Ana M. Jiménez-Lara

Association with Ets-1 Causes Ligand- and AF2-Independent Activation of Nuclear Receptors

ABSTRACT The vitamin D receptor (VDR) normally functions as a ligand-dependent transcriptional activator. Here we show that, in the presence of Ets-1, VDR stimulates the prolactin promoter in a ligand-independent manner, behaving as a constitutive activator. Mutations in the AF2 domain abolish vitamin D-dependent transactivation but do not affect constitutive activation by Ets-1. Therefore, in contrast with the actions of vitamin D, activation by Ets-1 is independent of the AF2 domain. Ets-1 also conferred a ligand-independent activation to the estrogen receptor and to peroxisome proliferator-activated receptor α. In addition, Ets-1 cooperated with the unliganded receptors to stimulate the activity of reporter constructs containing consensus response elements fused to the thymidine kinase promoter. There is a direct interaction of the receptors with Ets-1 which requires the DNA binding domains of both proteins. Interaction with Ets-1 induces a conformational change in VDR which can be detected by an increased resistance to proteolytic digestion. Furthermore, a retinoid X receptor-VDR heterodimer in which both receptors lack the core C-terminal AF2 domain can recruit coactivators in the presence, but not in the absence, of Ets-1. This suggests that Ets-1 induces a conformational change in the receptor which creates an active interaction surface with coactivators even in the AF2-defective mutants. These results demonstrate the existence of a novel mechanism, alternative to ligand binding, which can convert an unliganded receptor from an inactive state into a competent transcriptional activator.

Interaction of Vitamin D and Retinoid Receptors on Regulation of Gene Expression

Vitamin D and retinoic acid (RA) receptors (VDRs and RARs, respectively), bind as heterodimers with the retinoid X receptor (RXR) to hormone response elements (HREs) in target genes. In some cases RA and vitamin D can cooperate to stimulate transcription through the same HRE. However, VDR/RXR heterodimers bind in a transcriptionally unproductive manner and without a defined polarity on certain RA response elements, and under these circumstances vitamin D inhibits the response to RA. Although competition for binding to DNA may contribute to this inhibitory response, titration of common coactivators by VDR also appears to be involved in transrepression. Therefore, the transcriptional response to RA and vitamin D depends on a complex combinatory pattern of interaction among different receptors wih DNA and coactivators.

Lysine 246 of the vitamin D receptor is crucial for ligand-dependent interaction with coactivators and transcriptional activity.

Mutant K246A in the predicted helix 3 of the ligand-binding domain, as well as mutants L417S and E420Q in helix 12, which contains the core ligand-dependent transcriptional activation domain (AF-2), were generated to examine AF-2 activity of the vitamin D receptor (VDR). These mutations abolished vitamin D-dependent transactivation. In addition, VDR mediates a ligand-dependent repression of the response of the retinoic acid receptor β2 promoter to retinoic acid, and the helix 3 and helix 12 mutants were unable to mediate transrepression. Furthermore, the VDR mutants, but not the native receptor, enhanced phorbol ester induction of the activator protein-1-containing collagenase promoter. The helix 3 and helix 12 mutations strikingly reduced the ability of VDR to interact with the coactivators steroid receptor coactivator-1, ACTR, and the CREB-binding protein. As a consequence, overexpression of steroid receptor coactivator-1 increased vitamin D-dependent transactivation by VDR but not by the K246A mutant. These results indicate that the lysine 246 participates, together with residues in helix 12, in the recruitment of coactivators and that AF-2 activity is involved both in ligand-dependent transactivation and in transrepression by VDR.

The vitamin D receptor binds in a transcriptionally inactive form and without a defined polarity on a retinoic acid response element

Heterodimers of the vitamin D receptor (VDR) with the retinoid X receptor (RXR) bind in a transcriptionally unproductive manner to the retinoic acid response element present in the retinoic acid receptor‐β2 promoter. This element is composed of a direct repeat (DR) of the sequence PuGTTCA spaced by five nucleotides. However, the same sequence separated by three nucleotides (DR3) acts as a strong vitamin D response element. Here we show that the polarity of binding of the heterodimers to the DR3 was 5′‐RXR‐VDR‐3′, whereas on the DR5, both heterodimeric partners bind indistinctly to the 5′ or 3′ hemi‐sites. These results suggest that the response elements can allosterically regulate the conformation of the receptors to determine positive or negative regulation of gene expression. Despite the altered polarity, the DR5‐bound heterodimer was able to recruit the nuclear receptor coactivator ACTR in a vitamin D‐dependent fashion. Furthermore, binding of the corepressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) to the RXR/VDR heterodimer on a DR5 was not observed. Binding of RXR/VDR heterodimers to DRs with different transcriptional outcomes may generate selectivity and provide a greater complexity and flexibility to the vitamin D responses.—Jimenez‐Lara, A. M., Aranda, A. The vitamin D receptor binds in a transcriptionally inactive form and without a defined polarity on a retinoic acid response element. FASEB J. 13, 1073–1081 (1999)

Synergy between RA and TLR3 promotes type I IFN- dependent apoptosis through upregulation of TRAIL pathway in breast cancer cells

Due to its ability to regulate the growth, differentiation and apoptosis of cancer cells, retinoic acid (RA) is considered a signaling molecule with promising therapeutic potential in oncology. In this study, we show that RA is able to induce the intrinsic ability of breast cancer cells to recognize double-stranded RNA (dsRNA) through the upregulation of Toll-like receptor 3 (TLR3) expression. RA, co-administered with the dsRNA mimicker polyinosinic–polycytidylic acid (poly(I:C)), synergizes to mount a specific response program able to sense dsRNA through the concurrent upregulation of TLR3, the dsRNA helicases melanoma differentiation-associated antigen-5 (MDA-5) and RA-inducible gene-1 (RIG-1), and the dsRNA-activated protein kinase (PKR) expression, leading breast cancer cells to specifically express downstream transcriptional targets of dsRNA sensors, such as interferon-β (IFNβ), interleukin-8 (IL-8), chemokine (C-C motif) ligand 5 (CCL5), and C-X-C motif Chemokine 10 (CXCL10). A TLR3-dependent apoptotic program is also induced by RA and poly(I:C) co-treatment that correlates with the induction of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and contributes to block breast cancer cell proliferation. The mechanisms of apoptosis induced by RA/poly(I:C) in breast cancer cells involve type I IFN autocrine signaling, caspase-8 and caspase-3 activation, as well as TRAIL signaling. Our results reveal important links among RA, TLR3 and TRAIL and highlight the combined use of RA and poly(I:C) as a potential effective tumor therapy by improving the apoptotic response of cancer cells with low sensitivity to the action of synthetic dsRNA.

Retinoic acid protects human breast cancer cells against etoposide-induced apoptosis by NF-kappaB-dependent but cIAP2-independent mechanisms

BackgroundRetinoids, through their cognate nuclear receptors, exert potent effects on cell growth, differentiation and apoptosis, and have significant promise for cancer therapy and chemoprevention. These ligands can determine the ultimate fate of target cells by stimulating or repressing gene expression directly, or indirectly through crosstalking with other signal transducers.ResultsUsing different breast cancer cell models, we show here that depending on the cellular context retinoids can signal either towards cell death or cell survival. Indeed, retinoids can induce the expression of pro-apoptotic (i.e. TRAIL, TNF-Related Apoptosis-Inducing Ligand, Apo2L/TNFSF10) and anti-apoptotic (i.e. cIAP2, inhibitor of apoptosis protein-2) genes. Promoter mapping, gel retardation and chromatin immunoprecipitation assays revealed that retinoids induce the expression of this gene mainly through crosstalk with NF-kappaB. Supporting this crosstalk, the activation of NF-kappaB by retinoids in T47D cells antagonizes the apoptosis triggered by the chemotherapeutic drugs etoposide, camptothecin or doxorubicin. Notably apoptosis induced by death ligands (i.e. TRAIL or antiFAS) is not antagonized by retinoids. That knockdown of cIAP2 expression by small interfering RNA does not alter the inhibition of etoposide-induced apoptosis by retinoids in T47D cells reveals that stimulation of cIAP2 expression is not the cause of their anti-apoptotic action. However, ectopic overexpression of a NF-kappaB repressor increases apoptosis by retinoids moderately and abrogates almost completely the retinoid-dependent inhibition of etoposide-induced apoptosis. Our data exclude cIAP2 and suggest that retinoids target other regulator(s) of the NF-kappaB signaling pathway to induce resistance to etoposide on certain breast cancer cells.ConclusionsThis study shows an important role for the NF-kappaB pathway in retinoic acid signaling and retinoic acid-mediated resistance to cancer therapy-mediated apoptosis in breast cancer cells, independently of cIAP2. Our data support the use of NF-kappaB pathway activation as a marker for screening that will help to develop novel retinoids, or retinoid-based combination therapies with improved efficacy.

