Search PubMed⌕ Search

Biomedical subjects

M Pfahl

Publications and source records attributed to M Pfahl.

At least 37 records · Page 2Linked to original sources
PubMed1995

Conformational effects on retinoid receptor selectivity. 2. Effects of retinoid bridging group on retinoid X receptor activity and selectivity.

The natural retinoid 9-cis-retinoic acid is an activating ligand for both the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which are members of the retinoid/thyroid hormone/steroid hormone family of nuclear receptor proteins that activate gene transcription through specific response elements. The pharmacophoric groups necessary to confer RXR selectivity were established by evaluating the ability of 21 conformationally restricted retinoids to activate the TREpal retinoic acid receptor response element for gene transcription in the presence of one of the three RAR subtypes or RXR alpha. In contrast to those retinoids selective for the RARs, these RXR-selective retinoids have one less atom in the bridge linking the hydrophobic and carboxylic acid termini of the retinoid skeleton. Therefore, a one-carbon bridge replaces the 19-methyl group and 9E-double bond of 9-cis-retinoic acid and is further functionalized by inclusion in an isopropylidene group, a dioxolane ring, or a cyclopropane ring for optimal RXR alpha activity and selectivity. In addition, the beta-geranylidene and 20-methyl-(11E,13E)-dienoic acid groups of 9-cis-retinoic acid are replaced by a 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl ring and a 4-carboxylphenyl ring, respectively, for optimal activation and selectivity. RXR alpha selectivity is reduced on replacement of the 4-carboxylphenyl group by a 2-carboxyl-5-thienyl group or the 9-cis-retinoic acid methylpentadienoic acid terminus.

Cell Line↗
PubMed1995

The orphan receptor TAK1 acts as a repressor of RAR-, RXR- and T3R-mediated signaling pathways.

Recently, we reported the cloning and characterization of the novel orphan receptor TAK1. In this study, we analyze the interaction of TAK1 with a variety of response elements (RE's) and demonstrate that TAK1 binds effectively to RE's composed of the core motif PuGGTCA configured in direct repeats spaced by one or more nucleotides. TAK1 bound poorly to palindromic or inverted palindromic motifs and was unable to bind to a single core motif, suggesting that a dimeric site is required for binding. Transfection experiments with CV-1 cells revealed that TAK1 is able to repress retinoid- and thyroid-hormone-induced transactivation through a subset of retinoid and thyroid hormone RE's. Our studies indicate that the antagonism of RAR-mediated transactivation does not involve the formation of heterodimers between TAK1 and RAR or RXR but is due to the competition of TAK1 homodimers with RAR-RXR heterodimers and RXR homodimers for binding to RARE and RXRE, respectively. Our results suggest that the orphan receptor TAK1 can be a negative modulator of the regulation of gene expression mediated by retinoid and thyroid hormone signaling pathways.

Animals↗
PubMed1995

Similar ligand-induced conformational changes of thyroid hormone receptors regulate homo- and heterodimeric functions.

Thyroid hormone receptors (TRs) bind specific thyroid hormone response elements (TREs) as heterodimers with retinoid X receptors (RXRs) and act as transcriptional activators. As homodimers, TRs can bind a distinct set of sequences and function as ligand sensitive repressors. In our study, we compared the natural malic enzyme TRE (ME-TRE) as a model system for the TR/RXR heterodimer pathway to the chicken lysozyme silencer element F2-TRE which is strongly bound and regulated by TR/TR homodimers. Using electrophoretic mobility shift assays, transient transfections with a variety of natural and synthetic triiodothyronine and thyroxine derivatives as well as limited proteolytic analysis, we show that the natural homo- and heterodimeric pathways show similar ligand requirements. Furthermore, we observe that the ligand-induced conformational changes in the receptor proteins that either result in a loss of TR/TR homodimer binding and release of transcriptional repression or in transcriptional activation of TR/RXR heterodimers are indistinguishable. Therefore, we propose that in TR/TR homodimers and TR/RXR heterodimers very similar moieties of the receptors are involved in ligand binding and subsequent conformational changes that lead to loss of gene repression (TR/TR homodimer) and gain of gene activation (TR/RXR heterodimer).

Animals↗
PubMed1995

Regulation of H1(0) gene expression by nuclear receptors through an unusual response element: implications for regulation of cell proliferation.

