Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis.
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In response to different extracellular signals, Rel/NF-kappaB transcription factors are critical regulators of apoptosis in a variety of cell types. Here we show that in normal B and T cells, expression of the Bcl-2 prosurvival homolog, A1, is rapidly induced in a Rel-dependent manner by mitogens. In B-cell lines derived from c-rel-/- mice, which like primary cells lacking Rel undergo apoptosis in response to antigen receptor ligation, constitutive expression of an A1 transgene inhibits this pathway to cell death. These findings are the first to show that Rel/NF-kappaB regulates physiologically the expression of a Bcl-2-like protein that is critical for the control of cell survival during lymphocyte activation. (+info)
Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor.
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The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis. (+info)
WNT signaling in the control of hair growth and structure.
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Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways. (+info)
Wingless signaling leads to an asymmetric response to decapentaplegic-dependent signaling during sense organ patterning on the notum of Drosophila melanogaster.
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Wnt and Decapentaplegic cell signaling pathways act synergistically in their contribution to macrochaete (sense organ) patterning on the notum of Drosophila melanogaster. The Wingless-signaling pathway was ectopically activated by removing Shaggy activity (the homologue of vertebrate glycogen synthase kinase 3) in mosaics. Proneural activity is asymmetric within the Shaggy-deficient clone of cells and shows a fixed "polarity" with respect to body axis, independent of the precise location of the clone. This asymmetric response indicates the existence in the epithelium of a second signal, which we suggest is Decapentaplegic. Ectopic expression of Decapentaplegic induces extra macrochaetes only in cells which also receive the Wingless signal. Activation of Hedgehog signaling generates a long-range signal which can promote macrochaete formation in the Wingless activity domain. This signal depends upon decapentaplegic function. Autonomous activation of the Wingless signal response in cells causes them to attenuate or sequester this signal. Our results suggest a novel patterning mechanism which determines sense organ positioning in Drosophila. (+info)
BMP7 acts in murine lens placode development.
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Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation. (+info)
Antisense downregulation of a mouse mammary tumor virus activated protooncogene in mouse mammary tumor cells reverses the malignant phenotype.
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Activation of the protooncogene Wnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice. Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state. Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuous Wnt-1 expression for their ability to form tumors in vivo. To address this question, we constructed antisense and sense Wnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenous Wnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and sense Wnt-1 RNAs, changes in endogenous Wnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cells in vitro and in vivo were also examined. Our results show that (1) the levels of the expression of endogenous Wnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisense Wnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors ( approximately 0.2 cm in 16 weeks) compared to the tumors ( approximately 2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest that Wnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity. (+info)
Identification and localization of G protein subunits in human spermatozoa.
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Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility. (+info)
Cot protooncoprotein activates the dual specificity kinases MEK-1 and SEK-1 and induces differentiation of PC12 cells.
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Mitogenic signals initiated at the plasma membrane are transmitted to the nucleus through an intricate signalling network. We identified the protooncoprotein Cot as a new component of mitogenic signalling cascades, which activates both the classic cytoplasmic cascade and the SAPK stress pathway. Wildtype and activated Cot phosphorylate and activate MEK-1 and SEK-1 in vitro. These findings are consistent with the sequence homology between Cot and the rat gene Tpl-2. Expression of oncogenic Cot in 293, NIH3T3 and PC12 cells leads to in vivo phosphorylation of endogenous c-Jun and Erk-1/2 suggesting that the serine/threonine kinase Cot functions beside c-Raf-1 and Mos as a direct activator of MEK-1. Furthermore, we have examined the biological effects of Cot on the phenotype of fibroblastic and neuronal cells. In order to test a potential c-Raf-1 dependency of Cot transformation, the effect of oncogenic Cot on Raf revertant CHP25 cells was determined. Cot could restore the transformed phenotype indicating that Cot transformation is not dependent on active c-Raf-1 and that Cot is not a target for the putative Raf inhibitor, which is presumably active in the revertant cell line. Expression of oncogenic versions of Raf as well as v-Mos leads to differentiation of PC12 cells. Cot also induces neurite outgrowth of PC12 cells. These data are consistent with the role of Cot in the classic mitogenic cascade and suggest that the simultaneously activated JNK/SAPK stress pathway has no antagonistic effects in this context. (+info)