Search

We hypothesized that the brain-protective effect of hyperbaric oxygen (HBO) preconditioning in a transient global cerebral ischemia rat model is mediated by the inhibition of early apoptosis. One hundred ten male Sprague-Dawley (SD) rats (300-350 g body weight) were allocated to the sham group and three other groups with 10 min of four-vessel occlusion, untreated or preconditioned with either 3 or 5 hyperbaric oxygenations. HBO preconditioning improved neurobehavioral scores and reduced mortality, decreased ischemic cell change, reduced the number of early apoptotic cells and hampered a conversion of early to late apoptotic alterations. HBO preconditioning reduced the immunoreactivity of phosphorylated p38 in vulnerable neurons and increased the expression of brain derived neurotrophic factor (BDNF) in early stage post-ischemia. However, preconditioning with 3 HBO treatments proved less beneficial than with 5 HBO treatments. We conclude that HBO preconditioning may be neuroprotective by reducing early apoptosis and inhibition of the conversion of early to late apoptosis, possibly through an increase in brain BDNF level and the suppression of p38 activation.

Thyroid hormone receptors (TRs) form heterodimers with retinoid X receptors (RXRs). Heterodimerization is required for efficient TR DNA binding to most response elements and transcriptional activation by thyroid hormone. RXRs also function as auxiliary proteins for several other receptors. In addition, RXR alpha can be induced by specific ligands to form homodimers. Here we report that RXR-specific retinoids that induce RXR homodimers are effective repressors of the T3 response. We provide evidence that this repression by RXR-specific ligands occurs by sequestering of RXR from TR-RXR heterodimers into RXR homodimers. This ligand-induced squelching may represent an important mechanism by which RXR-specific retinoids and 9-cis retinoic acid mediate hormonal cross talk among a subfamily of nuclear receptors activated by structurally unrelated ligands.

Retinoid response pathways are mediated by two classes of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A central question is whether distinct response pathways are regulated by these two classes of receptors. The observation that the stereoisomer 9-cis-retinoic acid binds with high affinity to RXRs suggested that this retinoid has a distinct role in controlling RXR activity, but it was almost simultaneously discovered that RXRs function as auxiliary receptors for RARs and related receptors, and are essential for DNA binding and function of those receptors. Hence, although RARs seem to operate effectively only as heterodimeric RAR/RXR complexes, RXRs themselves apparently function predominantly, if not exclusively, as auxiliary receptors. Here we report that 9-cis-retinoic acid induces RXR homodimer formation. Our results demonstrate a new mechanism for retinoid action by which a ligand-induced homodimer mediates a distinct retinoid response pathway.

Thyroid hormones and retinoic acid function through nuclear receptors that belong to the steroid/thyroid-hormone receptor superfamily. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) require auxiliary nuclear proteins for efficient DNA binding. Here we report that retinoid X receptors RXR alpha is one of these nuclear proteins. RXR alpha interacts both with TRs and with RARs, forming heterodimers in solution that strongly interact with a variety of T3/retinoic acid response elements. Transfection experiments show that RXR alpha can greatly enhance the transcriptional activity of TR and RAR at low retinoic acid concentrations that do not significantly activate RXR alpha itself. Thus, RXR alpha enhances the transcriptional activity of other receptors and its own ligand sensitivity by heterodimer formation. Our studies reveal a new subclass of receptors and a regulatory pathway controlling nuclear receptor activities by heterodimer formation.

The biological response to retinoic acid (RA) and synthetic derivatives (retinoids) is mediated by three nuclear retinoic acid receptors, RAR alpha, beta and gamma. To explore the potential of retinoids as receptor subtype selective activators, we employed a transcriptional activation assay. Hybrid receptors that recognize an estrogen response element were used to avoid measuring activities of endogenous retinoic acid receptors. In response to retinoic acid, the three hybrid receptors ER-RAR alpha, ER-RAR beta and ER-RAR gamma exhibited the same induction profile as the corresponding wild type receptors RAR alpha, RAR beta, and RAR gamma. Three different retinoids, analogs of 6'-substituted naphthalene-2-carboxylic acid, elicited strong transcriptional activation of gamma receptor while no activation of alpha receptor was observed. Conversely, two retinobenzoic acid analogs showed a limited alpha selectivity. We conclude that retinoids with unique profiles of retinoic acid receptor subtype selectivity can be defined and tested for their impact on cellular differentiation and for therapeutical applications.

