transforming-growth-factor-beta and Carcinoma--Embryonal

beta-Catenin-mediated Wnt signaling is essential in embryonic development and in adult tissues. Recent studies have demonstrated that Axin not only plays an important inhibitory role in coordinating beta-catenin degradation, but is itself degraded by the low-density-lipoprotein receptor-related protein (LRP)5/6 Wnt co-receptor. Here, we demonstrate that the endocytic adaptor molecule Disabled-2 (Dab2), which we have previously demonstrated to act as an inhibitor of beta-catenin signaling, interacts with Axin and prevents its interaction with and degradation by the LRP5 co-receptor, thereby increasing its half-life and stabilization. Dab2 levels induced during retinoic acid-induced differentiation of F9, or during transforming growth factor-beta-induced epithelial-mesenchymal transdifferentiation of mouse mammary epithelial cells result in the stabilization of Axin and concomitant inhibition of beta-catenin signaling. Ectopic expression of Dab2 in F9 cells as well as in transformed cell lines results in increased Axin expression and attenuation of Wnt-mediated signaling. We conclude that Dab2 may play an important role in the maintenance of the differentiated state and restrain Wnt-mediated proliferation through its association with and modulation of Axin.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis Regulatory Proteins; Axin Protein; beta Catenin; Blotting, Western; Carcinoma, Embryonal; Cell Differentiation; Cell Proliferation; Epithelial Cells; Immunoprecipitation; Mesoderm; Mice; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tretinoin; Wnt Proteins

Nuclear receptor-mediated gene expression is proposed to be regulated by the ordered recruitment of large protein complexes in which activity depends on mutual interactions and posttranslational modifications. In contrast, relatively little attention has been given to mechanisms regulating the expression of the coregulator proteins themselves. Previously we have shown that the ligand-dependent corepressor, RIP140, is a direct transcriptional target of all-trans retinoic acid (RA). Here we demonstrate that RA induction of RIP140 constitutes a rate-limiting step in the regulation of retinoic acid receptor signaling. Silencing of the RA induction of RIP140 dramatically enhances and accelerates retinoid receptor transactivation, endogenous expression of other RA target genes, and RA-induced neuronal differentiation and cell cycle arrest in human embryonal carcinoma cells. The data suggest that RA induction of RIP140 constitutes a functional negative feedback loop that limits activation of retinoid receptors in the continued presence of RA and that acutely regulated expression of coregulators may be a general regulatory mechanism in hormonal signaling.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Embryonal; Cell Cycle; Cell Differentiation; Cell Line; Cell Nucleus; Cytochrome P-450 Enzyme System; Feedback, Physiological; G1 Phase; Gene Expression Regulation; Humans; Left-Right Determination Factors; Neurons; Nuclear Proteins; Nuclear Receptor Interacting Protein 1; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tretinoin

Human embryonal carcinoma (EC) cells represent the stem cells of testicular germ cell tumours (TGCTs) and are morphologically, antigenically and functionally related to the stem cells of early mammalian embryos. Despite the large capacity for differentiation displayed by TGCT stem cells, little is known of the factors controlling their developmental potency. We have analyzed the differentiation elicited in NT2D1 human embryonal carcinoma (EC) cells by Bone Morphogenetic Proteins (BMPs) and compared it with that elicited by retinoic acid (RA). We have found that while RA induced expression of neuronal, endodermal and epithelial markers in NT2D1 human EC cells, treatment with BMPs resulted in a predominantly epithelial phenotype. We also provide evidence to suggest that at least some of the effects elicited by RA in human EC cells might be mediated through RA-induced expression of BMP-7. Thus BMPs may play an important role in specifying the type of differentiation arising from human multipotent stem cells. The manipulation of BMP signalling in human embryonic multipotent stem cells may therefore prove a useful approach in attempts to generate specific differentiated cell types in vitro, and loss of the malignant and/or transformed phenotype.

Topics: Blotting, Northern; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Differentiation; Cells, Cultured; Endoderm; Epithelium; Humans; Immunohistochemistry; Neurons; Phenotype; RNA; Time Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tretinoin

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.