Cloning and sequence analysis of the 5'-flanking region of the human H1(0) histone gene, a differentiation-specific member of the H1 family, has revealed several potential regulatory elements. In this study, we have characterized the interactions of nuclear receptors with an unusual response element consisting of two half-sites arranged as a direct repeat with an 8-bp spacer (DR-8). Thyroid hormone receptors (TR) bind this DR-8 as homodimers and heterodimers with RXR. Retinoic acid receptors (RARs) also bind as heterodimers with RXR to the DR-8, and this binding is enhanced in the presence of retinoic acid (RA) and/or 9-cis RA. Reporter constructs containing the DR-8 allowed a several-fold induction by T3 in the presence of TRs. RAR alpha and RAR beta allowed RA-dependent transcriptional activation whereas RAR gamma mostly increased basal activity. 9-cis RA inhibited the T3 response, indicating a hormonal cross-talk among the subfamily of nuclear receptors. Two orphan receptors, COUP-TF and v-erbA, also bind the DR-8 sequence in the human H1(0) promoter. COUP-TF, which usually represses RAREs, enhances transcriptional activation through the DR-8 whereas v-erbA completely represses TR-RXR induction of the H1(0) gene. Thus, a number of signaling pathways that play important roles during development and differentiation are able to influence the transcription rate of this special H1 subtype directly through a DR-8 response element in its promoter. Because H1(0) expression levels inversely correlate with cell proliferation, our data suggest that several nuclear receptors and the v-erbA oncogene can influence cell proliferation via the regulation of H1(0) expression.

Base Sequence↗
PubMed1995

Thyroid hormone receptor-beta mutants associated with generalized resistance to thyroid hormone show defects in their ligand-sensitive repression function.

Thyroid hormone (T3) responses are mediated by two receptors, TR alpha and TR beta, that have been shown to require heterodimer formation with the retinoid-X receptors for effective interaction with most T3-responsive elements (TRE). In addition, it has been shown recently that one type of TRE, an inverted palindrome (IP) with a 4-, 5-, or 6-base pair spacer, can also bind TR homodimers with high affinity. This binding, however, is sensitive to T3, which suggests that TR homodimers could have important biological roles as T3-sensitive repressors. Here we have analyzed eight natural TR beta mutants associated with the syndrome of generalized resistance to thyroid hormone (GRTH). These receptor mutants are characterized by a variably decreased affinity for T3. We show here that their homodimer binding characteristics are altered. For example, kindred GH binds as a homodimer more weakly to DNA than wild-type (WT), whereas mutant PV forms clearly stronger homodimer complexes than WT even in the presence of TREs that bind WT receptor homodimers poorly. Although other mutants were able to bind IP-6 elements efficiently as homodimers, these homodimers showed a decreased sensitivity to T3 in accordance with their reduced affinities for the ligand. In vivo, six of the eight mutants were able to function as strong repressors on IP sites located 3' of the TATA box. Although T3 released repression by WT TR beta, the hormone did not release repression by some of the mutant receptors, and elevated concentrations of T3 were required to release repression by other mutants. Importantly, most of the GRTH-associated mutants were able to function as potent dominant negative repressors of WT in the homodimer pathway, whereas they showed little dominant negative activity in the heterodimer-dependent transcriptional activation pathway. Only one of the eight GRTH mutants, a deletion of the carboxy-terminus, was found to have a strong dominant negative activity on both T3 response pathways. Our data suggest a dominant negative mechanism of action for GRTH mutants that is consistent with their homodimer binding characteristics to IP-TREs and correlates well with T3 resistance in patients.

Animals↗
PubMed1995

TOR: a new orphan receptor expressed in the thymus that can modulate retinoid and thyroid hormone signals.

Vitamin A and other fat-soluble hormones and vitamins have important roles as modulators of essential biological processes such as homeostasis, development, differentiation, and oncogenesis and also as regulators of the immune system. The active form of vitamin A, retinoic acid, as well as vitamin D3 and thyroid hormones exert their actions by binding to specific nuclear receptors that represent one subfamily of the steroid/thyroid hormone receptor superfamily. To identify new members of the retinoid/thyroid hormone receptor subfamily that could play a role in the immune system, a screening of a T cell cDNA library was performed using a retinoid X receptor probe. A clone was isolated encoding a novel nuclear receptor expressed mainly in the thymus and T cell lines. This new receptor, TOR (thymus orphan receptor), is most closely related in both its DNA-binding domain and ligand-binding domain, 90% and 53%, respectively, to ROR alpha/RZR alpha and clusters with these two receptors and RZR beta in a phylogenetic tree, when both the DNA-binding domain and the ligand-binding domain sequences of nuclear receptors are compared. Thus, TOR is part of a subgroup of receptors, one of which has recently been reported to be activated by melatonin. TOR binds specifically to a direct repeat of the half-site sequence 5'-AGGTCA-3' with a four- or five-nucleotide spacer, DNA sequences that also serve as binding sites for thyroid hormone (TR), and retinoic acid receptors (RAR). In transient transfection experiments TOR does not activate a reporter gene carrying these sequences in the absence or the presence of any known nuclear receptor ligands. TOR, however, is able to repress TR and RAR activity on DR-4-TREs or DR-5-RAREs, respectively. Therefore, our data suggest that TOR, similar to COUP-TF, can negatively regulate retinoic acid and thyroid hormone signals. However, the response elements recognized by TOR and COUP-TF differ as do the expression patterns of these receptors. Thus, one important role of TOR could be to modulate retinoid and thyroid hormone signals in the thymus.

