A temporary compendium of thyroid hormone target genes in brain.

BACKGROUND: Thyroid hormone controls a number of developmental and physiological processes in the brain by directly acting on gene expression. Transcriptome analyses in rodent identified a number of thyroid hormone regulated genes in several brain areas at different stages. Genome wide analysis of chromatin occupancy in a neural cell line also identified a subset of genes which transcription is likely to be directly regulated by thyroid hormone receptors in neurons. However, the abundance of these data and apparent discrepancies between studies brought some confusion. RESULTS: We present here a meta-analysis of available data to identify recurrent themes in thyroid hormone action in brain cells. This provides a curated list of 734 regulated genes in rodent brain, and highlights a small number of likely direct target genes. Some of these genes are also regulated in amphibians during metamorphosis. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Late maternal hypothyroidism alters the expression of Camk4 in neocortical subplate neurons: a comparison with Nurr1 labeling.

Maternal thyroid hormones (THs) are essential for normal offspring's neurodevelopment even after onset of fetal thyroid function. This is particularly relevant for preterm children who are deprived of maternal THs following birth, are at risk of suffering hypothyroxinemia, and develop attention-deficit/hyperactivity disorder. Expression of neocortical Ca(2+)/calmodulin kinase IV (Camk4), a genomic target of thyroid hormone, and nuclear receptor-related 1 protein (Nurr1), a postnatal marker of cortical subplate (SP) cells, was studied in euthyroid fetuses and in pups born to dams thyroidectomized in late gestation (LMH group, a model of prematurity), and compared with control and developmentally hypothyroid pups (C and MMI groups, respectively). In LMH pups, the extinction of heavy Camk4 expression in an SP was 1-2 days delayed postnatally compared with C pups. The heavy Camk4 and Nurr1 expression in the SP was prolonged in MMI pups, whereas heavy Camk4 and Nurr1 expression in layer VIb remains at P60. The abnormal expression of Camk4 in the cortical SP and in layer VIb might cause altered cortical connectivity affecting neocortical function.

Identification of novel GH-regulated genes in C2C12 cells.

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons.

The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by approximately 1200 neurons whose cell bodies are located in the lateral hypothalamus. The hypocretins/orexins have been implicated in the regulation of rapid eye movement (REM) sleep and the pathophysiology of narcolepsy. In the present study, we investigated whether the locus coeruleus (LC), a structure receiving dense hcrtergic innervation, which is quiescent during REM sleep, might be a target for hcrt to regulate REM sleep. Local administration of hcrt1 but not hcrt2 in the LC suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep. These effects were blocked with an antibody that neutralizes hcrt binding to hcrt receptor 1. In situ hybridization and immunocytochemistry showed the presence of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC. Iontophoretic application of hcrt1 enhanced the firing rate of LC neurons in vivo, and local injection of hcrt1 into the LC induced the expression of c-fos in the LC area. We propose that hcrt receptor 1 in the LC is a key target for REM sleep regulation and might be involved in the pathophysiological mechanisms of narcolepsy.

The human RC3 gene homolog, NRGN contains a thyroid hormone-responsive element located in the first intron.

NRGN is the human homolog of the neuron-specific rat RC3/neurogranin gene. This gene encodes a postsynaptic 78-amino acid protein kinase substrate that binds calmodulin in the absence of calcium, and that has been implicated in dendritic spine formation and synaptic plasticity. In the rat brain RC3 is under thyroid hormone control in specific neuronal subsets in both developing and adult animals. To evaluate whether the human gene is also a target of thyroid hormone we have searched for T3-responsive elements in NRGN cloned genomic fragments spanning the whole gene. Labeled DNA fragments were incubated with T3 receptors (T3R) and 9-cis-retinoic acid receptors and immunoprecipitated using an anti T3R antibody. A receptor-binding site was localized in the first intron, 3000 bp downstream from the origin of transcription. Footprinting analysis revealed the sequence GGATTAAATGAGGTAA, closely related to the consensus T3-responsive element of the direct repeat (DR4) type. This sequence binds the T3R-9-cis-retinoic acid receptors heterodimers, but not T3R monomers or homodimers, and is able to confer regulation by T3R and T3 when fused upstream of the NRGN or thymidine kinase promoters. The data reported in this work suggest that NRGN is a direct target of thyroid hormone in human brain, and that control of expression of this gene could underlay many of the consequences ofhypothyroidism on mental states during development as well as in adult subjects.

Cell-specific effects of thyroid hormone on RC3/neurogranin expression in rat brain.

To identify thyroid hormone-sensitive neuronal populations in the forebrain, we studied the effects of thyroid hormone deficiency and replacement on the expression of RC3 messenger RNA (mRNA) in the rat brain by in situ hybridization. RC3/neurogranin is a brain-specific, calmodulin-binding, protein kinase C substrate that has been implicated in postsynaptic events involving calcium as a second messenger. We have previously shown that RC3 mRNA and protein concentrations are thyroid hormone dependent in developing and adult rats. In normal developing rats, RC3 expression occurs in two phases. Before postnatal day 10 (P10), RC3 mRNA was detected mainly in layers II/III and V of cerebral cortex and the CA fields of the hippocampus. From P10 to P15, it decreased in layer V and increased in layer VI, the retrosplenial cortex, the caudate-putamen nucleus, and the dentate gyrus. Expression in the caudate followed a lateral to medial gradient. Thyroid hormone deficiency interfered with the late phase of RC3 expression, such that developing hypothyroid rats showed lower RC3 expression in layer VI, the retrosplenial cortex, the dentate gyrus, and the caudate, and increased expression in layer V. These changes were reverted by T4 treatment. Adult- onset hyperthyroidism also reversibly decreased hybridization in the striatum. In contrast to other molecular targets of thyroid hormone in the brain, such as myelin genes, expression of RC3 was also affected by long term hypothyroidism in the absence of hormone replacement, indicating that thyroid hormone is a required factor for the cell-specific control of RC3 expression. In addition to identifying thyroid hormone-sensitive neurons, our results suggest that one action of thyroid hormone during brain development is the timely coordination of gene expression among phenotypically different, region-specific neuronal populations.