Topics: Animals; Base Sequence; beta-Galactosidase; Binding Sites; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Chloramphenicol O-Acetyltransferase; Drosophila; Gene Expression Regulation, Neoplastic; Genes, Reporter; Molecular Sequence Data; Plasmids; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Saccharomyces cerevisiae; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta (TGF-beta) superfamily have focused on Smad proteins, but have paid little attention to mitogen-activated protein (MAP) kinase cascades. Here we demonstrate that growth/differentiation factor-5 (GDF-5), but neither bone morphogenetic protein-2 (BMP-2) nor TGF-beta1, fully promotes the early phase of the chondrogenic response by inducing cellular condensation followed by cartilage nodule formation in a mouse chondrogenic cell line, ATDC5. We investigated which, if any, of the three major types of MAP kinase plays a functional role in the promotion of chondrogenesis induced by GDF-5. GDF-5 induced phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK). The phosphorylation of p38 MAP kinase was also induced by BMP-2 and TGF-beta1. An inhibitor of p38 and p38 beta MAP kinase, SB202190, showed complete inhibition of cartilage nodule formation but failed to affect alkaline phosphatase (ALP) activity induced by GDF-5. Expression of the type II collagen gene, a hallmark of chondrogenesis in vertebrates, was also induced by GDF-5 treatment and strongly suppressed by SB202190. On the other hand, although an inhibitor of MAP/ERK kinase, PD98059, inhibited the rapid phosphorylation of ERK by GDF-5, it inhibited neither ALP activity nor cartilage nodule formation induced by GDF-5. These results strongly suggest that the p38 MAP kinase cascade is involved in GDF-5 signaling pathways and that a role of the p38 MAP kinase pathway is necessary over a longer period to promote chondrogenesis in ATDC5 cells.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Embryonal; Cartilage; Cell Differentiation; Cell Size; Chondrogenesis; Collagen; Dose-Response Relationship, Drug; Enzyme Induction; Growth Differentiation Factor 5; Growth Substances; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type-beta receptors. Here, we show that the type II receptor (TbetaR-II) gene is activated at the transcriptional level when EC cells differentiate. We show that the differentiated cells, but not the parental EC cells, express transcripts for TbetaR-II. In addition, the expression of TbetaR-II promoter/reporter gene constructs are elevated dramatically when EC cells differentiate and we identify at least two positive and two negative regulatory regions in the 5' flanking region of the TbetaR-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the TbetaR-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the TbetaR-II promoter/reporter gene constructs when ATF-1 is overexpressed in EC-derived differentiated cells. Equally important, we identify a negative regulatory element in a 53-base pair region that had previously been shown to inhibit strongly the expression of TbetaR-II promoter/reporter gene constructs. Specifically, we demonstrate that this region, which contains an inverted CCAAT box motif, binds the transcription factor complex NF-Y (also referred to as CBF) in vitro. Furthermore, expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding by NF-Y, enhances TbetaR-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the TbetaR-II gene.

Topics: Activating Transcription Factor 1; Animals; Base Sequence; Carcinoma, Embryonal; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Line; DNA; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Mice; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate the localization of transforming growth factors (TGF-beta 1, -beta 2 and -beta 3) and their receptors (TGF-beta RI and RII).. The study included 26 paraffin-embedded tissues from human testicular neoplasms: 15 seminomas, 2 embryonal carcinomas, 1 immature teratoma, 4 immature teratomas with embryonal carcinoma, 1 immature teratoma with seminoma, 1 seminoma with embryonal carcinoma and 2 gonadal stromal tumors (Leydig cell tumors).. TGF-beta 1 immunoreactivity was cytoplasmic and was expressed in 22 (84.6%), TGF-beta 2 in 20 (77%), TGF-beta 3 in 11 (42.3%), TGF-beta-RI in 21 (80.8%) and TGF-beta-RII in 18 (69.2%) of the 26 neoplasms. The percentage of positive immunostained cells and the intensity of staining were significantly higher in tumor than in peritumor nonneoplastic testis. In the peritumor nonneoplastic testis, Leydig, Sertoli and germ cells coexpressed both the three TGF-beta isoforms and TGF-beta-RI and RII. The myoepithelial cells of the seminiferous tubules showed immunoreactivity for TGF-beta RI and RII but not for TGF-beta s. In tumor testis areas the pattern of TGF-beta and TGF-beta receptor expression and distribution varied according to the histologic type of testicular tumor. Seminomas showed a diffuse pattern of TGF-beta immunoreactivity, whereas immature teratomas had focal and patchy distribution. In teratomas, differentiated structures contained more TGF-beta s than undifferentiated structures.

Topics: Activin Receptors, Type I; Adolescent; Adult; Aged; Carcinoma, Embryonal; Germinoma; Humans; Immunohistochemistry; Leydig Cell Tumor; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Seminoma; Teratoma; Testicular Neoplasms; Transforming Growth Factor beta

Osteogenic protein-1 (OP-1, BMP-7), a bone morphogenetic protein in the transforming growth factor-beta superfamily, induces endochondral bone formation in vivo, but the mechanism of action of OP-1 in osteogenesis is not yet established. Three murine clonal cell lines in different stages of differentiation exhibit graded responses to recombinant human OP-1: the mouse embryonal carcinoma ATDC5 cell, with potential for chondroblastic differentiation; the osteoblast-like MC3T3-E1 cell derived from mouse calvaria; and the multipotent fibroblastic C3H10T1/2 cell derived from mouse embryo connective tissue. We show that OP-1 acts on early stage mesenchymal progenitor cells (ATDC5, C3H10T1/2) to induce chondroblastic differentiation, while OP-1 strongly enhances the osteoblastic phenotype of committed osteoblasts (MC3T3-E1), possibly explaining its induction of the endochondral ossification cascade in vivo. Markers of osteoblastic, chondroblastic, and adipocytic differentiation are compared. OP-1 is strongly mitogenic for ATDC5, showing dose-dependent (2.5-80 ng/ml) induction of Alcian blue staining, alkaline phosphatase activity, and mRNA expression for collagen types II and IX, and matrix Gla protein. MC3T3-E1 cells do not proliferate or stain with Alcian blue in response to OP-1, but express elevated levels of alkaline phosphatase and osteocalcin. While low-dose OP-1 treatment of C3H10T1/2 induces only adipocyte-like cells filled with lipid droplets, a high dose (500 ng/ml) causes the same cells to also exhibit chondrocytic properties. Thus, OP-1 can induce differentiation along elements of the endochondral ossification pathway according to the stage and potential of the target cell.

