Aurora Sánchez-Pacheco

Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

The thyroid hormone receptor antagonizes CREB-mediated transcription

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein–protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)‐containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein–protein interactions. In turn, expression of CREB reduces TR‐dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand‐dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross‐talk between CREB and TR signalling pathways, which can have important functional consequences.

A direct protein-protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF-1.

The nuclear receptors for thyroid hormone (TRs) and retinoic acid (RARs and RXRs) cooperate with the pituitary‐specific transcription factor GHF‐1 to activate the rat growth hormone (GH) gene. The GH promoter contains a hormone response element (HRE), which binds TR/RXR and RAR/RXR heterodimers, located close to two binding sites for GHF‐1. GHF‐1 inhibits binding of TR/ RXR and RAR/RXR heterodimers to an isolated HRE. Similarly, the receptors inhibit binding of GHF‐1 to its cognate site. These results suggest the existence of direct protein to protein interactions between the receptors and the pituitary transcription factor. This was confirmed by in vitro binding studies with GST fusion proteins, which demonstrated a strong association of GHF‐1 with RXR and a weaker interaction with RAR and TR. GHF‐1 and the receptor heterodimers form a ternary complex with a fragment of the rat GH promoter, which contains binding sites for both, and GHF‐1 increases receptor binding to the promoter when present in limiting conditions. These results suggest that the synergistic activation of the rat GH gene involves protein‐DNA interactions as well as a physical association between the nuclear receptors and the pituitary‐specific transcription factor GHF‐1.—Palomino, T., Sánchez‐Pacheco, A., Peña, P., Aranda, A. A direct protein‐to‐protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF‐1. FASEB J. 12, 1201–1209 (1998)

Thyroid hormone-mediated activation of the ERK/dual specificity phosphatase 1 pathway augments the apoptosis of GH4C1 cells by down-regulating nuclear factor-κB activity

Thyroid hormone (T3) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T3 on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T3 produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-kappaB (NF-kappaB)-dependent transcription, IkappaBalpha phosphorylation, translocation of p65/NF-kappaB to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T3, and inhibition of ERK alone abrogated T3-mediated apoptosis. In addition, T3 increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T3-induced inhibition of NF-kappaB-dependent transcription and p65/NF-kappaB translocation to the nucleus, as well as T3-mediated apoptosis. Overall, our results indicate that T3 induces apoptosis in rat pituitary tumor cells by down-regulating NF-kappaB activity through a mechanism dependent on the ERK/DUSP1 pathway.

Activation of the human immunodeficiency virus type I long terminal repeat by 1α,25-dihydroxyvitamin D3

The genetic predisposition of the host and the virus is the most important determinant for prediction of the course of human immunodeficiency virus type I (HIV-1) viral infection and acquired immune deficiency syndrome (AIDS) progression. Transcription from the HIV-1 long terminal repeat (LTR) is a crucial step for viral replication. Here, we describe a stimulatory role of the vitamin D receptor (VDR) on HIV-1 LTR transactivation. Transient transfections reveal that VDR activates the LTR in HeLa, U937, and Cos-1 cells in a ligand-dependent manner. 1alpha,25-Dihydroxyvitamin D3 (vitD3) promotes activation of a minimal LTR construct (from nucleotides -35 to +89), lacking a previously described hormone response element that binds several nuclear receptors. NF-kappaB (nuclear factor-kappa B) and Sp1-binding sites, which are responsible for most basal LTR activity in HeLa cells, are also dispensable for vitD3-dependent HIV-1 transcription. Although the tat response element element is not required for VDR-mediated HIV-1 gene expression, the viral protein Tat acts in a synergistic manner with the receptor to stimulate LTR activity. Furthermore, our data also show cooperation of the receptor with various cellular coactivators for HIV-1 transactivation by vitD3. Paradoxically, mutations in the VDR ligand-dependent transcriptional activation function-2 that abrogate vitD3-dependent stimulation through classical vitamin D response elements, do not reduce vitD3-mediated LTR transactivation. Furthermore, point mutations in the DNA-binding domain that abolish receptor binding to consensus DNA sequences do not affect ligand-dependent HIV-1 stimulation. These results show that VDR activates the HIV-1 LTR through different mechanisms, including non-classical nuclear receptor transcriptional actions that may ensure viral transcription under different physiological scenarios.

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression.

The rat growth hormone (GH) promoter was significantly activated in non‐pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand‐dependent activation was found in the presence of the pituitary‐specific transcription factor GHF‐1. When compared with GHF‐1, the splice variant GHF‐2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF‐2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over‐expression of GHF‐2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF‐1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

Binding of the Thyroid Hormone Receptor to a Negative Element in the Basal Growth Hormone Promoter Is Associated with Histone Acetylation

Nuclear thyroid hormone receptors (TRs) act as ligand-dependent activators, but paradoxically unliganded TRs can increase transcription of promoters containing negative response elements (nTRE), and hormone binding represses this activation. The rat growth hormone (GH) promoter contains a positive TRE and a nTRE. Ligand-dependent negative regulation mediated by the nTRE could play an important physiological role in restricting GH gene expression in non-pituitary cells that express TRs. With chromatin immunoprecipitation assays, we show here that the nTRE is responsible for binding of TR to the promoter in non-pituitary HeLa cells and that this element also governs transactivation by the unoccupied receptor and repression by triiodothyronine. Occupancy of the promoter by TR is concomitant with appearance of acetylated histone H3, and triiodothyronine causes release of the receptor as well as disappearance of the acetylated histone from the promoter. Although the nTRE overlaps the TATA box, the receptor does not exclude binding of TATA-binding protein, but could rather facilitate formation of the preinitiation complex. Furthermore, the proximal GH promoter is synergistically stimulated by unliganded TR and TATA-binding protein, whereas the ligand represses this cooperation. Constitutive receptor activity and synergism with TATA-binding protein require binding of corepressors. Furthermore, inhibitors of histone deacetylases enhance promoter activation by the unliganded receptor and reduce triiodothyronine-dependent repression, whereas expression of HDAC1 reverses promoter stimulation. This suggests that partitioning of histone acetylases and deacetylases between the receptors and basal transcription factors could be involved in regulation of the basal GH promoter by TRs.

Residues K128, 132, and 134 in the Thyroid Hormone Receptor-α Are Essential for Receptor Acetylation and Activity

The thyroid hormone receptor (TR)-alpha is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRalpha is posttranslationally modified by acetylation in response to its own ligand (T(3)). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRalpha decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRalpha mutant lost the ability to transactivate even at high T(3) concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRalpha interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRalpha suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRalpha.

The thyroid hormone response element is required for activation of the growth hormone gene promoter by nicotinamide analogs

N′‐Methylnicotinamide and nicotinamide, which decreased in vitro ADP‐ribosylation of nuclear proteins and/or cellular NAD+ content, selectively increased the basal expression of the rat growth hormone (GH) gene promoter and its response to triiodothyronine (T3). This increase was not found when the thyroid hormone response element (TRE) was deleted from the promoter. Transfection with an expression vector for the T3 receptor inhibited basal activity of the TRE‐containing promoter and repressed the stimulatory effect of N′‐methylnicotinamide. The addition of hormone relieved this inhibition and enhanced transcription above levels found in the absence of the transfected receptors. These results suggest a modulatory role of ADP‐ribosylation in hormonal regulation of gene expression.

Retinoic acid decreases thyroid hormone receptor expression in pituitary GH1 cells

Retinoic acid produced a time and dose-dependent depletion of thyroid hormone receptors in GH1 cells without modifying their affinity for triiodothyronine (T3). A maximal decrease (50-70%) was obtained after 24-48 h incubation with 5-10 microM retinoic acid. Treatment with 0.8 nM T3 for 24 h caused a similar reduction and did not potentiate the decrease produced by these concentrations of retinoic acid. However, the combination of sub-maximally effective doses of both ligands had an additive effect on receptor levels. The reduction of receptor caused by retinoic acid is accompanied by a decreased expression of c-erbA alpha 1 and alpha 2 mRNAs, but the retinoid did not reduce the abundance of c-erbA beta mRNA. In contrast, T3 decreased the levels of both alpha and beta transcripts.