Identification of a cis-acting element that interferes with thyroid hormone induction of the neurogranin (NRGN) gene.

The neuron-specific RC3/neurogranin gene is regulated by thyroid hormone at the transcriptional level in brain and in cultured neuronal cells. Regulation in vivo displays exquisite regional selectivity which is not due to differential distribution of thyroid receptors and is most probably related to region-specific trans-acting elements. We have previously identified an intronic thyroid hormone responsive element in the human RC3 gene homolog, NRGN. In a search for cis-acting elements that might contribute to the specificity of thyroid regulation, we have identified a novel sequence, TTCCAAAATGG, which binds to a developmentally regulated protein, and interferes with T3 transactivation.

Thyroid hormone regulation of rhes, a novel Ras homolog gene expressed in the striatum.

Thyroid hormone action on brain development is essentially exerted through regulation of the expression rate of a number of genes some of which have been identified in the past 10 years. In the present work we describe the thyroid hormone regulation of a novel Ras homolog which we have named Rhes (Ras homolog enriched in striatum). The rhes cDNA was previously isolated in subtractive hybridization experiments aimed at identifying cDNA clones corresponding to genes expressed preferentially in the rat striatum. The sequence was found to encode a small GTP-binding protein of the Ras family with highest homology to the dexamethasone-inducible Dexras1. Here we show that rhes mRNA and protein in the striatum are strongly dependent on the thyroidal status. Developmentally, Rhes was regulated such that in normal rats there was an increased rhes mRNA content in the striatum after postnatal day 5 (P5). Rhes concentration in hypothyroid rats was similar to that of normal rats at P5, but the subsequent age-dependent increase was blunted. The administration of a single T3 dose to hypothyroid rats normalized rhes mRNA concentration in 8 h, whereas it took 24 h, or more, to normalize the expression of rc3, another T3-dependent brain gene, involved in PKC signaling. Double in situ hybridization using rhes and rc3 riboprobes showed that the bulk of rhes signal was located in cells expressing rc3. Given the relevance of small GTPases in signal transduction it is very likely that control of rhes, in addition to rc3, is of relevance to explain the actions of thyroid hormone in the striatum, a region of the brain especially vulnerable in neurological cretinism.

Transcriptional induction of RC3/neurogranin by thyroid hormone: differential neuronal sensitivity is not correlated with thyroid hormone receptor distribution in the brain.

RC3/neurogranin is a calmodulin-binding protein kinase C substrate, located in dendritic spines of forebrain neurons. It has been implicated in post-synaptic signal transduction events involving Ca2+ and calmodulin leading to many forms of synaptic plasticity. RC3 gene expression is under developmental and physiological regulation. The main physiological regulator appears to be thyroid gland activity. Hypothyroidism decreased RC3 mRNA concentration in the brain of post-natal day 22 rats. The affected areas included layer 6 of cerebral cortex, layers 2-3 of retrosplenial cortex, dentate gyrus and the caudate whereas others were not affected by hypothyroidism, such as upper layers of cerebral cortex, the pyramidal layer of the hippocampus and the amygdala. A single administration of triiodothyronine (T3) induced a significant transcriptional increase of RC3 mRNA in hypothyroid rats, 24 h after administration. Differential sensitivity to thyroid hormone was not related to differential expression of T3 receptor isoforms or the T3 receptor inhibitory variant alpha2. Therefore, it is likely that cell sensitivity to thyroid hormone in the brain depends on T3 receptor-associated factors.

Characterization of the promoter region and flanking sequences of the neuron-specific gene RC3 (neurogranin).

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones.

Thyroid hormone-regulated expression of RC3/neurogranin in the immortalized hypothalamic cell line GT1-7.

The calmodulin-binding, protein kinase C substrate RC3/neurogranin is the product of a neuron-specific gene expressed in the forebrain that is under specific regional and temporal control by thyroid hormone (3,5,3'-triiodothyronine, T3). In vivo, some neuronal populations are sensitive and others are insensitive to T3. The goal of this study was to identify neuronal cell cultures that express RC3/neurogranin, to check whether they are sensitive to T3, and to examine the mechanism of regulation. We found that RC3 is induced by T3 in the hypothalamic cell line GT1-7 at the transcriptional level. The half-life of the mature mRNA was 20 h and was not affected by the hormone. Addition of T3 to the cell culture induces neurogranin mRNA after 6 h in the absence of new protein synthesis. These results suggest a direct transcriptional effect of T3 mediated through nuclear receptors. Indeed, GT1-7 cells express functional T3 receptors, as shown by northern blotting, nuclear T3-binding assays, and transactivation of reporter genes. The role of retinoic acid and glucocorticoids on RC3 expression was also evaluated, because we have previously noted the presence of consensus response elements for these hormones in the RC3 upstream promoter region. In contrast to T3, neither retinoic acid nor dexamethasone influences neurogranin expression despite the presence of respective functional receptors.