Topics: Adipocytes; Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cartilage; Cell Differentiation; Clone Cells; Collagen; Fibroblasts; Gene Expression; Glycerolphosphate Dehydrogenase; Glycosaminoglycans; Humans; Mice; Osteoblasts; Osteocalcin; Osteogenesis; Proteins; RNA, Messenger; Transforming Growth Factor beta

Retinoic acid (RA) affects the response of many cells to growth factors, including the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-beta, family of growth factors, originally identified by their bone-inducing activities. Their widespread expression suggests many roles other than that in osteogenesis. Because RA modulates the cell's response to growth factors, this may be a means by which the retinoids exert some of their known teratogenic effects. One such cellular response may be apoptosis. While apoptosis is required for normal development, the location and timing of its induction must be carefully controlled. Recently, several TGF-beta family members have been implicated in the induction of apoptosis in certain cell types. We show here, using P19 embryonal carcinoma cells, that the combination of RA and BMP2 or BMP4 synergistically induces apoptosis in 40% of the population within 24 hr. In contrast, RA alone induces apoptosis in only 10-15% of the population and each of the BMPs alone minimally induces apoptosis. Apoptosis depends on the dose of both the RA and the BMP as well as on new protein synthesis. Further, the induction of apoptosis prevents the formation of fully differentiated neurons and glial cells and instead leads to primarily smooth muscle cell differentiation. These results suggest that some of the malformations caused by retinoids may be due to the induction of inappropriate apoptosis in cells exposed to BMPs.

Topics: Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Drug Synergism; Flow Cytometry; Keratolytic Agents; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Embryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF-beta 2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF-beta 2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF-beta 2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF-beta 2 gene in the murine parietal endoderm-like cell line, PYS-2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF-beta 2 gene in PYS-2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF-beta 2 gene reduces significantly the activity of the TGF-beta 2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Gene Expression Regulation, Developmental; Genes, Regulator; Genes, Reporter; Humans; Mice; Molecular Sequence Data; Point Mutation; Promoter Regions, Genetic; Stem Cells; Transcription Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF-beta 2 gene and identified a CRE/ATF-like motif in the TGF-beta 2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF-like motif. To test this possibility, we performed gel mobility shift analysis using double-stranded oligodeoxynucleotides containing the TGF-beta 2 CRE/ATF-like motif and nuclear extracts prepared from F9 EC cells and F9-differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF-1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects AFT-1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF-like motif. Together, our studies identify a likely role for the CRE/ATF-like motif in the regulation of TGF-beta 2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed.

Topics: Activating Transcription Factor 1; Animals; Base Sequence; Carcinoma, Embryonal; Cell Differentiation; Cell Line; Cell Nucleus; DNA Probes; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Mice; Molecular Sequence Data; Nuclear Proteins; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Osteogenic protein-1 (OP-1) is a member of the transforming growth factor-beta super family closely related to the bone morphogenetic proteins and also known as bone morphogenetic protein-7. Other members of this family of growth factors influence cell differentiation as well as cell growth in a number of systems. The Drosophila homolog encoded by the decapentaplegic locus is involved in dorsal-ventral pattern formation during embryogenesis, whereas the expression of several bone morphogenetic proteins including OP-1 is developmentally regulated in mammalian embryos.. The effect of recombinant human OP-1 on the proliferation and differentiation of an established pluripotent human embryonal carcinoma (EC) cell line, NTERA2, and three nullipotent human EC cell lines, 2102Ep, 833KE and TERA-1, was investigated. These cells were grown under reduced serum conditions, and differentiation was monitored by morphology and expression of marker antigens.. OP-1 inhibited proliferation of NTERA2 and induced their differentiation, marked by changes in cellular morphology, the loss of EC cell antigens (SSEA3, SSEA4, the liver isozyme of alkaline phosphatase), and the appearance of new antigens, notably SSEA1 and class 1 major histocompatibility complex antigens. These changes were irreversible and did not involve significant cell degeneration or cell death. The OP-1-induced differentiation of NTERA2 appeared distinct from that induced by either retinoic acid or hexamethylene bisacetamide. Nevertheless, OP-1 did induce the homeobox gene, HOXA1. By contrast, OP-1 elicited only a limited and partial response from the nullipotent EC cell lines.. Our results suggest that pluripotent human EC cells differentiate in response to OP-1 and that this factor can modulate the differentiation induced by retinoic acid. Like other members of the transforming growth factor-beta super family, OP-1 might play an inductive role in the early embryo. The results also suggest a possible therapeutic value for OP-1 in the treatment of some germ cell tumors.

Topics: Acetamides; Antineoplastic Agents; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Gene Expression; Genes, Homeobox; Humans; Proteins; Recombinant Proteins; RNA; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured