Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone.

Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor beta. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.     

Regulation of intestinal epithelial cell growth by transforming growth factor type beta.

A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.     

Vgr-1, a mammalian gene related to Xenopus Vg-1, is a member of the transforming growth factor beta gene superfamily.

The transforming growth factor beta (TGF-beta)-related products of the Xenopus Vg-1 and Drosophila decapentaplegic (DPP) genes have been implicated in the control of growth and differentiation during embryogenesis. We have isolated a mouse cDNA, Vgr-1, that encodes a polypeptide structurally related to Xenopus Vg-1. Sequence comparisons indicate that the Vgr-1 protein belongs to a family of DPP-like gene products within the TGF-beta superfamily. The levels of Vgr-1 RNA were determined in embryos and tissues isolated at various stages of development. A 3.5-kilobase mRNA increases throughout development and into adulthood in many tissues and in F9 teratocarcinoma cells differentiating into endoderm in response to retinoic acid and cAMP. The amino acid homologies and patterns of expression suggest that, like the DPP gene product, Vgr-1 plays a role at various stages of development.     

Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene.

Transforming growth factor beta (TGF-beta) isoforms inhibit the growth of many cell types and block progression of the cell cycle by inhibiting events in late G1 phase. The retinoblastoma gene product, RB, also has properties of a cell-cycle regulatory factor. It remains underphosphorylated in the presence of TGF-beta and has been shown to repress the activity of the c-fos promoter, resulting in inhibition of transit through the cell cycle. These observations led us to examine effects of human RB on the expression of the human TGF-beta 1 gene. Using chimeric TGF-beta 1 promoter-chloramphenicol acetyltransferase gene constructs, we show that RB induces TGF-beta 1 gene expression in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma cells but represses its expression in NIH 3T3 and AKR-2B mouse cells. Several sequences homologous to the c-fos RB control element were identified in the TGF-beta 1 promoter. These results demonstrate that human RB can regulate TGF-beta 1 gene expression negatively or positively depending on the cell type.     

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.     

Expression and secretion of type beta transforming growth factor by activated human macrophages.

Alveolar macrophages activated with concanavalin A and peripheral blood monocytes activated with lipopolysaccharide secrete type beta transforming growth factor (TGF-beta). There is minimal TGF-beta secretion in unactivated monocytes, even though TGF-beta mRNA is expressed in these cells at a level similar to that in activated, lipopolysaccharide-treated cultures. U937 lymphoma cells, which have monocytic characteristics, also express mRNA for TGF-beta. Freshly isolated monocytes, both control and lipopolysaccharide-treated, secrete an acid-labile binding protein that inhibits TGF-beta action. We conclude the following: (i) that expression of TGF-beta mRNA is unrelated to monocyte activation, (ii) that secretion of TGF-beta is induced by monocyte activation, and (iii) that cosecretion of TGF-beta and its monocyte/macrophage-derived binding protein may modulate growth factor action. In contrast, monocytic expression of other growth factor genes, such as the B chain of platelet-derived growth factor, is not constitutive and requires activation.     

Seminalplasmin: recent evolution of another member of the neuropeptide Y gene family.

Seminalplasmin, the major basic protein of bull semen, an important regulator of calcium transport in bovine sperm and a positive modulator of the zona pellucida-induced acrosome reaction, is shown to be a recently created member of the neuropeptide Y gene family. Sequence analysis of the bovine peptide YY-pancreatic polypeptide gene cluster reveals an unexpected and extensive homology between seminalplasmin and the neuropeptide Y gene family, at the level of both gene structure and primary amino acid and nucleotide sequences. The extremely high degree of homology to the peptide YY gene, in both coding and especially noncoding regions, suggests that the seminalplasmin gene has arisen by a very recent gene duplication of the bovine peptide YY gene. Despite the more than 95% nucleotide sequence identity, a few specific mutations in the seminalplasmin gene have resulted in both the loss of the amino- and carboxyl-terminal cleavage sites characteristic of all other members of the neuropeptide Y family and the acquisition of a function apparently unrelated to the neurotransmitter/endocrine role of peptide YY.     

Modulation of type beta transforming growth factor activity in bone cultures by osteotropic hormones.

Type beta transforming growth factor (TGF-beta) activity was found in conditioned medium harvested from fetal rat and neonatal mouse calvariae by using the anchorage-independent growth of normal rat kidney fibroblasts as an indicator system. Calvariae incubated with parathyroid hormone, 1,25-dihydroxyvitamin D3, and interleukin 1, all factors that stimulate bone resorption showed a concentration-dependent increase in TGF-beta activity in the culture medium. Increases in TGF-beta activity persisted undiminished for 48 hr after removal of these factors. The increases in TGF-beta activity from the resorbing bone cultures were relatively greater than the increases in bone resorption. Calcitonin inhibition of bone resorption correlated with a decrease in TGF-beta activity. Thus, agents that modulate bone resorption also affect TGF-beta activity in the bone culture medium. Changes in local concentrations of TGF-beta activity by osteotropic hormones may be important in the regulation of normal bone remodeling.     