Vitamin D Represses Retinoic Acid-Dependent Transactivation of the Retinoic Acid Receptor-β2 Promoter: The AF-2 Domain of the Vitamin D Receptor Is Required for Transrepression1

Retinoic acid (RA)-dependent activation of the RA receptor b2 (RARb2) gene in embryonal carcinoma cells is mediated by binding of retinoid receptor heterodimers (RAR/RXR) to a RA response element (RARE) located closely to the TATA box. We have analyzed the effect of vitamin D on the response of the RARb2 promoter to RA in pituitary GH4C1 cells that coexpress receptors for retinoids and vitamin D. Incubation with vitamin D markedly reduced the response to RA caused by transcriptional interference of the vitamin D receptor (VDR) on the RARE. This DNA element binds VDR/RXR heterodimers with high affinity, and these inactive heterodimers can displace active RAR/RXR from the RARE. Overexpression of RXR in GH4C1 cells, as well as incubation with BMS649 (a RXR-specific ligand), increased the inhibitory effect of vitamin D, suggesting that the VDR/RXR heterodimer is the repressive species and that titration of RXR is not responsible for this inhibition. Although DNA binding could be required for full potency of the inhibitory activity of VDR, it is not absolutely required because a truncated receptor (VDR D1‐ 111), lacking the DNA binding domain, also displays repressor activity. Furthermore, the ability to mediate transrepression by vitamin D was strongly decreased when a mutant VDR in which the last 12 C-terminal aminoacids have been deleted (VDR DAF-2) was used. Because this region contains the domain responsible for liganddependent recruitment of coactivators, titration of common coactivators for VDR and RAR could be involved in the inhibitory effect of vitamin D. In agreement with this hypothesis, overexpression of E1A, which can act as a RARb2 promoter-specific coactivator, significantly reversed repression by vitamin D. (Endocrinology 140: 2898 ‐2907, 1999)

Retinoic acid regulates Schwann cell migration via NEDD9 induction by transcriptional and post-translational mechanisms

Schwann cell migration is essential during the regenerative response to nerve injury, however, the factors that regulate this phenomenon are not yet clear. Here we describe that retinoic acid (RA), whose production and signaling activity are greatly enhanced during nerve regeneration, increases Schwann cell migration. This is accompanied by the up-regulation of NEDD9, a member of the CAS family of scaffold proteins previously implicated in migratory and invasive behavior in gliomas, melanomas and the neural crest cells from which Schwann cells derive. This RA-induced NEDD9 accumulation is due to augmented mRNA levels, as well as an increase of NEDD9 protein stability. Although all NEDD9 phospho-isoforms present in Schwann cells are induced by the retinoid, the hormone also changes its phosphorylation status, thus altering the ratio between the different isoforms. Silencing NEDD9 in Schwann cells had no effect on basal migratory ability, but completely abrogated RA-induced enhanced migration. Collectively, our results indicate that RA could be a major regulator of Schwann cell migration after nerve injury, thus offering a new insight into peripheral nerve repair.

Pro-apoptotic signaling induced by Retinoic acid and dsRNA is under the control of Interferon Regulatory Factor-3 in breast cancer cells

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic–polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.