Gene regulation by thyroid hormones is mediated through multiple nuclear receptors. Only some of these thyroid hormone receptor (TR) isoforms become transcriptional enhancers in the presence of the thyroid hormone T3. Here we analyze the regulatory function of the human TR alpha 2 isoform. This protein does not bind T3 and is not a transcriptional activator of thyroid hormone-responsive elements (TRE). Transfected TR alpha 2 functions as a constitutive repressor of the transcriptional activators TR alpha 1 and TR beta 1 but also represses heterologous receptors, including the retinoic acid receptor and the estrogen receptor, which can activate TRE-controlled genes. TR alpha 2 protein showed strongly reduced DNA binding to a palindromic TRE when compared with the active TRs. Hybrid receptor analysis revealed that the special properties of the TR alpha 2 protein, including its repressor function and DNA binding characteristics, are intrinsic properties of its carboxyterminus and can be transferred to other receptors. Although it has been shown that the active TRs can act as repressors and silencers due to their strong DNA binding in the absence of hormone, our data show that TR alpha 2 is unlikely to inhibit TRs and other receptors through a competitive DNA binding mechanism. Antibody gel shift experiments suggest that repression by TR alpha 2 might result from interaction with active receptors. Thus, the receptor-like TR alpha 2 isoform differs from typical nuclear receptors in its DNA-binding and ligand-binding properties and appears to regulate the activity of other receptors via protein-protein interaction.

The identification of hormone response elements in the promoter regions of hormonally regulated genes has revealed a striking similarity between the estrogen response element (ERE) and a palindromic thyroid hormone response element (TRE) derived from the GH gene promoter. In addition, this TRE was described as a strong retinoic acid receptor response element for all three subtypes: alpha, beta, and gamma. We show here that the TRE in the absence of thyroid hormone receptor (TR) behaves similarly to imperfect EREs, which can synergize to mediate a strong estrogen-dependent activation of transcription. However, in the presence of TR, but the absence of T3, activation of the TRE constructs by estrogen receptor (ER) is inhibited. In vitro, ER and TR were found to bind to the TRE in the absence and presence of their respective ligands; however, TRs form a more stable complex with the TRE than does ER. To examine whether repression of ER activity on the TRE constructs by TR was due to heterodimer formation, we employed truncated TR mutants (tTR) that lacked the DNA-binding domain, but contained the ligand-binding/dimerization domain. The tTRs were shown to be efficient inhibitors of TR, but not of ER. Thus, inhibition of ER activity on TREs by TRs does not result from heterodimer formation. We discuss a mechanism in which TRs, in the absence of thyroid hormone, control TRE activation by related receptors by preventing their access to the TRE. This mechanism can greatly enhance the fidelity of the ligand-specific response from a TRE.

Retinoids regulate many biological processes, including differentiation, morphogenesis and cell proliferation. They are also important therapeutic agents, but their clinical usefulness is limited because of side effects. Retinoid activities are mediated by specific nuclear receptors, the RARs and RXRs, which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 (refs 12-15), which usually mediates cell proliferation signals. Because the two types of receptor actions are mechanistically distinct, we investigated whether conformationally restricted retinoids, selective for each type of receptor action, could be identified. Here we describe a new class of retinoids that selectively inhibits AP-1 activity but does not activate transcription. These retinoids do not induce differentiation in F9 cells but inhibit effectively the proliferation of several tumour cell lines, and could thus serve as candidates for new retinoid therapeutic agents with reduced side effects.

The morphogen retinoic acid (RA) regulates gene transcription by interacting with specific nuclear receptors that recognize DNA sequences near responsive promoters. While much has recently been learned about the nuclear receptor proteins, little is known about the genes that are directly regulated by RA and their cis-acting response elements recognized by these receptors. Here we have analyzed the RA receptor-beta (RAR beta) gene promoter that is controlled by RA. We find that a RA-responsive element (RARE) is located adjacent to the TATA box. The RARE shows a direct repeat symmetry which is essential for its function. While thyroid hormone-responsive elements can also function as RAR response elements, we show here that this RARE is activated by endogenous RARs and RAR beta, but cannot be regulated by thyroid hormone receptors and other known nuclear receptors. In addition, we find that RAR gamma is a poor activator of this RARE. However, the response element is bound with high affinity by both RAR beta and RAR gamma as well as by thyroid hormone receptors. Thus, interaction between specific response elements and receptors is insufficient for gene activation.

