Biomedical subjects

M Pfahl

Publications and source records attributed to M Pfahl.

At least 19 recordsLinked to original sources

PubMed1992

Retinoids selective for retinoid X receptor response pathways.

Retinoids have a broad spectrum of biological activities and are useful therapeutic agents. Their physiological activities are mediated by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). RARs, as well as several related receptors, require heterodimerization with RXRs for effective DNA binding and function. However, in the presence of 9-cis-retinoic acid, a ligand for both RARs and RXRs, RXRs can also form homodimers. A series of retinoids is reported that selectively activates RXR homodimers but does not affect RAR-RXR heterodimers and thus demonstrates that both retinoid response pathways can be independently activated.

Animals↗

PubMed1992

A retinoic acid response element from the rat CRBPI promoter is activated by an RAR/RXR heterodimer.

The expression of the rat cellular retinol binding protein I (rCRBPI) can be upregulated in vivo by retinoic acid (RA). Here we have analyzed the rCRBPI promoter region and compared it to the corresponding mouse sequence. We find that the CRBPI 5' flanking region has been highly conserved between rat and mouse, including a RA response element (RARE) approximately 1 kb upstream of the start of transcription. The RARE is of the direct repeat type with a two nucleotide spacer. Like other direct repeat RAREs, this response element is activated by RAR alpha and beta but not by RAR gamma 1. Furthermore, the rCRBPI-RARE is most effectively activated when both RAR and RXR are present. In addition RAR/RXR heterodimers are required for efficient binding to the rCRBPI-RARE, while RARs or RXR alone do not interact effectively with this response element. The rCRBPI gene is therefore most likely activated in vitro by a RAR/RXR heterodimer.

Animals↗

PubMed1992

Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid.

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.

Animals↗

PubMed1992

Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors.

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.

Animals↗

PubMed1992

COUP orphan receptors are negative regulators of retinoic acid response pathways.

The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number of orphan receptors whose biological roles have not yet been determined. Although much has been learned in recent years about RA receptor (RAR) functions, little is known about how specific RA response programs are restricted to certain tissues and cell types during development and in the adult. It has been recently shown that RAR activities are regulated by retinoid X receptors (RXR) through heterodimer formation. In an effort to isolate and further characterize nuclear receptors that modulate RAR and/or RXR activities, we have screened cDNA libraries by using a RXR alpha cDNA probe. Two clones, COUP alpha and COUP beta, identical and closely related to the orphan receptor COUP-TF, were obtained. We show that COUP proteins dramatically inhibit retinoid receptor activities on certain response elements that are activated by RAR/RXR heterodimers or RXR homodimers. COUP alpha and -beta bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell- or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.

Base Sequence↗

PubMed1992

RAR gamma 2 expression is regulated through a retinoic acid response element embedded in Sp1 sites.

At the level of transcription, all signals of the vitamin A derivative retinoic acid (RA) are mediated by the RA receptors (RARs) as well as the retinoid X receptors (RXRs). The control of expression of the various receptor subtypes and their specific isoforms appears to be strictly regulated and can be assumed to play a pivotal role during development and in the adult tissue. It has previously been shown that the RAR beta 2 isoform can regulate its own synthesis through an RA response element (RARE) in its promoter. Recent evidence suggests that the expression of other RAR isoforms, including that of RAR gamma 2, are also regulated by RA. We present evidence that expression of the RAR gamma 2 isoform can be regulated through the RARE in its own promoter region. Similar to the beta 2 RARE, the gamma 2 RARE consists of a 6-bp direct repeat with a 5-nucleotide spacer, but it has different functional features, including receptor specificity, basal-level activity, and affinity for RAR. In agreement with recent observations, this response element is bound most effectively by RAR/RXR heterodimers. Single-base-pair mutations had different effects on the activity of this RARE. The gamma 2 RARE is surrounded by several binding sites for the transcription factor Sp1. Cotransfected Sp1 enhanced strongly the activity of gamma 2 promoter reporter constructs in Drosophila cells. Our data suggest an important role for RAR-containing heterodimers and Sp1 in the regulation of RAR gamma 2 expression.

Animals↗

PubMed1992

Heterodimeric receptor complexes determine 3,5,3'-triiodothyronine and retinoid signaling specificities.

Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) have been shown to interact with nuclear auxiliary proteins resulting in heteromeric complexes that bind strongly to their responsive elements. Recently the retinoid X receptors (RXRs) have been identified as one class of these nuclear proteins. RXRs strongly increase binding of TRs and RARs to a synthetic thyroid hormone (and retinoic acid) responsive element. Here results show that the binding of the heteromeric complexes to various natural response elements is highly specific and dictated by the partner of RXR in the complex. TR alpha and TR beta formed complexes with RXR alpha that strongly and selectively bound to natural thyroid hormone responsive elements, i.e. those from the rat alpha-myosin heavy chain gene and the rat malic enzyme gene. RXR alpha complexes with RAR alpha, RAR beta, and RAR gamma bound selectively to retinoic acid responsive elements from the human RAR beta 2 gene (hRAR beta 2), the gene of the rat cellular retinol binding protein I and the human apolipoprotein A1 gene. Under the conditions used here RXR alpha by itself did not bind to any of the responsive elements tested. Although TRs and RARs formed heterodimers with RXR in solution, these complexes were strongly stabilized by specific, high affinity response elements, but not by low affinity response elements. Transfection analyses showed strong synergism between receptors that formed effective heterodimers in transcriptional activation on several but not all response elements. Overall, these data demonstrate that RARs and TRs are unlikely to function as monomers or homodimers on the response elements investigated here and require RXRs or comparable proteins for effective response element activation.

Animals↗

PubMed1991

Differential effects of all-trans and 13-cis-retinoic acid on mRNA levels of nuclear retinoic acid receptors in rat lung and liver.

The effects of three retinoids, all-trans-retinoic acid (all-trans-RA), 13-cis-RA, and etretin were examined on mRNA abundance of nuclear retinoic acid receptors (RAR-alpha, beta, and gamma) in lung and liver of retinol deficient and chow fed rats. All-trans-RA increased lung RAR-beta mRNA levels 5 or 11-fold in chow fed and retinol deficient rats, respectively. Similarly to lung, liver RAR-beta mRNA levels were 3-fold higher in retinol deficient rats fed all-trans-RA than the rats fed cottonseed oil. Lung RAR-gamma mRNA levels were also induced 2-fold by all-trans-RA. In contrast to this, 13-cis-RA and etretin at equimolar doses failed to enhance lung or liver RAR-beta or lung RAR-gamma mRNA levels in retinol deficient rats. These data for the first time show that all-trans-RA is more effective than its 13-cis-isomer in regulating the expression of RAR-beta and gamma transcripts in adult animal.

Animals↗

PubMed1991

6'-substituted naphthalene-2-carboxylic acid analogs, a new class of retinoic acid receptor subtype-specific ligands.

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.

Animals↗

PubMed1991

Retinoic acid affects the expression of nuclear retinoic acid receptors in tissues of retinol-deficient rats.

The multitude of biological effects of the vitamin A metabolite, retinoic acid, are mediated by nuclear retinoic acid receptors (RARs), which are members of the steroid/thyroid hormone receptor superfamily. RAR-alpha, -beta, and -gamma are encoded by three genes from which multiple isoforms can be generated. Recent studies suggest that the expression of at least some RAR isoforms can be regulated by retinoic acid in certain cell lines. Here we examined regulation of RAR expression in the adult animal. RARs were analyzed by Northern blots from lung, liver, and testes of retinol-deficient rats. Retinol deficiency caused a 65-70% decrease in the mRNA levels of lung and liver RAR-beta, whereas no change was observed in RAR-alpha and -gamma mRNA levels in these organs. In the testes of retinol-deficient animals, two transcripts, RAR-alpha 1 (3.7 kb) and RAR-alpha 2 (2.8 kb), were detected as compared with one RAR-alpha 1 (3.7 kb) transcript in retinol-sufficient testes. When retinol-deficient rats were orally administered 1 dose of retinoic acid (100 micrograms per rat), lung RAR-beta mRNA levels started to increase after 1 hr and reached a 16-fold higher level after 4 hr; after 4 hr these retinoic acid-fed rats also showed a 7-fold increase in liver RAR-beta mRNA levels as compared with levels in the retinol-deficient rats. In contrast, liver, lung, and testes RAR-alpha transcripts remained either unchanged or showed only a slight increase in response to retinoic acid. RAR-gamma was constitutively expressed in lung, and its mRNA levels were induced 2-fold by retinoic acid. These results show tissue diversity in the rapid induction of RAR-beta and RAR-gamma by retinoic acid in the adult animal and suggest distinct roles for the various receptor isoforms in the control of the retinoid response.

Animals↗

PubMed1991

Identification of retinoids with nuclear receptor subtype-selective activities.

Retinoic acid (RA) and its synthetic analogues, retinoids, have shown promising results in the prevention of epithelial carcinogenesis and in the treatment of acute promyelocytic leukemia and various proliferative skin disorders. Retinoid action on gene regulation is mediated by three distinct nuclear retinoic acid receptor subtypes, RA receptors alpha, beta, and gamma. The existence of multiple RA receptors has raised the possibility that receptor subtype-specific retinoids with reduced side effects can be developed. To analyze the activity of retinoids at the molecular level, we used a receptor activation assay. RA and 22 retinoids were compared on the three receptor subtypes. We found the alpha receptor to be least sensitive to activation by RA and the gamma receptor to be most sensitive. Compared with RA, one of the retinoids showed increased activity for the alpha and beta receptors. Three retinoids revealed no gene activation activity and showed no antagonistic effects when assayed in the presence of RA. Surprisingly, several of the retinoids were efficient activators of the beta and gamma receptors but poor activators or nonactivators of the alpha receptor. Our data demonstrate that the three RA receptor subtypes have differential ligand activation specificities and that the design of receptor subtype-selective retinoids is possible.