转化生长因子β1-509C／T基因多态性与大肠癌的关系

目的研究转化生长因子β1（TGF—β1）基因-509位点C／T的多态性,并探讨其与大肠癌易感性的关系。方法采用聚合酶链反应-限制性片段长度多态性方法,检测70例大肠癌组和102例对照组TGF—β1基因-509位点C／T等位基因及基因型分布,并对该基因多态性与大肠癌临床病理特征之间的关系进行分析。同时采用酶联免疫吸附试验（ELISA）检测大肠癌组和对照组血清TGF—β1水平。结果TGF—β1等位基因频率及基因型频率在大肠癌组和对照组的总体分布比较无显著性差异。大肠癌组按Dukes分期后,发现DukesC＋D期大肠癌患者-509CT／TT基因型频率明显高于DukesA＋B期患者（64．9％比39．4％,P=0．033,OR=2．840,95％CI：1．075～7．501）。DukesC＋D期大肠癌患者与DukesA＋B期相比,-509T等位基因频率有增高趋势（41．9％比27．3％,P=0．07,OR=1．922,95％CI：0．943～3．917）,但差异无显著性。大肠癌患者血清TGF—β1水平显著高于对照组。结论TGF—β1-509位点基因多态性与大肠癌无关,可能与大肠癌临床分期有关。     

Purification and initial characterization of a type beta transforming growth factor from human placenta.

A polypeptide transforming growth factor (TGF) that induces anchorage-dependent rat kidney fibroblasts to grow in soft agar has been isolated from human placenta and purified to homogeneity. This polypeptide is classified as a type beta TGF because it does not compete with epidermal growth factor (EGF) for membrane receptor sites but does require EGF for induction of anchorage-independent growth of indicator cells. Purification of this peptide was achieved by acid/ethanol extraction of the placenta, followed by gel filtration, cation exchange, and HPLC of the acid-soluble proteins. Homogeneity of the TGF-beta from the final column was shown by its constant specific activity and amino acid composition across the peak of soft agar colony-forming activity and by its migration as a single band at Mr 23,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. Under reducing conditions, the protein migrated on a gel as a single band at Mr 13,000. The purified placental TGF-beta caused half-maximal growth stimulation of indicator cells in soft agar at 64-72 pg/ml (3 pM) in the presence of EGF at 2 ng/ml (0.34 nM).     

Differential effects of transforming growth factor type beta on the growth and function of adrenocortical cells in vitro.

Transforming growth factor type beta (TGF-beta) suppresses basal as well as corticotropin (ACTH)-stimulated steroid formation by bovine adrenocortical cells in culture. The effect is dose dependent and is not accompanied by any change in adrenocortical cell growth. The minimum effective dose of TGF-beta is 4 X 10(-13) M (10 pg/ml), and maximal inhibition is observed at a concentration of 4 X 10(-11) M (1 ng/ml). A 16- to 20-hr incubation with TGF-beta is required to decrease steroidogenesis, and 12-18 hr are required before cells treated with TGF-beta recover complete responsiveness to corticotropin. Increases in cAMP mediated by corticotropin, forskolin, and isobutylmethylxanthine are not modified by the addition of TGF-beta; thus adenylate cyclase activity is unaffected by TGF-beta. Although TGF-beta inhibits the formation of all of the delta 4-steroids measured (including cortisol, corticosterone, aldosterone, and androstenedione), its effect can be completely reversed by the addition of 25-hydroxycholesterol, pregnenolone, or progesterone to the cells. In contrast, the addition of low density lipoprotein has no effect suggesting that TGF-beta targets the conversion of cholesterol precursors to cholesterol. The results demonstrate a highly potent effect of TGF-beta on the differentiated function of the adrenocortical cell. The inhibition of steroidogenesis can be dissociated from any effect on cell proliferation, and it occurs distal to the formation of cAMP but proximal to the formation of cholesterol. The results suggest that in the adrenal, TGF-beta or TGF-beta-like proteins may be playing an important role in modifying the differentiated state of the adrenocortical cell.     

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.     

Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.

The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.     

BSC-1 growth inhibitor/type beta transforming growth factor is a strong inhibitor of thymocyte proliferation.

Growth inhibitor/type beta transforming growth factor purified from BSC-1 cells and human platelets is shown to strongly inhibit the proliferation of Con A-stimulated mouse thymocytes. The inhibition can be achieved with growth inhibitor/type beta transforming growth factor concentrations approximately equal to 1/10th those necessary to inhibit keratinocyte cultures. The inhibitory effect in thymocyte cultures can be reversed by the addition of interleukin 2. These findings suggest that growth inhibitor/type beta transforming growth factor is a naturally occurring immunoregulator.     

Coronary vessel development is dependent on the type III transforming growth factor beta receptor

Transforming growth factor (TGF)beta receptor III (TGFbetaR3), or beta-glycan, binds all 3 TGFbeta ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGFbetaR1, TGFbetaR2). TGFbetaR3 facilitates signaling via TGFbetaR1/TGFbetaR2 but also has been suggested to play a unique and nonredundant role in TGFbeta signaling. Targeted deletion of Tgfbr3 revealed a requirement for Tgfbr3 during development of the coronary vessels. Coronary vasculogenesis is significantly impaired in null mice, with few vessels evident and numerous, persistent blood islands found throughout the epicardium. Tgfbr3-null mice die at embryonic day 14.5, the time when functional coronary vasculature is required for embryo viability. However, in null mice nascent coronary vessels attach to the aorta, form 2 coronary ostia, and initiate smooth muscle recruitment by embryonic day 14. Analysis of earlier developmental stages revealed defects in the epicardium. At embryonic day 13.5, these defects include an irregular and hypercellular epicardium with abundant subepicardial mesenchyme and a thin compact zone myocardium. Tgfbr3-null mice also displayed other defects in coronary development, including dysmorphic and distended vessels along the atrioventricular groove and subepicardial hemorrhage. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild-type littermates. These data demonstrate a requirement for Tgfbr3 during coronary vessel development that is essential for embryonic viability.