Publisher Summary This chapter describes rapid and sensitive methods procedures for the probing, cloning, and analysis of nuclear retinoic acid receptors and their ligands. The procedures can be easily adapted for the general analysis of other proteins. Conventional cDNA cloning approaches are time-consuming and require a multitude of skills; this chapter presents an easy to follow polymerase chain reaction (PCR) procedure for the isolation of retinoic acid receptor (RAR) cDNA that can be accomplished within a few days. The chapter discusses two analysis systems can be used to determine quickly the overall integrity of the PCR clone. One is the in vitro transcription/translation assay. The second analysis system evaluates the functional integrity of the encoded receptor protein in the transient transfection system. The chapter describes an efficient procedure that allowed comparing the gene activation capacity of a large number of individual synthetic retinoids. This system will be extremely useful for the evaluation of optimal, receptor-specific agonists and antagonists.

Both thyroid hormone (T3) and retinoic acid signal essential steps in development, differentiation and morphogenesis. Specific nuclear receptors for these ligands have recently been cloned. Previously we have noted a close homology between the DNA-binding domains of the epsilon-retinoic acid receptor (RAR-epsilon, also designated RAR-beta), the thyroid hormone receptors and the oestrogen receptor. We have now found that RAR-epsilon is very efficient at inducing transcription from two distinct thyroid-hormone responsive elements (TREs). Transcription induced by ligand-activated RAR-epsilon from a TRE can, however, be repressed by thyroid-hormone receptor in the absence of its ligand. Conversely, in the presence of its ligand, thyroid-hormone receptor will activate transcription from a TRE irrespective of the presence of unbound RAR. The use of hybrid receptors has shown that the DNA-binding domain of RAR is the essential target for inhibition by thyroid-hormone receptors. These data, together with in vitro DNA-binding studies, suggest that thyroid-hormone receptors may have dual regulatory roles: in the presence of hormone they function as TRE-specific transcriptional activators; in the absence of hormone, however, they can function as TRE-specific repressors.

As a step in the characterization of development and ageing in the nematode Caenorhabditis elegans, the activities of different groups of enzymes that supposedly exert modulating functions in and after protein synthesis have been determined. From embryonic (E), the four juvenile larval stages (L1-L4) and the gravid adult (A,A+), the selection of defined developmental stages extends to two different preparations of aged nematodes (S10, S12). Some aminoacyl-tRNA synthetase activities remain nearly unchanged in all stages up to the adult, some increase continuously during the larval stages and the remaining activities show stage-specific alterations. Upon ageing all activities except the one for tryptophan decrease sharply, tRNA methylase activities increase from E to L4, decrease from L4 to adult and to aged nematodes with only qualitative alterations in substrate specificity. The activity of tubulin: tyrosine ligase exhibits a parallel pattern, while arginyltransferase activity has a plateau between L2 and L4. The results are consistent with the idea of a modulation of protein synthesis and other cellular processes by quantitative activity changes during development and ageing.

The specific activities of 17 aminoacyl-tRNA synthetases from liver, lung, heart, spleen, kidney, small intestine and skeletal muscle of young (2 months) and aged (39 months) female Han:NMRI mice were determined under standard conditions of sample preparation and assay. The average reduction of total activity during ageing is 70% for liver, 50% for lung and spleen, nearly 40% for heart and kidney and nearly 20% for intestine and skeletal muscle. Detailed comparison reveals no general, but an organ-specific pattern. Aminoacyl-tRNA synthetases were, furthermore, found to be ribosome-associated in higher proportions in liver tissue from aged mice.

The specific activities of 17 aminoacyl-tRNA synthetases in liver, lung, heart, spleen, kidney and small intestine of old female normal and leukemic (reticulum -cell sarcoma, type A) mice have been monitored. No difference appears for lung, heart and kidney; small increases with varying particular changes for liver and marked increases in spleen and small intestine of the tumor bearing mice have been found, following a similar pattern. This finding suggests a coordinated adaptation to modulation of the requirements of protein synthesis imposed by histiocytic sarcoma.