responsive ALK-5/TbetaR-I. Although levels of TGF-beta1, TGF-beta2, and TGF-beta3 messenger RNA (mRNA) were marginally altered, the level of activin mRNA was strongly upregulated during the inflammatory response. Our data illustrate the usefulness of antiphosphorylated Smad antibodies in demonstrating active TGF- beta/activin-mediated signaling in vivo and strongly suggest that activin/Smad-mediated signaling could be a critical contributor in the pathophysiology of allergic pulmonary diseases.
Activins↗
Smad regulation in TGF-beta signal transduction.
Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-beta. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.
Activin Receptors, Type I↗
Phosphorylation of Smad signaling proteins by receptor serine/threonine kinases.
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Animals↗
Signal transduction: multiple pathways, multiple options for therapy.
Many aspects of cell behavior, such as growth, motility, differentiation, and apoptosis, are regulated by signals cells receive from their environment. Such signals are important, e.g., during embryonal development, wound healing, hematopoiesis, and in the regulation of the immune response, and may come from interactions with other cells or components of the extracellular matrix, or from binding of soluble signaling molecules to specific receptors at the cell membrane. Hereby different signaling pathways are initiated inside the cell. Perturbations of such signaling pathways are seen in several types of diseases, e.g., cancer, inflammatory conditions, and atherosclerosis. Thus, antagonists of several signaling pathways have potential clinical utility. Several such compounds are currently used or are in clinical trials; others are currently being analyzed in animal models.
Diabetes Mellitus↗
Retention of PDGFR-beta function in mice in the absence of phosphatidylinositol 3'-kinase and phospholipase Cgamma signaling pathways.
Signal transduction by the platelet-derived growth-factor receptor beta (PDGFR-beta) tyrosine kinase is required for proper formation of vascular smooth muscle cells (VSMC). However, the importance of individual PDGFR-beta signal transduction pathways in vivo is not known. To investigate the role of two of the pathways believed to be critical for PDGF signal transduction, we have generated mice that bear a PDGFR-beta that can no longer activate PI3kinase or PLCgamma. Although these mutant mice have normal vasculature, we provide multiple lines of evidence in vivo and from cells derived from the mutant mice that suggest that the mutant PDGFR-beta operates at suboptimal levels. Our observations indicate that although loss of these pathways can lead to attenuated PDGF-dependent cellular function, certain PDGFR-beta-induced signal cascades are not essential for survival in mice.
Animals↗
Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis.
Tissue factor (TF) is the cellular receptor for factor FVIIa (FVIIa), and the complex is the principal initiator of blood coagulation. The effects of FVIIa binding to TF on cell migration and signal transduction of human fibroblasts, which express high amounts of TF, were studied. Fibroblasts incubated with FVIIa migrated toward a concentration gradient of PDGF-BB at approximately 100 times lower concentration than do fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis induced by FVIIa/TF. Moreover, a pronounced suppression of chemotaxis induced by PDGF-BB was observed with active site-inhibited FVIIa (FFR-FVIIa). The possibility that hyperchemotaxis was induced by a putative generation of FXa and thrombin activity was excluded. FVIIa/TF did not induce increased levels of PDGF beta-receptors on the cell surface. Thus, the hyperchemotaxis was not a result of this mechanism. FVIIa induced the production of inositol-1,4, 5-trisphosphate to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB were additive. FFR-FVIIa did not induce any release of inositol-1,4,5,-trisphosphate. Thus, binding of catalytically active FVIIa to TF can, independent of coagulation, modulate cellular responses, such as chemotaxis.
Amino Acid Chloromethyl Ketones↗