Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity.
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During Drosophila embryogenesis, the dorsal transcription factor activates the expression of twist, a transcription factor required for mesoderm formation. We show here that the mammalian twist proteins, twist-1 and -2, are induced by a cytokine signaling pathway that requires the dorsal-related protein RelA, a member of the NF-kappaB family of transcription factors. Twist-1 and -2 repress cytokine gene expression through interaction with RelA. Mice homozygous for a twist-2 null allele or doubly heterozygous for twist-1 and -2 alleles show elevated expression of proinflammatory cytokines, resulting in perinatal death from cachexia. These findings reveal an evolutionarily conserved signaling circuit in which twist proteins regulate cytokine signaling by establishing a negative feedback loop that represses the NF-kappaB-dependent cytokine pathway. (+info)
Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.
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Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. We addressed the role of basic helix-loop-helix (bHLH) proteins in SMC differentiation by first determining the role of two E-box (CAnnTG) motifs, binding sites for bHLH proteins, in the transcriptional regulation of the SMC differentiation marker gene, smooth muscle alpha-actin (SM alpha-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM alpha-actin promoter/LacZ reporter gene in vivo in transgenic mice. We then determined the potential role of class I bHLH proteins, E12, E47, HEB, and E2-2, in SM alpha-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM alpha-actin promoter. Activation by HEB and E2-2 was synergistic with serum response factor. Additionally, the dominant-negative/inhibitory HLH proteins, Id2, Id3, and Twist, inhibited both the E12 and serum response factor-induced activations of the SM alpha-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box-containing region of the SM alpha-actin promoter in vivo in the context of intact chromatin in SMCs. Taken together, these results provide the first evidence of E-box-dependent regulation of a SMC differentiation marker gene in vivo in transgenic mice. Moreover, they demonstrate a potential role for class I bHLH factors and their inhibitors, Id and Twist, in SM alpha-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation. (+info)
Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation.
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Wnt1, initially identified as a mammary oncogene, can activate transcription via beta-catenin/TCF complexes. Twist, a transcription factor of the basic helix-loop-helix class, has also been suggested to have oncogenic properties. The aim of this study was to determine whether Twist is regulated by Wnt1 and might thus be a novel mediator of Wnt signaling. We found that Twist was up-regulated in C57MG and HC11 murine mammary epithelial cells in response to Wnt1 expression. Additionally, we detected Twist expression in normal mammary gland and found elevated Twist expression in approximately 70% of mammary tumors from Wnt1 transgenic mice. A murine Twist promoter fragment was shown to be responsive to beta-catenin, and its activity was enhanced by coexpression of c-jun and Ets factors of the PEA3 family. Both PEA3 factors and c-jun were highly expressed in tumors from Wnt1 transgenic mice and may therefore contribute to the increased Twist expression observed in these tumors. To evaluate functional consequences of Twist induction, we examined the effect of Twist on mammary cell differentiation. Strikingly, overexpression of either Wnt1 or Twist in HC11 mammary epithelial cells completely suppressed induction of the milk protein beta-casein in response to lactogenic hormones. Additionally, Wnt1, but not Twist, partially abrogated induction of WDNM1, another marker of lactogenic differentiation. Taken together, our data indicate that Twist expression is regulated by Wnt/beta-catenin signaling and that both Wnt1 and Twist can function as inhibitors of lactogenic differentiation, an effect that could contribute to mammary tumorigenesis. (+info)
Molecular mechanisms in calvarial bone and suture development, and their relation to craniosynostosis.
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The development and growth of the skull is a co-ordinated process involving many different tissues that interact with each other to form a complex end result. When normal development is disrupted, debilitating pathological conditions, such as craniosynostosis (premature calvarial suture fusion) and cleidocranial dysplasia (delayed suture closure), can result. It is known that mutations in the fibroblast growth factor receptors 1, 2, and 3(FGFR1, 2, and 3), as well as the transcription factors MSX2 and TWIST cause craniosynostosis, and that mutations in the transcription factor RUNX2 (CBFA1) cause cleidocranial dysplasia. However, relatively little is known about the development of the calvaria: where and when these genes are active during normal calvarial development, how these genes may interact in the developing calvaria, and the disturbances that may occur to cause these disorders. In this work an attempt has been made to address some of these questions from a basic biological perspective. The expression patterns of the above-mentioned genes in the developing mouse skull are detailed. The microdissection and in vitro culture techniques have begun the task of identifying Fgfrs, Msx2, and Twist interacting in intricate signalling pathways that if disrupted could lead to craniosynostosis. (+info)
Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth.
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Human craniosynostosis syndromes, resulting from activating or neomorphic mutations in fibroblast growth factor receptor 2 (FGFR2), underscore an essential role for FGFR2 signaling in skeletal development. Embryos harboring homozygous null mutations in FGFR2 die prior to skeletogenesis. To address the role of FGFR2 in normal bone development, a conditional gene deletion approach was adopted. Homologous introduction of cre recombinase into the Dermo1 (Twist2) gene locus resulted in robust expression of CRE in mesenchymal condensations giving rise to both osteoblast and chondrocyte lineages. Inactivation of a floxed Fgfr2 allele with Dermo1-cre resulted in mice with skeletal dwarfism and decreased bone density. Although differentiation of the osteoblast lineage was not disturbed, the proliferation of osteoprogenitors and the anabolic function of mature osteoblasts were severely affected. (+info)
A Drosophila model to study the functions of TWIST orthologs in apoptosis and proliferation.
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The twist gene has been characterized for its role in myogenesis in several species. In addition, in mammalian cultured cells, it has been shown that twist is a potential oncogene antagonizing p53-dependent apoptosis. To study, in vivo, the role of twist in apoptosis and proliferation, we constructed transgenic Drosophila lines allowing ectopic expression of different twist orthologs. We report that: (i) Drosophila twist induces apoptosis and activates the reaper promoter, (ii) nematode twist induces arrest of proliferation without apoptosis, and (iii) human twist retains its potentialities observed in mammalian cultured cells and antagonizes Drosophila p53-dependent apoptosis. In addition, we show that human twist is able to induce cell proliferation in Drosophila. Data suggest that the pathway by which human twist antagonizes Drosophila p53 could be conserved. These transgenic lines thus constitute a powerful tool to identify targets and modifiers of human twist. (+info)
A Twist-like bHLH gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos.
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Ascidian larvae develop mesenchyme cells in their trunk. A fibroblast growth factor (FGF9/16/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. We have identified two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 as downstream factors of this FGF. These two genes are phylogenetically closely related to each other, and were expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrated that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream factor of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos. (+info)
Control of apterous by vestigial drives indirect flight muscle development in Drosophila.
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Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vg(null) mutant, we report that a total absence of vg leads to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. We show that vg and scalloped (sd), the only known VG transcriptional coactivator, are coexpressed during IFM development. Moreover, we observed an ectopic expression of ct and ap, two markers of DFM development, in developing IFMs of vg(null) pupae. In addition, in vg(null) adult flies, degenerating DLMs express twist (twi) ectopically. We provide evidence that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, we were able to rescue the muscle phenotype of vg(null) flies by using the activity of ap promoter to drive VG expression. Thus, vg appears to be a key regulatory gene of IFM development. (+info)