雌激素诱发大鼠原位与移植垂体催乳素瘤中催乳素基因与TGFα和TGF …

利用本实验室建立的１７β－雌二醇诱致Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ（ＳＤ）大鼠原位垂体和异体移植于肾囊的垂体同时形成催乳素瘤的动物模型,采用Ｎｏｒｔｈｅｍ印迹杂交方法,我们观察了Ｅ２长期作用（１２０ｄ）后诱发的原位与移植垂体ＰＲＬ瘤中ＰＲＬ基因和两种转化生长因子ＴＧＦα和ＴＧＦβ１基因表达水平的改变。结果表明：在Ｅ２长期作用后,原位垂体与异体移植于肾囊,从而远离下丘脑的垂体均可形成垂体ＰＲＬ瘤;原位     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response.

Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family. Intracellular proteins that associate with these receptors are likely to play an important role in signaling. 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI. We now describe the identification of S. mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay. S. mansoni eIF2 alpha also interacts with human TGF beta receptors. Strongest association was demonstrated with kinase inactive receptors, particularly the type II TGF beta receptor (T beta RII). Both T beta RI and T beta RII phosphorylate eIF2 alpha in vitro, at sites other than the previously described eIF2 alpha phosphorylation sites. EIF2 alpha also modulates signaling by TGF beta receptors; however, in contrast to 14-3-3 epsilon, eIF2 alpha overexpression inhibits the TGF beta-driven response. These data suggest a novel function for eIF2 alpha in the TGF beta signaling pathway. In addition, we have demonstrated an independent interaction between eIF2 alpha and 14-3-3 epsilon. Coexpression of 14-3-3 epsilon with eIF2 alpha leads to the abrogation of the inhibitory effect of eIF2 alpha on TGF beta-mediated signaling. The interaction of these two regulatory proteins with each other and with the TGF beta receptors and their relative expression levels are likely to be important in fine-tuning the regulation of TGF beta signal transduction.     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells.

Transforming growth factor-beta (TGF beta) is a potent growth inhibitor in most epithelial cells. We evaluated the effects of norethindrone (which in combination with estrogen is commonly used in oral contraceptives) and other progestins [medioxyprogesterone acetate (MPA) and R5020, which are not used in oral contraceptives] on cell growth and the expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs in MCF-7 human breast cancer cells. Growth of MCF-7 cells was stimulated by norethindrone (10(-8)-10(-5) M), with maximal growth stimulation at 10(-7) M norethindrone after 7 days of treatment. However, the growth of MCF-7 cells was not affected by MPA (10(-8) M) or R5020 (10(-8) M). Treatment with the antiestrogen 4-hydroxytamoxifen at a concentration of 10(-7) M blocked the growth stimulation induced by norethindrone. The norethindrone-induced growth stimulation was accompanied by a dramatic decrease in TGF beta 2 and TGF beta 3 mRNA levels, whereas the level of TGF beta 1 mRNA was not affected by any of the compounds tested. In addition, treatment with MPA or R5020 did not affect TGF beta 2 and TGF beta 3 mRNA levels. The inhibitory effect of norethindrone on TGF beta 2 and TGF beta 3 mRNA levels could be blocked by the addition of 10(-7) M 4-hydroxytamoxifen. Norethindrone as well as estradiol decreased estrogen receptor mRNA levels and increased progesterone receptor mRNA levels. This is the first report which demonstrates that norethindrone stimulates estrogen-responsive human breast cancer cell growth and inhibits the expression of TGF beta 2 and TGF beta 3 mRNAs. These results suggest that the differential regulation of TGF beta expression by norethindrone may be at least partly responsible for the growth stimulation induced by norethindrone. Thus, the norethindrone component of some oral contraceptives may be sufficiently estrogenic to facilitate the development of breast cancer.     

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.     

Developmental expression of the TGF beta s in the mouse cochlea.

Mice with targeted disruption of the TGF beta 2 gene display defects in epithelial-mesenchymal tissue interactions in several tissues including the developing cochlea. Specifically, the region of the spiral limbus and the overlying interdental cells, structures putatively involved in endolymphatic fluid homeostasis, display morphogenetic abnormalities. These findings prompted us to explore the pre-natal and post-natal expression of all three mammalian TGF beta genes in the developing mouse inner ear. TGF beta 2 mRNA expression was identified throughout the cochlear epithelium at all of the developmental stages examined. TGF beta 3 mRNA expression was identified in the mesenchymal tissues of the cochlea surrounding the otic epithelium. We found no evidence for compensation by the other two TGF beta isoforms in the cochleas of the TGF beta 2 mutants.     