3T3 Cells↗
PubMed1995

Thyroid hormone receptor homodimers can function as ligand-sensitive repressors.

Unlike the steroid hormone receptors that bind their response elements as homodimers, thyroid hormone receptor (TRs) as well as retinoic acid receptors and several other receptors have been shown to require heterodimerization with retinoid X receptors (RXR) for efficient binding to most response elements. In this article we have compared in detail TR DNA binding and its gene-regulatory characteristics in the presence and absence of RXR. We observe that in the absence of RXR, TRs are able to bind with high affinity as homodimers to a subset of thyroid hormone response elements consisting of two AGGTCA motifs arranged as inverted palindromes. This binding is inhibited by T3, which prevents TR homodimers from functioning as ligand-dependent transcriptional activators. We demonstrate that TR homodimers can act as potent ligand-responsive repressors, in particular when binding to sites 3' of the TATA box. Thus, TRs appear to have important regulatory functions in the absence of RXRs. This is strongly supported by our observations that some naturally occurring TR beta mutants that have been associated with generalized thyroid hormone resistance as well as the v-erbA oncogene are defective in this activity. Thus ligand-sensitive repression by TRs is an important regulatory mechanism.

Animals↗
PubMed1994

A new class of retinoids with selective inhibition of AP-1 inhibits proliferation.

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.

Animals↗
PubMed1994

A specific defect in the retinoic acid response associated with human lung cancer cell lines.

The effects of retinoic acid (RA) are mainly mediated by its nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs) that regulate target gene expression by binding to specific RA-response elements (RAREs). RAR beta is the best characterized RA-responsive gene. Due to the presence of a RARE (beta RARE) in its promoter, the expression of the RAR beta 2 is markedly increased in response to RA in most epithelial tissues, including lung. Recently, it was observed that the RAR beta gene is not expressed in a number of human lung cancer cell lines, suggesting a possible correlation between abnormal expression of the RAR beta gene and lung cancer development. In this study, we investigate the RA response in human lung cancer cell lines. Here we report that the expression of the RAR beta gene cannot be regulated by RA in the majority of human lung cancer cell lines examined, while the general response to RA is intact. The nonresponsiveness of the RAR beta gene results from different defects in the response mechanism. Interestingly, we find in some cell lines a differential responsiveness of the beta RARE such that the element is inactive in its natural promoter context but active when linked to the heterologous tk promoter. Importantly, we also observe that the presence of retinoid receptors is not sufficient for the induction of the RAR beta gene. This suggests that specific factors determine the RA responsiveness in the context of its natural promoter. Our observation that the RA nonresponsiveness of the RAR beta promoter is a common feature of human lung cancer cell lines suggests that balanced RAR beta expression is an essential feature for the maintenance of a normal state of lung tissue.

Blotting, Northern↗
PubMed1994

A synthetic retinoid antagonist inhibits the human immunodeficiency virus type 1 promoter.

Retinoids regulate a broad range of biological processes and affect cell growth and differentiation of many cell types, including the immune system. Recently, it was reported that human immunodeficiency virus type 1 (HIV-1) expression in macrophages is enhanced by retinoic acid (RA). Retinoid signals are mediated by the RA receptors (RARs) and retinoid X receptors (RXRs) that bind to specific RA responsive elements (RAREs) in the promoter region of susceptible genes. Here, we report on a RARE in the long terminal repeat (LTR) region that allows activation of the HIV-1 LTR. The RARE is composed of two consensus RARE half-sites (A/GGGTCA) arranged as a palindrome separated by 9 nucleotides and is activated by both RAR/RXR heterodimers and RXR homodimers. We show that the COUP (chicken ovalbumin upstream promoter) orphan receptors also bind to the HIV-1 RARE and repress the retinoid response of the HIV-1 RARE or the HIV-1 LTR. Furthermore, a newly discovered synthetic retinoid is shown to be a potent inhibitor of retinoid-induced activation of the HIV-1 RARE. These observations suggest additional approaches for the inhibition of HIV replication.

