Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors

The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.

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The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4280-4285.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4280-4285

Author(s):

A Bhushan ◽

H Y Lin ◽

H F Lodish ◽

C R Kintner

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Full Range ◽

Type Ii ◽

Tgf Beta ◽

Mesodermal Cell ◽

Xenopus Embryos ◽

Growth Factor Beta

The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells.

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Dcdc42 acts in TGF-beta signaling during Drosophila morphogenesis: distinct roles for the Drac1/JNK and Dcdc42/TGF-beta cascades in cytoskeletal regulation

Journal of Cell Science ◽

10.1242/jcs.112.8.1225 ◽

1999 ◽

Vol 112 (8) ◽

pp. 1225-1235 ◽

Cited By ~ 13

Author(s):

M.G. Ricos ◽

N. Harden ◽

K.P. Sem ◽

L. Lim ◽

W. Chia

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Dorsal Closure ◽

Tgf Beta ◽

Epithelial Migration ◽

Amino Terminal ◽

Kinase Cascade ◽

Continuous Sheet ◽

Growth Factor Beta

During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted transforming-growth-factor-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative transforming-growth-factor-beta-like receptors Thickveins and Punt activates a transforming-growth-factor-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the transforming-growth-factor-beta-like signaling pathway can disrupt dorsal closure. Our findings show that although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the transforming-growth-factor-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK.

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Transforming growth factor beta inhibits endomitosis in the Dami human megakaryocytic cell line

Blood ◽

10.1182/blood.v76.3.533.bloodjournal763533 ◽

1990 ◽

Vol 76 (3) ◽

pp. 533-537 ◽

Cited By ~ 1

Author(s):

SM Greenberg ◽

C Chandrasekhar ◽

DE Golan ◽

RI Handin

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cell Line ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Mrna Levels ◽

Dependent Manner ◽

Tgf Beta ◽

Growth Factor Beta ◽

Megakaryocytic Cell

Megakaryocyte development is a carefully controlled process that is at least partially regulated by cytokines. Previous investigations of megakaryocyte development have focused primarily on defining growth factors that induce or enhance differentiation. In this study we demonstrate that a specific cytokine, transforming growth factor beta 1 (TGF beta 1), inhibits the phorbol myristate acetate (PMA)-induced differentiation of the Dami human megakaryocytic cell line. The addition of purified platelet TGF beta 1 inhibits PMA-induced endomitosis in a dose-dependent manner. Inhibition of endomitosis occurs with as little as 0.4 pmol/L TGF beta 1, is half-maximal at 6.4 pmol/L, and is maximal between 40 and 200 pmol/L TGF beta 1. Inhibition does not require other growth factors or nonmegakaryocytic cells. Removal of TGF beta 1 from the cultures decreases inhibition, suggesting that the continuous presence of TGF beta 1 is required and that its effects are reversible. This effect occurs even though the Dami cells constitutively express TGF beta 1 messenger RNA (mRNA) and the TGF beta 1 mRNA levels are increased by PMA. TGF beta 1 also has been shown to inhibit endomitosis during short-term culture of primary human megakaryocytes. These results suggest a model in which negative as well as positive regulatory factors modulate a critical stage of megakaryocyte development.

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The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4280 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4280-4285 ◽

Cited By ~ 20

Author(s):

A Bhushan ◽

H Y Lin ◽

H F Lodish ◽

C R Kintner

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Full Range ◽

Type Ii ◽

Tgf Beta ◽

Mesodermal Cell ◽

Xenopus Embryos ◽

Growth Factor Beta

The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells.

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Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.661 ◽

1989 ◽

Vol 108 (2) ◽

pp. 661-669 ◽

Cited By ~ 280

Author(s):

N L Thompson ◽

K C Flanders ◽

J M Smith ◽

L R Ellingsworth ◽

A B Roberts ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Histochemical Staining ◽

Tgf Beta ◽

Amino Terminal ◽

Neonatal Mice ◽

Glandular Cells ◽

Growth Factor Beta ◽

Immunohistochemical Techniques

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

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Transforming growth factor beta inhibits endomitosis in the Dami human megakaryocytic cell line

Blood ◽

10.1182/blood.v76.3.533.533 ◽

1990 ◽

Vol 76 (3) ◽

pp. 533-537 ◽

Cited By ~ 24

Author(s):

SM Greenberg ◽

C Chandrasekhar ◽

DE Golan ◽

RI Handin

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cell Line ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Mrna Levels ◽

Dependent Manner ◽

Tgf Beta ◽

Growth Factor Beta ◽

Megakaryocytic Cell

Abstract Megakaryocyte development is a carefully controlled process that is at least partially regulated by cytokines. Previous investigations of megakaryocyte development have focused primarily on defining growth factors that induce or enhance differentiation. In this study we demonstrate that a specific cytokine, transforming growth factor beta 1 (TGF beta 1), inhibits the phorbol myristate acetate (PMA)-induced differentiation of the Dami human megakaryocytic cell line. The addition of purified platelet TGF beta 1 inhibits PMA-induced endomitosis in a dose-dependent manner. Inhibition of endomitosis occurs with as little as 0.4 pmol/L TGF beta 1, is half-maximal at 6.4 pmol/L, and is maximal between 40 and 200 pmol/L TGF beta 1. Inhibition does not require other growth factors or nonmegakaryocytic cells. Removal of TGF beta 1 from the cultures decreases inhibition, suggesting that the continuous presence of TGF beta 1 is required and that its effects are reversible. This effect occurs even though the Dami cells constitutively express TGF beta 1 messenger RNA (mRNA) and the TGF beta 1 mRNA levels are increased by PMA. TGF beta 1 also has been shown to inhibit endomitosis during short-term culture of primary human megakaryocytes. These results suggest a model in which negative as well as positive regulatory factors modulate a critical stage of megakaryocyte development.

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Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.653 ◽

1989 ◽

Vol 108 (2) ◽

pp. 653-660 ◽

Cited By ~ 242

Author(s):

K C Flanders ◽

N L Thompson ◽

D S Cissel ◽

E Van Obberghen-Schilling ◽

C C Baker ◽

...

Keyword(s):

Amino Acids ◽

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Human Colon ◽

Matrix Proteins ◽

Differential Staining ◽

Tgf Beta ◽

Amino Terminal ◽

Growth Factor Beta

We have localized transforming growth factor-beta (TGF-beta) in many cells and tissues with immunohistochemical methods, using two polyclonal antisera raised to different synthetic preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF-beta 1. These two antibodies give distinct staining patterns; the staining by anti-CC(1-30) is intracellular. This differential staining pattern is consistently observed in several systems, including cultured tumor cells; mouse embryonic, neonatal, and adult tissues; bovine fibropapillomas; and human colon carcinomas. The extracellular staining by anti-CC(1-30) partially resembles that seen with an antibody to fibronectin, suggesting that extracellular TGF-beta may be bound to matrix proteins. The intracellular staining by anti-LC(1-30) is similar to that seen with two other antibodies raised to peptides corresponding to either amino acids 266-278 of the TGF-beta 1 precursor sequence or to amino acids 50-75 of mature TGF-beta 1, suggesting that anti-LC(1-30) stains sites of TGF-beta synthesis. Results from RIA and ELISAs indicate that anti-LC(1-30) and anti-CC(1-30) recognize different epitopes of this peptide and of TGF-beta 1 itself.

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