Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.

We have investigated the regulation of the activin-inducible distal element (DE) of the Xenopus goosecoid promoter. The results show that paired-like homeodomain transcription factors of the Mix family, Mixer and Milk, but not Mix.1, mediate activin/TGF-β-induced transcription through the DE by interacting with the effector domain of Smad2, thereby recruiting active Smad2/Smad4 complexes to the Mixer/Milk-binding site. We identify a short motif in the carboxyl termini of Mixer and Milk, which is demonstrated to be both necessary and sufficient for interaction with the effector domain of Smad2 and is required for mediating activin/TGF-β-induced transcription. This motif is not confined to these homeodomain proteins, but is also present in the Smad2-interacting winged-helix proteins Xenopus Fast-1, human Fast-1, and mouse Fast-2. We demonstrate directly that transcription factors of different DNA-binding specificity recruit activated Smads to distinct promoter elements via a common mechanism. These observations, together with the temporal and spatial expression patterns ofMixer and Milk, lead us to propose a model for mesoendoderm formation in Xenopus in which these homeodomain transcription factor/Smad complexes play a role in initiating and maintaining transcription of target genes in response to endogenous activin-like signals.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor f3 (TRf3) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis. 3,3',5-Triiodothyronine (

regulation of transcriptionThe Smads are responsible for transducing signals from receptors for members of the TGF-P superfamily to the nucleus, where they participate in transcriptional regulation. Smads have an inherently low affinity for DNA, requiring other transcription factors to recruit them to DNA. We have identified paired-like homeodomain transcription factors of the Xenopus Mix family, Mixer and Milk, as new partners for activated Smad2/Smad4 complexes. We have identified and characterized a short Smad interaction motif in the C-termini of Mixer and Milk, which is also present in completely unrelated transcription factors that interact with Smad2, the winged-helix proteins human Fast-1, mouse Fast-2 and Xenopus Fast-1. This motif is both necessary and sufficient for interaction with Smad2 and is required for mediating TGF-p/activin-induced transcription. Our demonstration that transcription factors of different DNA-binding specificity recruit activated Smads to distinct promoter elements through a common mechanism explains the molecular basis of the specificity of TGF-P/activin signalling. FROM INFLAMMATION T O SKIN BIOLOGY: PROTEIN KINASE CAS-283 CADES THAT CONTROL NF-KB ACTIVITY kmnoffice@ucrd.eduExposure to proinflammatory cytokines (TNF, IL-I), or hyproducts of bacterial and viral infections, such as endotoxin or dsRNA results in activation of signalling cascades that eventually lead to stimulation of NF-KB activity and induction of NF-KB target genes. Such genes code for cytokines, chemokines, adhesion molecules and enzymes, such as COX2, that produce secondary inflammatory mediators. Although chemically unrelated, the TNF and IL-I receptors use members of the TRAF family (TlZAF2 and TRAF6) as signal transducers that activate protein kinase cascades that regulate NF-KB activity by inducing the phosphorylarion of IrBs through IKK, which is a complex protein kinase composed of m o catalytic subunits (IKKa, IKKP) and a regulatory subunit (IKKy). IKKB is the recipient of proinflammatory signals that are recxuited to the complex via IKKy and is directly responsible for IKB phosphorylation and NF-KB activation. By contrast, IKKa responds to devclopmental signals and it controls key steps in the differenriation of ectodermal derivatives, including the epidermis, cornea and conjunctiva. Although the physiological activators of IKK in response to different proinflammatory stimuli remain to he identified, not all of them have to function as IKK kinases. For instance, PKR which mediates the response to dsRNA activates IKK via protein-protein interaction rather than direct phosphorylation. In addition to positive control, IKK activity is subject to negative control. Some of the negative regulation of IKK activity is mediated via C-terminal autophosphorylation and some is due to a novel mechanism based on the synthesis of cyclopentenone prostaglandins (cyPGs). These compounds, whose synthesis is catalyzed by COX2, react with a particular cysteine residue in the activation loop of the IKK catalytic subunits to ...