Animals↗
PubMed1994

Recombinant human retinoic acid receptor beta. Binding of synthetic retinoids and transcriptional activation.

All-trans-retinoic acid mediates cell growth and differentiation by binding to and then activating nuclear retinoid receptor proteins that regulate gene transcription. Recombinant human retinoic acid receptor beta was cloned and expressed in Escherichia coli as a fusion protein rMBP-RAR beta with maltose-binding protein to facilitate purification. After isolation from bacterial lysates, rMBP-RAR beta was used for binding with selected retinoids. Scatchard analysis with [11,12-3H2]all-trans-retinoic acid gave a Kd of 0.34 nM. Competitive binding studies with a series of conformationally restricted aromatic retinoids indicated that the Ki values for binding to rMBP-RAR beta correlated with the logs of the EC50 values for gene transcriptional activation (p < or = 0.05) and with those for the relative activation compared to that of all-trans-retinoic acid (p < or = 0.01). Inspection of binding-activation correlation diagrams indicates candidate structures for improved retinoid agonists or antagonists.

Amino Acid Sequence↗
PubMed1994

Vertebrate receptors: molecular biology, dimerization and response elements.

The nuclear receptors are a large family of ligand sensitive transcriptional regulators. They bind specific DNA sequences (response elements) in the promoter region of responsive genes. While the steroid hormone receptors bind as homodimers just two basic response elements, the retinoid and thyroid hormone receptor family interacts with a diverse set of response elements as homo- and heterodimers. The retinoid X receptors have a central role, since they form heterodimers with a whole subclass of receptors, consistent with the pleiotropic effects of retinoids. Differences in the mechanism of actions between steroid hormone receptors and other members of the family are discussed.

Animals↗
PubMed1994

A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily.

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides (DR-4). Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/RXR alpha heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.

Amino Acid Sequence↗
PubMed1994

Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor.

The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.

Amino Acid Sequence↗
PubMed1994

An evolutionary conserved COUP-TF binding element in a neural-specific gene and COUP-TF expression patterns support a major role for COUP-TF in neural development.

The COUP transcription factors (COUP-TF and ARP-1) are the most highly conserved members of the nuclear receptor superfamily throughout evolution. Previous studies indicated that COUP orphan receptors may be involved in early neurogenesis in Drosophila and zebrafish. Here we identified a neural-specific gene, arrestin, whose transcription can be regulated by endogenous COUPs through a DR-7 element (direct repeat with a 7-base pair spacer) located upstream of the transcription start site. Importantly, the COUP binding site of the arrestin gene promoter is conserved among mouse, bovine, and human. However, the mouse element is also capable of responding to retinoic acid while the element in the human gene does not. Expression of COUP-TF correlates with the known expression sites of the arrestin gene in vivo, notably during the differentiation of the retina. We also show that COUP-TF is expressed in a spatio-temporally defined pattern in the murine central nervous system during embryogenesis. It appears that the expression pattern of COUP-TF is unique in certain regions of the developing brain, which would indicate a novel role for COUP-TF and/or ARP-1, distinct from their role in restricting other hormonal signaling pathways. Together our data suggest that COUPs play a crucial role in controlling a subset of neural-specific programs during development.

Animals↗
PubMed1993

Conformational effects on retinoid receptor selectivity. 1. Effect of 9-double bond geometry on retinoid X receptor activity.

A major challenge is the development of retinoids with selective biological activities. Recently, studies on retinoid response mechanisms indicate that retinoids activate two classes of nuclear receptor proteins, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Here, we analyze the activity of a series of (E)- and (Z)-stilbenecarboxylic acids for gene transcriptional activation of the RARs and RXR-alpha to determine the optimum pharmacophore for receptor activation. The data obtained indicate that RAR and RXR response pathways can be separated by using the appropriate ligand. The conformations of (Z)-4-[2-(5-,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prop en-1-yl]benzoic acid (Z)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)propen-2-yl]benzoic acid were examined by experimental and theoretical methods to establish the appropriate conformation of the latter that specifically activated the retinoid RXR. A palladium(0)-catalyzed aryl bromide-arylboronic acid coupling under nonanhydrous conditions was used to construct a biaryl bond in the conformationally restricted retinoid 2'- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthaleny)biphenyl-4-c arboxylic acid, which had RXR activity.

Binding Sites↗