Susana Álvarez

A unique secondary-structure switch controls constitutive gene repression by retinoic acid receptor

In the absence of ligand, some nuclear receptors, including retinoic acid receptor (RAR), act as transcriptional repressors by recruiting corepressor complexes to target genes. This constitutive repression is crucial in metazoan reproduction, development and homeostasis. However, its specific molecular determinants had remained obscure. Using structural, biochemical and cell-based assays, we show that the basal repressive activity of RAR is conferred by an extended β-strand that forms an antiparallel β-sheet with specific corepressor residues. Agonist binding induces a β-strand–to–α-helix transition that allows for helix H11 formation, which in turn provokes corepressor release, repositioning of helix H12 and coactivator recruitment. Several lines of evidence suggest that this structural switch could be implicated in the intrinsic repressor function of other nuclear receptors. Finally, we report on the molecular mechanism by which inverse agonists strengthen corepressor interaction and enhance gene silencing by RAR.

Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state

A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro.

Aldo-keto reductases from the AKR1B subfamily: retinoid specificity and control of cellular retinoic acid levels.

NADP(H)-dependent cytosolic aldo-keto reductases (AKRs) have been added to the group of enzymes which contribute to oxidoreductive conversions of retinoids. Recently, we found that two members from the AKR1B subfamily (AKR1B1 and AKRB10) were active in the reduction of all-trans- and 9-cis-retinaldehyde, with K(m) values in the micromolar range, but with very different k(cat) values. With all-trans-retinaldehyde, AKR1B10 shows a much higher k(cat) value than AKR1B1 (18 min(-1)vs. 0.37 min(-1)) and a catalytic efficiency comparable to that of the best retinaldehyde reductases. Structural, molecular dynamics and site-directed mutagenesis studies on AKR1B1 and AKR1B10 point that subtle differences at the entrance of their retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinoid cyclohexene ring, analyzed here further, also influence such specificity. Overall it is suggested that the rate-limiting step in the reaction mechanism with retinaldehyde differs between AKR1B1 and AKR1B10. In addition, we demonstrate here that enzymatic activity of AKR1B1 and AKR1B10 lowers all-trans- and 9-cis-retinoic acid-dependent trans-activation in living cells, indicating that both enzymes may contribute to pre-receptor regulation of retinoic acid and retinoid X nuclear receptors. This result supports that overexpression of AKR1B10 in cancer (an updated review on this topic is included) may contribute to dedifferentiation and tumor development.

Retinoic acid and Wnt/β-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

Retinoid receptors and therapeutic applications of RAR/RXR modulators.

Retinoic acid receptors (RARs) are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth, differentiation, survival and death. Due to their regulatory potential, these nuclear receptors (NRs) are major drug targets for a variety of pathologies, including cancer and metabolic diseases. A large amount of RAR- and RXR-selective ligands, ranging from (partial) agonists to antagonists and inverse agonists, have been designed and the corresponding structural and functional analyses have provided deep insight into the molecular basis of ligand action. Ligands regulate, via allosteric conformational changes, the ability of these NRs to interact with different sets of coregulators, which in turn recruit enzymatically active complexes/machineries. Here, we describe strategies in the design of selective RXR and RAR modulators and review the structural mechanisms by which the diverse pharmacological classes of compounds modulate receptor functions. Finally, we discuss the perspectives for retinoid- and rexinoid-based therapies.

Inhibition of IκB Kinase-β and Anticancer Activities of Novel Chalcone Adamantyl Arotinoids

On the basis of the observations that chalcone 7 (MX781) and some related adamantyl arotinoids (AdArs) inhibit IkappaB alpha kinase beta (IKKbeta) activity, inhibit cell growth, and induce apoptosis in cancer cells, a new series of AdArs structurally related to 7 have been designed and synthesized. Modifications were intended to reduce or eliminate RAR activity, and we evaluated the effect of the novel analogues of 7 on IKKbeta activity and proliferation of a variety of cancer cell lines (leukemia, Jurkat; prostate, PC-3; breast carcinomas, T47D, MDA-MB-468). Consistent with the design principles, the biological activities of these AdArs do not appear to be RAR-mediated, since most analogues are unable to activate RAR-mediated transactivation and exhibit significantly diminished antagonist activity. All compounds are capable of inducing apoptosis in Jurkat cells, as demonstrated by elevated DEVDase activity and externalization of phosphatidylserine. Several of the analogues elicit stronger growth inhibitory activity against prostate (PC-3) and breast (MDA-MB-468) carcinoma cells, which contain elevated basal IKK activity; this antiproliferative activity correlates with increased inhibition of recombinant IKKbeta in vitro, suggesting that the anticancer activities of these AdArs might be related to the inhibition of IKK/NFkappaB signaling.

Understanding abnormal retinoid signaling as a causative mechanism in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a frequently occurring source of severe neonatal respiratory distress. It has been hypothesized that abnormal retinoid signaling contributes to the etiology of this developmental anomaly. Here, we use rodent models toward specifically understanding the role of retinoid signaling in the developing diaphragm and how its perturbation is a common mechanism in drug-induced CDH. This includes monitoring of retinoic acid (RA) response element (RARE) activation with RARE-lacZ mice, RA supplementation studies, systematic analyses of the expression profile of key elements in the RA signaling pathway within the developing diaphragm, and the in utero delivery of a RA receptor (RAR) antagonist. These data demonstrate the timing of RARE perturbation by CDH-inducing teratogens and the efficacy of RA supplementation. Furthermore, a detailed profile of retinoid binding proteins, synthetic enzymes, and retinoid receptors within primordial diaphragm cells was obtained. The expression profile of RAR-alpha was particularly striking in regard to its overlap with the regions of primordial diaphragm affected in multiple CDH models. Blocking of RAR signaling with the pan-RAR antagonist BMS493 induced a very high degree of CDH, with a marked left-right sidedness that depended on the timing of drug delivery. Collectively, these data demonstrate that retinoid signaling is essential for normal diaphragm development, providing further support to the hypothesis that abnormalities related to the retinoid signaling pathway cause diaphragmatic defects. This study also yielded a novel experimental model that should prove particularly useful for further studies of CDH.

HIV-1 infection and neurocognitive impairment in the current era.

Brain HIV‐1‐infection may result in a syndrome of profound cognitive, behavioral and motor impairment known as AIDS dementia complex (ADC) in adults and HIV‐related encephalopathy in children. Although the introduction of highly active antiretroviral therapy (HAART) has prolonged and improved the lives of infected individuals, it is clear that HAART does not provide complete protection against neurological damage in HIV/AIDS. HIV‐1 associated dementia is a complex phenomenon, which could be the result of several mechanisms caused by those players using different intracellular signaling pathways. Understanding the causes of neurodegeneration during HIV‐1 infection and the factors which certain individuals develop disease can provide researches on new therapeutic targets to positively affect disease outcomes. Controlling CNS viral replication with HAART is an essential primary approach, but it should be complemented with adjunctive CNS‐directed therapeutics. Understanding the nature of HIV‐1 infection within the CNS as well as inflammatory responses will ultimately lead to the elimination of HIV‐associated neurocognitive disorders. Copyright

Retinoid receptor subtype-selective modulators through synthetic modifications of RARγ agonists

A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify novel RARgamma antagonists based on the structure of known RARgamma agonists. The transcriptional activities of the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR subtype-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a selective RARgamma antagonist, some of them are endowed with interesting activities. In particular 24a acts as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RARalpha than TTNPB and synergizes at low concentration with TTNPB-bound RARalpha but not RARbeta or RARgamma. Similarly, 24c synergizes with TTNPB-bound RARgamma and exhibits RARalpha,beta antagonist activity. Compounds 24b and 25b are strong RARalpha,beta-selective antagonists without agonist or antagonist activities for RARgamma. Compounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial agonist/antagonists have been defined.

Retinoic acid signaling targets Hox genes during the amphioxus gastrula stage: insights into early anterior-posterior patterning of the chordate body plan.

Previous studies of vertebrate development have shown that retinoic acid (RA) signaling at the gastrula stage strongly influences anterior-posterior (A-P) patterning of the neurula and later stages. However, much less is known about the more immediate effects of RA signaling on gene transcription and developmental patterning at the gastrula stage. To investigate the targets of RA signaling during the gastrula stage, we used the basal chordate amphioxus, in which gastrulation involves very minimal tissue movements. First, we determined the effect of altered RA signaling on expression of 42 genes (encoding transcription factors and components of major signaling cascades) known to be expressed in restricted domains along the A-P axis during the gastrula and early neurula stage. Of these 42 genes, the expression domains during gastrulation of only four (Hox1, Hox3, HNF3-1 and Wnt3) were spatially altered by exposure of the embryos to excess RA or to the RA antagonist BMS009. Moreover, blocking protein synthesis with puromycin before adding RA or BMS009 showed that only three of these genes (Hox1, Hox3 and HNF3-1) are direct RA targets at the gastrula stage. From these results we conclude that in the amphioxus gastrula RA signaling primarily acts via regulation of Hox transcription to establish positional identities along the A-P axis and that Hox1, Hox3, HNF3-1 and Wnt3 constitute a basal module of RA action during chordate gastrulation.