Interactions between retinoids and TGF beta s in mouse morphogenesis.

Using immunocytochemical methods we describe the distribution of different TGF beta isoforms and the effects of excess retinoic acid on their expression during early mouse embryogenesis (8 1/2 - 10 1/2 days of development). In normal embryos at 9 days, intracellular TGF beta 1 is expressed most intensely in neuroepithelium and cardiac myocardium whereas extracellular TGF beta 1 is expressed in mesenchymal cells and in the endocardium of the heart. At later stages, intracellular TGF beta 1 becomes very restricted to the myocardium and to a limited number of head mesenchymal cells; extracellular TGF beta 1 continues to be expressed widely in cells of mesenchymal origin, particularly in head and trunk mesenchyme, and also in endocardium. TGF beta 2 is widely expressed at all stages investigated while TGF beta 3 is not expressed strongly in any tissue at the stages examined. Exposure of early neural plate stage embryos to retinoic acid caused reduced expression of TGF beta 1 and TGF beta 2 proteins but had no effect on TGF beta 3. Intracellular TGF beta 1 expression was reduced in all tissues except in the myocardium, while extracellular TGF beta 1 was specifically reduced in neuroepithelium and cranial neural crest cells at early stages. TGF beta 2 was reduced in all embryonic tissues. The down-regulation of intracellular TGF beta 1 was observed up to 48 hours after initial exposure to retinoic acid while some down-regulation of TGF beta 2 was still seen up to 60 hours after initial exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.     

Turning it up a Notch: cross-talk between TGF beta and Notch signaling

Signaling through both the transforming growth factor beta (TGF beta) superfamily of growth factors and Notch play crucial roles during embryonic pattern formation and cell fate determination. Although both pathways are able to exert similar biological responses in certain cell types, a functional interaction between these two signaling pathways has not been described. Now, three papers provide evidence of both synergy and antagonism between TGF beta and Notch signaling. These reports describe a requirement for Notch signal transducers in TGF beta- and BMP-induced expression of Notch target genes, as well as in BMP-controlled cell differentiation and migration. These papers uncover a direct link between the Notch and TGF beta pathways and suggest a critical role for Notch in some of the biological responses to TGF beta family signaling.     

Differential expression of TGF beta isoforms in murine palatogenesis

We have studied the expression of genes encoding transforming growth factors (TGFs) beta 1, beta 2 and beta 3 during development of the secondary palate in the mouse from 11.5 to 15.5 days postcoitum using in situ hybridisation. The RNA detected at the earliest developmental stage is TGF beta 3, which is localised in the epithelial component of the vertical palatal shelf. This expression continues in the horizontal palatal shelf, predominantly in the medial edge epithelium, and is lost as the epithelial seam disrupts, soon after palatal shelf fusion. TGF beta 1 RNA is expressed with the same epithelial pattern as TGF beta 3, but is not detectable until the horizontal palatal shelf stage. TGF beta 2 RNA is localised to the palatal mesenchyme underlying the medial edge epithelia in the horizontal shelves and in the early postfusion palate. The temporal and spatial distribution of TGF beta 1, beta 2 and beta 3 RNAs in the developing palate, together with a knowledge of in vitro TGF beta biological activities, suggests an important role for TGF beta isoforms in this developmental process.     

Differential expression of genes encoding TGFs beta 1, beta 2, and beta 3 during murine palate formation.

Transforming growth factor beta 1 (TGF beta 1) has been shown to have multiple effects on primary cultures of palate-derived cell types. We report the analysis, by in situ hybridization, of RNA expression for three different TGF beta isoforms (TGF beta 1, beta 2, and beta 3) during murine embryonic palate development. Differential expression of the three TGF beta genes is seen in the palatal shelves in mesenchymal and epithelial cells known to be involved in the morphogenesis of this organ. Taken together, these results suggest that the TGF beta s act as endogenous factors involved in the formation of the mammalian palate.