Transformation of intestinal epithelial cells by chronic TGF-beta1 treatment results in downregulation of the type II TGF-beta receptor and induction of cyclooxygenase-2.
(1/2312)
The precise role of TGF-beta in colorectal carcinogenesis is not clear. The purpose of this study was to determine the phenotypic alterations caused by chronic exposure to TGF-beta in non-transformed intestinal epithelial (RIE-1) cells. Growth of RIE-1 cells was inhibited by >75% following TGF-beta1 treatment for 7 days, after which the cells resumed a normal growth despite the presence of TGF-beta1. These 'TGF-beta-resistant' cells (RIE-Tr) were continuously exposed to TGF-beta for >50 days. Unlike the parental RIE cells, RIE-Tr cells lost contact inhibition, formed foci in culture, grew in soft agarose. RIE-Tr cells demonstrated TGF-beta-dependent invasive potential in an in vitro assay and were resistant to Matrigel and Na-butyrate-induced apoptosis. The RIE-Tr cells were also tumorigenic in nude mice. The transformed phenotype of RIE-Tr cells was associated with a 95% decrease in the level of the type II TGF-beta receptor (TbetaRII) protein, a 40-fold increase in cyclooxygenase-2 (COX-2) protein, and 5.9-fold increase in the production of prostacyclin. Most RIE-Tr subclones that expressed low levels of TbetaRII and high levels of COX-2 were tumorigenic. Those subclones that express abundant TbetaRII and low levels of COX-2 were not tumorigenic in nude mice. A selective COX-2 inhibitor inhibited RIE-Tr cell growth in culture and tumor growth in nude mice. The reduced expression of TbetaRII, increased expression of COX-2, and the ability to form colonies in Matrigel were all reversible upon withdrawal of exogenous TGF-beta1 for the RIE-Tr cells. (+info)
Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12.
(2/2312)
Activation of the type I TGFbeta receptor (TbetaR-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated TbetaR-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. TbetaR-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the TbetaR-II phosphorylation sites and further stabilizing the inactive conformation of TbetaR-I. Certain structural features at the catalytic center of TbetaR-I are characteristic of tyrosine kinases rather than Ser/Thr kinases. (+info)
Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells.
(3/2312)
Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling. (+info)
Microsatellite instability, Epstein-Barr virus, mutation of type II transforming growth factor beta receptor and BAX in gastric carcinomas in Hong Kong Chinese.
(4/2312)
Microsatellite instability (MI), the phenotypic manifestation of mismatch repair failure, is found in a proportion of gastric carcinomas. Little is known of the links between MI and Epstein-Barr virus (EBV) status and clinicopathological elements. Examination of genes mutated through the MI mechanism could also be expected to reveal important information on the carcinogenic pathway. Seventy-nine gastric carcinomas (61 EBV negative, 18 EBV positive) from local Hong Kong Chinese population, an intermediate-incidence area, were examined. Eight microsatellite loci, inclusive of the A10 tract of type II transforming growth factor beta receptor (TbetaR-II), were used to evaluate the MI status. MI in the BAX and insulin-like growth factor II receptor (IGF-IIR) genes were also examined. High-level MI (>40% unstable loci) was detected in ten cases (12.7%) and low-level MI (1-40% unstable loci) in three (3.8%). High-level MI was detected in two EBV-associated cases (11%) and the incidence was similar for the EBV-negative cases (13%). The high-level MIs were significantly associated with intestinal-type tumours (P = 0.03) and a more prominent lymphoid infiltrate (P = 0.04). Similar associations were noted in the EBV-positive carcinomas. The high-level MIs were more commonly located in the antrum, whereas the EBV-associated carcinomas were mostly located in body. Thirteen cardia cases were negative for both high-level MI and EBV. All patients aged below 55 were MI negative (P = 0.049). Of the high-level MIs, 80% had mutation in TbetaR-II, 40% in BAX and 0% in IGF-IIR. Of low-level MIs, 33% also had TbetaR-II mutation. These mutations were absent in the MI-negative cases. Of three lymphoepithelioma-like carcinomas, two cases were EBV positive and MI negative, one case was EBV negative but with high-level MI. In conclusion, high-level MIs were present regardless of the EBV status, and were found in a particular clinicopathological subset of gastric carcinoma patient. Inactivation of important growth regulatory genes observed in these carcinomas confirms the importance of MI in carcinogenesis. (+info)
Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.
(5/2312)
Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation. (+info)
The cytoplasmic carboxy-terminal amino acid determines the subcellular localization of proTGF-(alpha) and membrane type matrix metalloprotease (MT1-MMP).
(6/2312)
Transforming growth factor alpha (TGF-(alpha)) is synthesized as a precursor transmembrane molecule (proTGF-(alpha)) whose ectodomain is shed from the cell surface generating mature, soluble, growth factor. In agreement with recent reports, here we show that the structural determinant that targets proTGF-(alpha) to the cell surface maps to the very C-terminal cytoplasmic amino acid, valine. The primary localization of proTGF-(alpha) C-terminal mutants is a perinuclear area that colocalizes with ER markers. Since the ectodomain shedding machinery that acts on proTGF-(alpha) is known to be located at the cell surface, deficient transport provides an explanation for the previously reported lack of PKC activated ectodomain shedding of proTGF-(alpha) C-terminal mutants. The transport of wild-type proTGF-(alpha) to the cell surface was found to be mediated by a mechanism that includes a specific component saturable by wild-type proTGF-(alpha) but not by cell surface transmembrane proteins whose trafficking is independent of their cytoplasmic tail such as betaglycan. C-terminal valines are likely to be a general determinant of the subcellular location of cell surface transmembrane proteins since the maturation and trafficking of MT1-MMP C-terminal mutants are severely impaired. Our data suggest the existence of a targeting mechanism that acts on cell surface transmembrane molecules as diverse as proTGF-(alpha) and MT1-MMP and that the interaction with such a mechanism depends on the identity of the C-terminal amino acid of the targeted molecules. (+info)
Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators.
(7/2312)
Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways. (+info)
Interactions of high affinity insulin-like growth factor-binding proteins with the type V transforming growth factor-beta receptor in mink lung epithelial cells.
(8/2312)
High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (TbetaR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with TbetaR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TbetaR-V as demonstrated by the detection of the approximately 400-kDa 125I-IGFBP.TbetaR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TbetaR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to TbetaR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (TbetaR-I.TbetaR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TbetaR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TbetaR-I and TbetaR-II heterocomplex. (+info)