Animals↗

PubMed1991

Genomic organization of the retinoic acid receptor gamma gene.

The retinoic acid receptors (RAR) belong to the large family of ligand responsive gene regulatory proteins that includes receptors for steroid and thyroid hormones. These proteins contain two highly conserved domains, involved in determining their DNA and ligand binding activities. Three distinct RARs have been identified (RAR alpha, beta, and gamma) which are encoded by genes on separate chromosomes. Additional isoforms of the three receptors have been described that all differ in the N-terminal regions. To gain insight into the genomic organization and mechanisms of RAR isoform generation, we have analyzed the genomic structure of the RAR gamma gene. The major portion of the RAR gamma protein, including DNA and ligand binding domains, is encoded by seven exons that are identical for all RARg isoforms and are represented by a relatively small portion of the RAR gamma gene. The major portion of this gene encodes separate N-terminal exons for gamma 1 and gamma 2 isoforms and several exons for gamma 1 untranslated regions. We show that RAR gamma 2 transcription is regulated by its own promoter. In comparison with the steroid receptor subfamily, various splice sites of RAR gamma occur at altered positions, suggesting that the RAR subfamily has diverged early during evolution.

Amino Acid Sequence↗

PubMed1991

Regulatory functions of a non-ligand-binding thyroid hormone receptor isoform.

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.

Base Sequence↗

PubMed1991

Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities.

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

Base Sequence↗

PubMed1991

Antagonism between retinoic acid receptors.

In the developing mouse, retinoic acid receptors (RARs) beta and gamma 1 are expressed in characteristic spatiotemporal patterns which are correlated with different developmental fates of the respective tissues. Understanding the cues that regulate the expression of the various RARs may therefore provide insights into the process of tissue diversification. Transcription of RAR beta is rapidly upregulated through a retinoic acid-responsive element (here referred to as the beta RARE) in its promoter. Like RAR alpha and RAR beta, RAR gamma 1 has been implicated in the activation of the beta RARE. Therefore, it is puzzling that RAR beta and RAR gamma 1 appear to be expressed in reciprocal patterns. In the present report, we show that RAR gamma 1, one of the two predominant RAR gamma isoforms, can inhibit the activity of RAR gamma 2, RAR beta, and endogenous RAR on the beta RARE. In contrast, the three RAR gamma isoforms tested and RAR beta activated a palindromic thyroid hormone response element with similar levels of efficiency. The differential activity of RAR gamma 1 compared with that of RAR beta appears to reside in both the N-terminal and the C-terminal halves of RAR gamma 1. RAR gamma 1-mediated inhibition of other RARs may involve competition for the response element as well as direct interaction with other receptors and might be part of a regulatory system contributing to the characteristic tissue distribution of the various RARs.

Animals↗

PubMed1991

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.

Amino Acids↗

PubMed1991

Inhibition of estrogen receptor activity by the tumor promoter 12-O-tetradeconylphorbol-13-acetate: a molecular analysis.

Cell proliferation and phenotype of cells from female reproductive tissues are regulated by estrogens. It is therefore important to understand how estrogen action can be modulated. It recently has been reported that certain nuclear receptors can antagonize the tumor promoter 12-O-tetradeconylphorbol-13-acetate (TPA) by direct interaction with the transcription factor AP-1, and that the AP-1 constituents cJun and cFos can inhibit receptor activity. This mutual antagonism appears to be based on direct protein-protein interaction. In the human breast cancer cell line MCF-7, TPA leads to growth arrest and altered cell morphology. We have investigated here whether in MCF-7 cells and other cell lines AP-1 and estrogen receptors (ERs) can inhibit each other's activity. We find that TPA or the AP-1 components cJun and cFos can inhibit estradiol-dependent estrogen receptor activity in most cell lines investigated. In addition, ER mRNA is rapidly down-regulated in MCF-7 cells. Gel retardation experiments show that ER DNA binding is inhibited in vitro by cJun protein, while ER also can inhibit cJun DNA binding. However, in vivo we do not observe inhibition of AP-1 activity by ER in the cell lines investigated here. On the contrary, we observed an enhancing effect at low ER concentrations on AP-1. Together our data suggest a new regulatory pathway by which ER activity can be modulated by AP-1. Several mechanisms including ER-AP-1 protein interaction appear to be involved.

Base Sequence↗

PubMed1991

Thyroid hormone receptors repress estrogen receptor activation of a TRE.

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.

Base Sequence↗