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Molecular Endocrinology, doi:10.1210/me.2007-0062
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Molecular Endocrinology 21 (10): 2391-2402
Copyright © 2007 by The Endocrine Society

Rapid, Nongenomic Actions of Retinoic Acid on Phosphatidylinositol-3-Kinase Signaling Pathway Mediated by the Retinoic Acid Receptor

Susana Masiá, Susana Alvarez, Angel R. de Lera and Domingo Barettino

Biology of Hormone Action Unit (S.M., D.B.). Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia [Consejo Superior de Investigaciones Cientificas (CSIC)], E-46010 Valencia, Spain; Universidade de Vigo (S.A., A.R.d.L.), Departamento de Química Orgánica, Facultad de Ciencias, E-36200 Vigo, Spain

Address all correspondence and requests for reprints to: Dr. Domingo Barettino, Instituto de Biomedicina de Valencia (Consejo Superior de Investigaciones Cientificas). Jaime Roig, 11, E-46010 Valencia, Spain. E-mail: dbarettino{at}ibv.csic.es.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR({alpha}ß{gamma})L–/L– null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RAR{alpha} and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RAR{alpha} (Myr-RAR{alpha}) was targeted to plasma membrane. Transfection of Myr-RAR{alpha} to mouse embryo fibroblast-RAR({alpha}ß{gamma})L–/L– null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RETINOIC ACID (RA), the biologically active form of vitamin A, plays a major role as physiological regulator of important biological processes, such as early embryonic development, and the development of certain organs and systems, etc. (1). RA is an important signaling molecule for the development of the nervous system, especially by controlling proliferation and differentiation of neural cells (2, 3). The actions of RA and its derivatives are mediated by two types of receptors, retinoic acid receptors (RARs) and retinoic X receptors (RXRs), belonging to the nuclear hormone receptor superfamily, which encompasses receptors for steroid and thyroid hormones, vitamins A and D, and many other signaling molecules (4). Nuclear hormone receptors have been traditionally considered as ligand-regulated transcription factors, acting through binding to specific DNA elements on the promoters of target genes and ultimately leading to changes in the expression levels of those genes. However, novel nongenomic mechanisms of signal transduction through nuclear hormone receptors have been described. These rapid, nongenomic actions do not rely on gene transcription or protein synthesis, but involve ligand-induced modulation of signal transduction pathways (5, 6, 7, 8).

Administration of RA to neuroblastoma cells in vitro leads to proliferative arrest and neuronal differentiation (9, 10, 11). These results prompted the introduction of RA and its derivatives into therapeutic protocols for neuroblastoma patients, with success especially in the treatment of minimal residual disease (12, 13). In a previous paper we have described that RA treatment of SH-SY5Y neuroblastoma cells results in activation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation (14). Although the mechanisms by which RA regulates gene transcription are well understood, very little is known about the molecular mechanisms underlying the activation of PI3K/Akt signaling pathway by RA. We report here that activation of PI3K by RA occurs through a nongenomic action of the classical nuclear RAR. In contrast to what has been reported for steroid receptors, where interaction of the receptor with the regulatory subunit of PI3K (p85) was under the control of ligand, (15, 16, 17), we show that RAR forms a stable complex with p85-PI3K. Activation of PI3K by ligand-bound RAR is mediated by a novel mechanism in which the binding of RAR by its cognate ligand results in the formation of a signaling complex by recruiting the catalytic subunit of PI3K. In addition, the experiments shown here suggest a role for RA in regulating the intracellular location of that complex including RAR and the two subunits of PI3K.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rapid, Nongenomic Activation of PI3K and ERK1/2 MAPK Pathways by RA
Administration of RA to SH-SY5Y neuroblastoma cells results in activation of the PI3K signaling pathway (14). When this phenomenon was analyzed in its correct timeframe, we observed that RA rapidly activated the PI3K signaling pathway, and specific phosphorylation of Akt kinase in Ser473 could be detected within 5 min of RA treatment (Fig. 1AGo). Rapid activation of the PI3K signaling pathway by RA argued for an atypical, nongenomic activation event and against a classical transcriptional action of RA. In fact, RA treatment could induce phosphorylation of Akt kinase in the absence of newly synthesized proteins in cycloheximide (CHX)-treated cells as well as in the absence of new transcription in actinomycin D (AMD)-treated cells (Fig. 1BGo). Activation of signaling pathways by RA is not limited to PI3K/Akt, but also the ERK1/2 MAPK pathway was rapidly activated in RA-treated neuroblastoma cells, as detected by Western blot with phosphorylation-specific ERK1/2 antibodies (Fig. 1CGo).


Figure 1
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  		Fig. 1. Rapid, Nongenomic Activation of PI3K/Akt and ERK1/-2 Signaling Pathways by RA Treatment in SH-SY5Y Neuroblastoma Cells

A, Rapid activation of PI3K signaling pathway by RA. Cells were treated with RA (1 µM) for the times indicated in the figure, and total cell extracts were prepared. The phosphorylation state of Akt was analyzed by Western blot with specific antibodies against Akt phosphorylated in Ser473 (AKT-P). The filter was reprobed with antibodies against total Akt (AKT). Each lane contains 25 µg total protein. B, Rapid activation of PI3K by RA did not required new gene transcription or newly synthesized proteins. Neuroblastoma cells were pretreated for 30 min with the protein synthesis inhibitor CHX (10 µg/ml) or the transcription inhibitor AMD (1 µg/ml). Afterward, they were treated with RA (1 µM) or vehicle for 5 min, and the phosphorylation state of Akt was analyzed by Western blot as described in the legend of panel A. C, Rapid activation of ERK1/2 signaling pathway by RA. Cells were treated with RA (1 µM) for the times shown in the figure, and total cell extracts were prepared. The phosphorylation state of ERK1/2 MAPKs was analyzed by Western blot with specific antibodies against the phosphorylated (Tyr204) form of ERK 1/2 (ERK-P). The filter was reprobed with antibodies against ERK2 (ERK2). Each lane contains 25 µg total protein.

 
Rapid activation of signaling pathways by RA appeared to be not restricted to neuroblastoma cells. Mouse embryo fibroblast NIH-3T3 cells expressed considerable amounts of RAR{gamma} (Fig. 2AGo) and responded to RA treatment by activating PI3K (Fig. 2BGo) and ERK1/2 (Fig. 2DGo) signaling pathways (see also Ref. 18). Activation of the PI3K signaling pathway in response to RA treatment occurred in serum-deprived NIH-3T3 cells (Fig. 2BGo), as well as in cells growing in the presence of 10% newborn calf serum (Fig. 2CGo). Although in serum-fed cells Akt phosphorylation basal levels were higher than in serum-starved cells, RA treatment resulted in increased Akt phosphorylation (Fig. 2CGo). As was the case for neuroblastoma cells, activation of PI3K by RA in NIH-3T3 cells was not prevented by CHX or AMD treatments. As expected, RA-induced phosphorylation of Akt was impaired by treatment with the specific PI3K inhibitor LY294002 (Fig. 2CGo).


Figure 2
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  		Fig. 2. Activation of PI3K/Akt and ERK1/2 Signaling Pathways by RA in NIH-3T3 Cells

A, RAR{gamma} is expressed in NIH-3T3 cells. RAR{gamma} (~50 kDa, arrow) was detected in total cell extract by Western blot with specific antibodies. The positions of the corresponding marker bands and their molecular masses (in kilodaltons) are shown on the left side of the blot. Gel lane contained 25 µg total proteins. B, Rapid activation of PI3K by RA in NIH-3T3 cells. Cells were serum starved for 14 h and then treated with RA (1 µM) for the times indicated. The phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. C, Rapid activation of PI3K by RA did not required new gene transcription or newly synthesized proteins. NIH-3T3 cells were pretreated for 30 min with the transcription inhibitor AMD (1 µg/ml), the protein synthesis inhibitor CHX (10 µg/ml), or the PI3K-specific inhibitor LY294002 (LY, 10 µM). Afterward they were treated with RA (1 µM) or vehicle for 10 min, and the phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. D, Rapid activation of ERK1/2 signaling pathway by RA in NIH-3T3 cells. Cells were serum starved for 14 h and then treated with RA (1 µM) for the times indicated. Total cell extracts were prepared, and the phosphorylation state of ERK1/2 MAPKs was analyzed by Western blot as described in the legend of Fig. 1CGo. AKT-P, Phosphorylated Akt; ERK-P, phosphorylated ERK.

 
Involvement of Nuclear Receptor RAR in RA-Induced Rapid Nongenomic Activation of PI3K
The first type of evidence supporting the involvement of the nuclear RAR in RA-induced nongenomic actions came from the pharmacological features of the activation of PI3K by RA in neuroblastoma cells. First, the concentrations of RA required for activation of the PI3K signaling pathway (as low as 10 nM; see Fig. 3AGo) are compatible with the observed Kd of RARs for RA in the nanomolar range (19). Second, all the different RAR ligands tested up to now, including RAR-specific synthetic retinoids, have been able to activate PI3K similarly to all-trans-RA. As shown in Fig. 3BGo, both natural and synthetic RAR-selective agonists [all-trans-RA, 13-cis-RA, 4[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)-1-propenyl] benzoic acid (TTNPB), Am580, N-(4-hydroxyphenyl)-retinamide (4-HPR), and ALRT1550] as well as pan-RAR agonists (9-cis-RA and LG 100567) induced phosphorylation of Akt in Ser473 (Fig. 3BGo). Next, we tested whether the activation of PI3K signaling pathway by RA or a RAR-selective agonist like TTNPB could be prevented by the pan-RAR antagonist AGN193109 (20). Surprisingly, treatment of cells with AGN193109 (5 µM) could not block the activation of PI3K by RA or TTNPB (0.5 µM). Moreover, AGN193109 itself acts as an agonist and activated Akt phosphorylation (Fig. 3CGo). However, in the same concentrations AGN193109 prevented RA-induced transcriptional activation of RARB, one of the prominent RA-regulated genes in neuroblastoma cells, as shown by Northern Blot hybridization (Fig. 3DGo). Therefore, AGN193109 showed a dissociated antagonist/agonist profile, having a potent transcriptional antagonistic activity, but acting as an agonist in respect to PI3K activation.


Figure 3
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  		Fig. 3. Pharmacological Profile of the Activation of the PI3K/Akt Signaling Pathway by RA and Its Derivatives in Neuroblastoma Cells

A, Neuroblastoma cells were treated during 10 min with different RA concentrations as indicated in the figure, and the phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. B, Activation of PI3K by different RAR agonists. Neuroblastoma cells were treated during 10 min with all-trans-RA (RA), 9-cis-RA (9c-RA), 13-cis-RA (13c-RA), TTNPB, Am580, 4-HPR (fenretinide), ALRT1550 (ALRT), and LG100567 (LG) at 1 µM. Afterward, the phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. C, Effect of the RAR transcriptional antagonist AGN193109 on the activation of PI3K by RAR agonists. Neuroblastoma cells were treated during 10 min with 0.5 µM RA (RA), 0.5 µM TTNPB (TTNPB), 0.5 µM RA, and 5 µM AGN193109 (RA+AGN), 0.5 µM TTNPB and 5 µM AGN193109 (TTNPB+AGN), and 5 µM AGN193109 alone (AGN). Afterward, the phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. D, As a control, the effect of the RAR antagonist AGN193109 on the transcriptional induction of RARB, encoding the RARß receptor, in neuroblastoma cells was tested. Cells were treated with 0.5 µM RA (RA), or 0.5 µM RA and 5 µM AGN193109 (RA+AGN) during 24 h. Afterward, total RNA was prepared and the expression of RARB transcript (~ 3 kb) was analyzed by Northern blot with a 32P-labeled RARB probe. Each lane contained 15 µg total RNA. The 28S rRNA band in the blot stained with methylene blue is shown as internal loading control (rRNA). AKT-P, Phosphorylated Akt.

 
The second type of evidence supporting the involvement of nuclear RAR in the activation of PI3K was obtained by loss and gain of function experiments. Cultured mouse embryo fibroblasts (MEFs) from wild-type animals responded to RA treatment by activating the PI3K/Akt pathway (Fig. 4AGo). However, activation of the PI3K/Akt pathway by RA was abolished in a mouse embryo fibroblast cell line devoid of all the tree RAR isoforms, named MEF(RAR{alpha}ß{gamma})L–/L– (21) (Fig. 4BGo). Reintroduction of a functional RAR{alpha} gene through a retroviral expression vector resulted in restoration of RA-induced PI3K/Akt activation (Fig. 4CGo).


Figure 4
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  		Fig. 4. RA-Induced PI3K Activation Was Abolished in RAR Null MEF Cells but Was Restored by Reintroduction of RAR Expression

A, RA treatment rapidly activates PI3K in wild-type MEF cells. Wild-type MEF cells were serum starved for 14 h and treated with 1 µM RA for the times indicated on the figure and total cell extracts were prepared. The phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. B, RA-induced PI3K activation is abolished in MEF(RAR{alpha}ß{gamma})L–/L– cells. MEF(RAR{alpha}ß{gamma})L–/L– cells were serum starved for 14 h and treated with 1 µM RA for the times indicated on the figure, and total cell extracts were prepared. As control for the integrity of the PI3K signaling pathway, cells were serum stimulated with 10% FBS for 10 min (lane labeled SE). The phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. C, Retrovirus-mediated reexpression of RAR{alpha} restored RA-induced PI3K activation. MEF(RAR{alpha}ß{gamma})L–/L– cells and a derivative cell line expressing RAR{alpha} through retroviral transfection, MEF(RAR{alpha}ß{gamma})L–/L–+RAR{alpha}, were serum starved for 14 h and treated with 1 µM RA for the times indicated on the figure, and total cell extracts were prepared. The phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. The filter was reprobed additionally with antibodies against RAR{alpha}. MEF-wt, Wild-type MEF cells; P-AKT, phosphorylated Akt.

 
A third type of evidence supporting the involvement of RAR in RA-induced activation of the PI3K/Akt signaling pathway was obtained by assaying PI3K activity on immunoprecipitated fractions of control and RA-treated SH-SY5Y cells (Fig. 5Go). Anti-RAR{alpha} immunoprecipitated fractions were assayed together with anti-PI3K-p110 immunoprecipitates as positive control and control IgG immunoprecipitates as negative control. PI3K activity could be detected associated with RAR{alpha} immunocomplexes only in RA-treated cells, whereas no activity above background levels was detected in RAR{alpha}-immunoprecipitated fractions from untreated cells.


Figure 5
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  		Fig. 5. PI3K Activity Was Physically Associated with Ligand-Bound RAR{alpha}

PI3K activity was assayed in vitro on immunoprecipitated fractions of control (–) and RA-treated (1 µM, 10 min.) (+) SH-SY5Y neuroblastoma cells. Immunoprecipitation reactions were set with anti-RAR{alpha} and anti-p110-PI3K antibodies, and parallel control reactions with a nonrelated antibody (n. r.) were set as controls. PI3K activity was assayed on the immunoprecipitates in the presence of phosphatidylinositol and [{gamma}-32P]-TP as described in Materials and Methods section. The caption shows a detail of the autoradiogram of the TLC plate showing the generation of phosphatidylinositol-3-phosphate (PIP) in vitro. Activity data (in arbitrary units) were obtained by quantification of the radioactivity in the spots with Phosphor capture screens in a Fujifilm FLA5000 laser scanner. IP, Immunoprecipitation.

 
All these experiments together strongly support a role for the classical nuclear RAR in rapid, nongenomic activation of PI3K signaling pathway by RA.

Ligand Differentially Regulates the Association between RAR and PI3K Subunits
The kinase assays performed on immunoprecipitates shown in Fig. 5Go suggest the idea of a physical association between RAR and PI3K that could be regulated by ligand binding to RAR, and prompted us to analyze the interactions of the PI3K subunits with RAR in SH-SY5Y neuroblastoma cells. Surprisingly, immunoprecipitation experiments followed by Western blot detection demonstrated the existence of a stable physical interaction between RAR{alpha} and the p85 regulatory subunit of PI3K in SH-SY5Y neuroblastoma cells. Antibodies against RAR{alpha} immunoprecipitated p85-PI3K from SH-SY5Y cell extract, but not an irrelevant antibody used as control (Fig. 6AGo). Conversely, a p85-PI3K antiserum immunoprecipitated RAR{alpha} (Fig. 6BGo). However, association of p85 and RAR{alpha} was observed independently of the presence of RA (Fig. 6Go, A and B). This association could be detected in the cell nucleus, as demonstrated by immunoprecipitation experiments carried out in nuclear extracts (Fig. 6CGo). In contrast, RA-induced association of p110-PI3K to RAR{alpha} was observed in immunoprecipitates from RAR{alpha} antibodies (Fig. 6AGo). Immunoprecipitation experiments with an antibody against p110-PI3K showed association between p110, p85, and RAR{alpha}, and ligand binding increased notably complex formation between p110 and p85 and RAR{alpha} (Fig. 6DGo). Taken together, the results of the immunoprecipitation experiments support the idea of a stable complex between RAR{alpha} and p85, the regulatory subunit of PI3K. Ligand binding to RAR would facilitate the formation of a signaling complex, by increasing the association of the p110 catalytic subunit of PI3K.


Figure 6
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  		Fig. 6. Interactions between RAR{alpha} and PI3K Subunits

A, Total cell extracts were prepared from neuroblastoma cells treated with 1 µM RA or vehicle for 10 min. About 150 µg protein from control or RA-treated cell extracts were incubated with 5 µg RAR{alpha} antibody. As control, parallel reactions were set with an unrelated antibody. Immunocomplexes were precipitated using antirabbit IgG beads (eBioscience), following the manufacturer’s instructions, and suspended in sample buffer containing freshly added 50 mM DTT. After Western blot the filter was sequentially developed with anti-p85-PI3K and anti-RAR{alpha}, using horseradish peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. The filter was reprobed additionally with an anti-p110-PI3K monoclonal antibody. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. B, Total cell extracts were prepared from neuroblastoma cells treated with 1 µM RA or vehicle for 10 min. About 150 µg protein from control or RA-treated cell extracts was incubated with 5 µg p85-PI3K antibody. As control, parallel reactions were set with an unrelated antibody. Immunocomplexes were precipitated as described in the legend of panel A. After Western blot, the filters were sequentially developed with anti-RAR{alpha} and anti-p85-PI3K, using horseradish peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. C, Nuclear extract was prepared from untreated neuroblastoma cells, and 150 µg protein from nuclear extract were incubated with 5 µg p85-PI3K antibody. As control, a parallel reaction was set with an unrelated antibody. Immunocomplexes were precipitated, and Western blot was carried out as described in the legend of panel A. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. D, RA administration increases association of p110-PI3K to RAR{alpha}-p85-PI3K complex in neuroblastoma cells. Total cell extracts were prepared from neuroblastoma cells treated with 1 µM RA or vehicle for 10 min. About 150 µg protein from control or RA-treated cell extracts was incubated with 5 µg p110-PI3K antibody. As control, parallel reactions were set with an unrelated antibody. Immunocomplexes were precipitated as described in the legend of panel A. After Western blot, the filters were sequentially developed with anti-RAR{alpha} and anti-p85-PI3K using horseradish peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. IP, Immunoprecipitation; n.r., nonrelated antibody; WB, Western blot.

 
We wanted to know whether a direct protein-protein interaction determines the association between RAR and p85-PI3K. This was tested in vitro by glutathione-S-transferase (GST) pull-down assay, using recombinant GST-RAR{alpha} fusion protein expressed in Escherichia coli and extracts from cells overexpressing p85-PI3K tagged with the hemagglutinin (HA) epitope. As shown in Fig. 7AGo, binding of p85-PI3K to GST-RAR{alpha} could not be detected, independently of the presence of RA. As positive control experiment, the assay readily detected the interaction of GST-RAR{alpha} and RXR{alpha}, which are known to form heterodimers in vitro (22). Consistently with that, immunoprecipitation experiments performed after cotransfection of expression plasmids for RAR{alpha} and p85{alpha}-HA into COS-7 cells did not result in the formation of a binary complex (Fig. 7BGo). Although most of p85-PI3K was in the precipitated fraction, RAR{alpha} was recovered quantitatively from the supernatant of the immunoprecipitation. Taken together, the results pointed out that the interaction between RAR and p85-PI3K is not direct. Therefore, other yet unknown proteins mediating or stabilizing RAR-p85 interactions may be involved in complex formation, which may became limiting in overexpression experiments.


Figure 7
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  		Fig. 7. The Interaction between RAR and p85-PI3K Does Not Appear to Be Direct

A, RAR{alpha} and p85-PI3K do not interact in vitro in GST pull-down assays. Recombinant GST-RAR{alpha} and GST protein were expressed in E. coli and bound to glutathione-agarose beads. GST-RAR{alpha} or GST beads were incubated with cell lysates of COS-7 cells expressing p85-PI3K-HA (HA-p85) or human RXR{alpha} (RXR{alpha}). Extract from nontransfected cells is used as negative control (C). Where indicated 1 µM RA (+) or vehicle (–) was added to the pull-down reaction. Bound proteins were detected by Western blot, and the filter was sequentially developed with anti-HA and anti-RXR{alpha} antibodies. For comparison, the amount of extracts loaded in the lanes labeled input corresponds to 25% of the amount loaded from the pull-down assays. B, RAR{alpha} and p85-PI3K did not form a complex when coexpressed in COS-7 cells. COS-7 cells were cotransfected with RAR{alpha} and p85-HA expression plasmids. Total cell extract (150 µg protein) was incubated with 5 µg anti-p85-PI3K antibody. As control a parallel immunoprecipitation was set with an unrelated IgG (n. r.). Immunocomplexes were precipitated as described in the legend of Fig. 6AGo. The supernatant of the p85 immunoprecipitation (snat) was also included in the Western blot, and the filters were sequentially developed with anti-RAR{alpha} and anti-p85-PI3K, using horseradish-peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. IP, Immunoprecipitation; WB, Western blot.

 
Influence of RAR Intracellular Localization on RA-Induced PI3K Activation
A paradox arises from the fact that PI3K activity takes place at the plasma membrane, whereas RAR, as other members of the nuclear receptor superfamily, is located mainly in the nucleus. Therefore, intracellular localization of RAR might be relevant for RA-induced activation of PI3K. To address this point, we have examined whether ectopic localization of RAR at the plasma membrane would affect the activation of PI3K by RA. For this purpose we have constructed a chimerical receptor fusing c-Src myristylation domain (23) to the N terminus of RAR{alpha} (Myr-RAR{alpha}). We have overexpressed this chimerical Myr-RAR{alpha} and wild-type RAR{alpha} in NIH-3T3 cells through retroviral vectors and examined their intracellular localization in conventional cell fractionation experiments. The results showed that most of the Myr-RAR{alpha} could be located in the nuclear fraction, as was the case for wild-type RAR{alpha}. However, considerable amounts of Myr-RAR{alpha} could be detected in the microsomal fraction and in purified plasma membrane, whereas wild-type RAR{alpha} was not detected in membrane fractions (Fig. 8AGo). In detergent-partition experiments Myr-RAR{alpha} could be detected in the detergent-resistant fraction, whereas wild-type RAR{alpha} could not (data not shown). As compared with those of wild-type RAR{alpha}, the levels of Myr-RAR{alpha} were strongly increased in detergent-resistant membrane microdomain fractions (lipid rafts), comigrating with caveolin 1 in sucrose gradients (Fig. 8BGo). Therefore, these experiments demonstrated that Myr-RAR{alpha} shows an increased presence in plasma membrane fractions, as compared with wild-type RAR{alpha}.


Figure 8
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  		Fig. 8. Ectopic Expression of RAR{alpha} in the Plasma Membrane Results in Increased Activation of the PI3K/Akt Signaling Pathway

A, Myr-RAR{alpha} is targeted to plasma membrane. Whole-cell extract, nuclear fraction, microsomal fraction, and plasma membrane fraction purified from sucrose gradients were obtained from retrovirally transfected NIH-3T3 cells overexpressing wild-type RAR{alpha} or Myr-RAR{alpha} chimerical receptor. RAR{alpha} and Myr-RAR{alpha} were detected by Western blot with antibodies against RAR{alpha}. Equal amounts of protein from RAR{alpha}- and Myr-RAR{alpha}-expressing cells were loaded. B, Myr-RAR{alpha} localizes in detergent-resistant membrane microdomains (lipid rafts). Triton X-100 extracts from NIH-3T3 cells overexpressing RAR{alpha} and Myr-RAR{alpha} were fractionated in parallel sucrose step gradients as described in Materials and Methods. Fractions were collected from each gradient (ten 0.4-ml fractions from the top to the bottom, fractions 1–10; fraction 10 included the pellet). The presence of RAR{alpha} in total cell extract (T) and the different fractions (1 2 3 4 5 6 7 8 9 10 ) were analyzed by Western blotting with specific antibodies. The filter was reproved with antibodies against caveolin-1 (cav1) to mark the low-density lipid rafts-containing fractions. C, Expression of Myr-RAR{alpha} increases the activity of PI3K/Akt signaling pathway. Parental MEF(RAR{alpha}ß{gamma})L–/L– cells and its derivatives expressing RAR{alpha} and Myr-RAR{alpha} through retroviral vectors were serum starved for 14 h, treated with vehicle (–) or RA (1 µM, 10 min) (+) as indicated on the figure, and total cell extracts were prepared. The phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. The filter was reprobed additionally with antibodies against RAR{alpha}. D, Transfection of Myr-RAR{alpha} increases the activity of PI3K/Akt signaling pathway. COS-7 cells were transfected with expression vectors encoding RAR{alpha}, Myr-RAR{alpha}, or empty vector (mock) by the calcium phosphate protocol. Medium was replenished after transfection, and 24 h later the cells were treated with vehicle or 1 µM RA for 10 min. Total cell extracts were prepared and the phosphorylation state of Akt was analyzed by Western blot as described in the legend of Fig. 1AGo. The filter was reprobed additionally with antibodies against RAR{alpha}. P-AKT, Phosphorylated Akt.

 
The ability of Myr-RAR{alpha} to activate PI3K signaling pathway was tested in retrovirally transfected MEF(RAR{alpha}ß{gamma})L–/L– cells. As compared with wild-type RAR{alpha}, Myr-RAR{alpha} was a potent activator of PI3K in the absence of RA, and, surprisingly, the addition of RA did not further increase PI3K activity (Fig. 8CGo). Similar results were obtained in transient transfection experiments in COS-7 cells (Fig. 8DGo). These results showed that the presence of the receptor at the plasma membrane results in increased activation of PI3K, although in those conditions binding of the ligand appears to be dispensable.

The above results are compatible with a model in which the binding of the ligand may play a role in the localization of RAR to the plasma membrane, allowing interactions with components of the signal transduction machinery leading to PI3K activation. To test that hypothesis, we have examined the intracellular location of RAR. Attempts to show the presence of RAR in plasma membrane from SH-SY5Y or NIH-3T3 cells by immunofluorescent confocal microscopy with a variety of commercially available anti-RAR antibodies failed (data not shown). A green fluorescent protein-tagged RAR{alpha} version (GFP-RAR{alpha}) was generated, and its expression vector was transiently or stably transfected to COS-7 cells. When analyzed by fluorescent confocal microscopy, the cells showed strong nuclear staining with few cells showing plasma membrane localization of GFP-RAR{alpha} fusion protein. However, a consistent plasma membrane pattern could not be detected, and the addition of RA did not affect GFP-RAR{alpha} intracellular distribution (data not shown). As an alternative approach, we have analyzed the distribution of endogenous RAR{alpha} in SH-SY5Y neuroblastoma cells by means of conventional biochemical cell fractionation experiments. RAR{alpha} could not be detected in microsomal or purified plasma membrane fractions by Western blot immunodetection (data not shown). Similar fractionation experiments were performed in NIH-3T3 cells. As expected, most of the RAR{gamma} was detected in the nuclear fraction. Administration of RA to NIH-3T3 cells resulted in a strong increase in the levels of RAR{gamma} into the membrane fractions (microsomal and purified plasma membranes) (Fig. 9AGo). Similarly, RAR{alpha}-p85 complexes could be detected in the microsomal fraction of SH-SY5Y neuroblastoma cells in experiments involving a combination of cell fractionation and immunoprecipitation. A RA-induced increase in the levels of those complexes in membrane fractions could be observed (Fig. 9BGo). Both experiments suggest the idea of a ligand-induced redistribution of the receptor to plasma membrane.


Figure 9
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  		Fig. 9. Ligand-Induced Localization of RAR to Plasma Membrane

A, Liganded RAR{gamma} is targeted to plasma membrane in RA-treated NIH-3T3 cells. Cells were treated with vehicle (–) or 1 µM RA (+) for 10 min. Whole-cell extract, nuclear fraction, microsomal fraction, and plasma membrane fraction purified from sucrose gradients were obtained as described in Materials and Methods and analyzed by Western blotting. Equal amounts of protein from untreated and RA-treated cells were loaded. The filters were probed sequentially with antibodies against RAR{gamma}, caveolin-1 (Cav1), and Lamin A (LamA). n.d., Not done. B, Detection of RAR{alpha}-p85 complexes in membrane fractions from SH-SY5Y cells. Microsomal fractions were prepared from neuroblastoma cells treated for 10 min with 1 µM RA (+) or vehicle (–). About 150 µg protein from microsomal fractions from control or RA-treated cells was incubated with 5 µg p85-PI3K antibodies as indicated. Immunocomplexes were precipitated as described in the legend of Fig. 6AGo. After Western blot, the filters were sequentially developed with anti-RAR{alpha} and anti-p85-PI3K, using horseradish peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. Note the anomalous migration of RAR{alpha} (~50 kDa) in the immunoprecipitate lanes that is due to comigration with IgG heavy chain (~50 kDa), which in its reduced form was not detected by the TrueBlot secondary antibodies. Total cell extract from SH-SY5Y cells was included as reference (T). IP, Immunoprecipitation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although the classical transcriptional effects of RA have been extensively studied (see Refs. 4 and 24 for recent reviews), little information is available about the nonclassical, rapid nongenomic effects of RA on signal transduction pathways and their physiological implications. A considerable amount of information on nongenomic actions of steroid hormones has accumulated during the last few years (see Refs. 6, 7, 8 and 25 for recent reviews), but only a few examples of nongenomic actions of RA have been reported (14, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34).

We have obtained insight into the mechanism of activation of PI3K by RA, and the results reported here support a novel mechanism, in which a stable complex including RAR, the regulatory subunit p85-PI3K, and other unknown proteins occurs in the absence of RA. Ligand binding to RAR facilitates the association of p110, the catalytic subunit of PI3K, to that complex and therefore promotes PI3K activity. In addition, we have shown experiments suggesting a role for RA in the regulation of the intracellular location of that signaling complex that could be relevant for PI3K activation, and recruitment of the signaling complex to plasma membrane resulted in increased PI3K activity. We believe that this novel mechanism could be extended to other members of the nuclear receptor superfamily, especially for those belonging to the retinoic/thyroid hormone /vitamin D subfamily of receptors.

The involvement of the classical nuclear hormone receptors in the nongenomic actions of steroid hormones and other ligands has been controversial over the past decade. However, considerable evidence has accumulated supporting that nuclear hormone receptors are also responsible for the rapid nongenomic actions of steroid hormones, especially for estrogen receptor (ER) (35, 36, 37, 38, 39). The participation of nuclear RAR in RA-mediated nongenomic actions is supported by the pharmacological profile of PI3K activation, by loss and gain function experiments performed in MEF(RAR{alpha}ß{gamma})L–/L– cells, and by the physical association between liganded RAR{alpha} and PI3K activity, as demonstrated in immunoprecipitation/PI3K assays. Although the failure of the RAR antagonist AGN193109 in preventing the activation of PI3K by RA or TTNPB could argue against the participation of RAR, AGN193109 itself is able to activate PI3K, suggesting a dissociated transcriptional antagonist/nongenomic agonist profile, as has been reported for other compounds such as the specific ER modulators estren and raloxifen (40, 41) or synthetic vitamin D3 analogs (42).

PI3K is an important signaling node on the control of a variety of cellular responses, including survival, growth, and differentiation (Ref. 43 for review). The mechanism depicted here involves RA-induced formation of a signaling complex, probably through stimulation of the association of p85 and p110 subunits of PI3K. The impact of the regulatory subunit p85 on PI3K activity has been controversial, and evidence supporting both positive and negative effects of p85 on PI3K activity exists in the literature (44, 45). Our results suggest that heterodimer formation between p85 and p110 results in increased PI3K activity. In addition, recruitment of p110 subunit to plasma membrane in the proximity of its substrate could affect its activation, as previously reported (46). A mechanism involving cellular retinol-binding protein I in the regulation of PI3K subunit heterodimerization has been reported recently in breast cancer cells, where RA signaling would have negative effects on PI3K subunit association and PI3K activity. However, the different context between adoptive long-term effects (47) and the rapid effects analyzed here could account for the controversy.

The presence of steroid hormone receptors at the plasma membrane and in caveolae and its importance for the cellular effects of steroid hormones are increasingly accepted. However, how the receptors are tethered to the membrane is still unclear, although the few reports addressing this point suggest that membrane localization could result from receptor interaction with specific membrane proteins and/or lipid modifications (for review see Ref. 25). Because RAR, as other members of the nuclear receptor superfamily, is located mainly in the nucleus, an intriguing paradox arises from the fact that rapid nongenomic actions of nuclear receptors appear to be initiated at the plasma membrane. Any model attempting to explain the mechanism of PI3K activation by RAR needs to address this point. The importance of the localization of the receptor at the plasma membrane became evident because ectopic expression of RAR{alpha} to plasma membrane by the introduction of a myristylation domain resulted in increased PI3K activation. Increased nongenomic signaling was also observed by targeting ER to the plasma membrane, but full activation required the addition of hormone (48). Conversely, chimerical or mutant ERs with reduced plasma membrane localization show reduced nongenomic activity (40, 49, 50). Some experiments shown here suggest that ligand binding may control the localization of the receptor, promoting the presence of ligand-bound RAR at the plasma membrane. However, we have failed to obtain formal demonstration for this hypothesis by confocal microscopy. Probably only a minor part of the RAR pool is engaged in nongenomic actions (<5%, as estimated from immunoprecipitation experiments). This could be close to the threshold level for conventional detection methodologies. As suggested here for RAR, a rapid ligand-induced association of vitamin D3 receptor to plasma membrane has been also reported (51, 52).

The mechanism proposed here shows striking differences with the prevalent model proposed to explain the nongenomic actions of steroid receptors as the ER (for a review see Ref. 25). In that case a pool of resident plasma membrane ER molecules interact there with the components of the signal transduction machinery in such a way that those interactions are under the control of the ligand, as described for the interactions between ER, glucocorticoid receptor, or androgen receptor and p85, the regulatory subunit of PI3K (15, 16, 17, 53). The different dynamics of the two subfamilies of nuclear hormone receptors and their peculiarities may have imposed different regulatory solutions to ultimately obtain the ligand-dependent regulation of PI3K activation. The unliganded receptors of the retinoic/thyroid subfamily are mainly nuclear, and they do not form inactive complexes with heat shock protein 90 (hsp90) and other proteins. A stable interaction of p85-PI3K with the receptor, shown here for RAR, was recently reported for thyroid hormone receptor (54, 55). If the presence of the complex including the receptor and p85-PI3K at the plasma membrane may increase upon ligand binding, as indicated by the experiments shown here, this may facilitate the recruitment of the catalytic subunit p110-PI3K to the complex and could allow interactions with other components of the signal transduction machinery. On the contrary, unliganded steroid receptors form an inactive complex with hsp90 and other proteins that keep the receptor in an inactive form. It appears conceivable that these interactions with hsp90 and/or other proteins within the unliganded steroid receptor complex could preclude an interaction with p85-PI3K, which only takes place once the complex is dissociated upon ligand binding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Treatments
SH-SY5Y human neuroblastoma cells, COS-7 cells, MEFs from wild-type animals, MEF(RAR{alpha}ß{gamma})L–/L– cells, and BOSC23 cells were cultured in DMEM with 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. NIH-3T3 cells were cultured in DMEM with 10% newborn calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cell cultures were kept in a humidified incubator at 37 C with 5% CO2. The medium was replaced every 3 d, and the cells were split before they reached confluence. CHX, AMD, Puromycin, LY294002, all-trans-RA, 9-cis-RA, 13-cis-RA, TTNPB, Am580, and 4-HPR (fenretinide) were purchased from Sigma Chemical Co. (St. Louis, MO). AGN193109 (UVI2109) (20), ALRT1550 (UVI2103) (56), and LG100567 (UVI2104) (57) were synthesized, and their spectroscopic and analytical data matched those reported in the literature. The different compounds were dissolved in ethanol or dimethylsulfoxide and added to the culture medium at the indicated concentrations.

RNA Analysis
Northern blot analysis of total RNA from SH-SY5Y cells with a 32P-labeled probe was performed as previously described (14). RARB probe consisted of a 1.4-kb DNA fragment containing the human RARß2 cDNA (58).

Western Blot and Immunoprecipitation
Whole-cell extracts were obtained by lysis of the cells in RIPA buffer (50 mM HEPES, pH 7.4; 150 mM NaCl; 1 mM EGTA) containing 0.5% Nonidet P-40, protease [1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptin] and phosphatase inhibitors (1 mM sodium ortovanadate, 1 mM NaF). After 10 min incubation on ice, the lysate was cleared by centrifugation (16,100 x g, 10 min, 4 C), and protein concentration was determined. Western blot analysis of proteins from whole-cell extracts was performed as described elsewhere (14). Antibodies against RAR{alpha}, RAR{gamma}, RXR{alpha}, caveolin 1, ERK2, Akt 1/2, and the phosphorylated (Tyr204) form of ERK1/2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated Akt (Ser473), RAR{alpha}, and lamin A were obtained from Cell Signal Technologies (Beverly, MA). Antibodies against PI3K-p85 and -p110 were from Upstate Biotechnology, Inc. (Lake Placid, NY). HA.11 monoclonal antibody was purchased from Covance Laboratories, Inc. (Madison, WI). Monoclonal antibody against p110-PI3K was a gift of Dr. A. C. Carrera (Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientificas-Centro Nacional de Biotecnologia, Madrid). Horseradish-peroxidase-conjugated secondary antibodies were obtained from GE Healthcare (Chalfont St. Giles, UK), Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and Santa Cruz Biotechnology. Chemiluminescent signals were developed with enhanced chemiluminescence (ECL) or ECL Plus (GE Healthcare).

For immunoprecipitation, cells were treated with RA (1 µM, 10 min) or vehicle, and cell extracts were prepared in 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40, and protease (1 mM PMSF, 40 µg/ml aprotinin, and 40 µg/ml leupeptin) and phosphatase inhibitors (1 mM sodium ortovanadate, 1 mM NaF). Alternatively, nuclear extract or microsomal fractions were prepared as described below in Subcellular Fractionation. Protein (~150 µg) from control or RA-treated cell extracts was incubated with 5 µg of antibodies against p85-PI3K, RAR{alpha}, or p110-PI3K as indicated. As control, parallel reactions were set with an unrelated antibody. Immunocomplexes were precipitated using antirabbit IgG beads (eBioscience, San Diego, CA), following the manufacturer’s instructions, and suspended in sample buffer containing freshly added 50 mM dithiothreitol (DTT). After Western blot as above, the filters were sequentially developed with the antibodies indicated in the figure, using horseradish peroxidase-conjugated Rabbit TrueBlot (eBioscience) as secondary antibodies. Chemiluminiscent signals were developed with ECL (GE Healthcare).

GST Pull-Down Assay
GST-RAR{alpha} (59) was expressed in E. coli BL21 cells and purified by using standard techniques. For the pull-down assays, 20 ng GST-RAR{alpha} or GST protein immobilized in glutathione-agarose beads was added to cell lysates (160 µg protein) from COS-7 cells expressing p85-PI3K-HA or RXR{alpha} in 500 µl 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM PMSF, 40 µg/ml aprotinin, 40 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM NaF, and 1 mM sodium ortovanadate. Extract from nontransfected COS-7 cells was used as negative control. Where indicated, 1 µM RA was included in the incubation mixture. After incubation (18 h, 4 C), the beads were washed three times with the same buffer, and the bound proteins were detected by Western blot.

PI3K Assay
Extracts from control or RA-treated (1 µM RA, 10 min) SH-SY5Y cells, containing 150 µg protein, were incubated with 5 µg RAR{alpha} antibodies, p110-PI3K antibodies, or an unrelated control IgG, and the immunocomplexes were recovered with protein A/G Plus-Agarose (Santa Cruz Biotechnology). The complexes were washed three times with 10 mM HEPES (pH 7.5), 0.1 mM EGTA. Kinase reactions were set by mixing 20 µl immunoprecipitates, 20 µl of 0.5 mg/ml L-{alpha}-phosphatidylinositol (Sigma) in 10 mM HEPES (pH 7.4), 0.1 mM EGTA, and 10 µl of 50 mM HEPES (pH 7.4), 25 mM MgCl2 containing 10 µCi [{gamma}-32P]ATP (PerkinElmer Life Science Products, Boston, MA; SA, 3000 Ci/mmol). Kinase assays were performed as described elsewhere (14). Quantification of the radioactivity on the spots was carried out with Phosphor capture screens in a Fujifilm FLA5000 laser scanner.

Transient and Retrovirus-Mediated Transfection
Myr-RAR{alpha} expression vector was generated by in-frame fusion of RAR{alpha} (2–462) to the 21 residues of the chick c-Src myristylation domain present in pCEFL-Myr expression vector (a gift of Dr. P. Crespo, IIB-University de Cantabria). Semiconfluent COS-7 cells were transfected by the calcium phosphate method with expression vectors for RAR{alpha} and Myr-RAR{alpha}. After 8–14 h of exposure to the precipitates, the medium was replenished. RA treatments took place after 24 h, and cells were lysed and cell extracts processed for Western blotting. Retroviral particles for the expression of RAR{alpha} and Myr-RAR{alpha} and its derivatives were prepared by transfecting retroviral vector expression vectors based on pBABE (60) into BOSC23 packaging cells (61) by the calcium phosphate method. After 2 d of incubation, the supernatants containing retrovirus were used to infect semiconfluent MEF(RAR{alpha}ß{gamma})L–/L– or NIH-3T3 cells, and pools of transfected cells were selected with puromycin (2.5 µg/ml). For immunoprecipitation, semiconfluent COS-7 cells were cotransfected with expression vectors for human RAR{alpha} and p85-PI3K-HA (62) by the calcium phosphate method. After 8–14 h of exposure to the precipitates, the medium was replenished, and after an additional 24 h the cells were lysed and cell extracts were processed for immunoprecipitation.

Subcellular Fractionation
Membrane fractions were obtained according to Ref. 52 with some modifications. SH-SY5Y neuroblastoma cells, wild-type, or retrovirally transfected NIH-3T3 cells overexpressing RAR{alpha} or Myr-RAR{alpha} were washed with PBS and harvested. Cells were pelleted by centrifugation and resuspended in TEDK buffer (10 mM Tris-HCl, pH 7.4; 0.3 M KCl; 1 mM EDTA; 1 mM DTT) containing protease (1 mM PMSF, 40 µg/ml aprotinin, 40 µg/ml leupeptin) and phosphatase inhibitors (1 mM sodium ortovanadate, 1 mM NaF). Cell suspension was homogenized in a glass-Teflon homogenizer, and the lysate was centrifuged first at 200 x g for 10 min to eliminate cell debris, and later to 16,100 x g for 20 min to pellet the nuclei and mitochondria. The cleared lysate was centrifuged at 100,000 x g for 45 min in a Beckmann SW60 rotor. The pellet containing the microsomal fraction was resuspended in 1.5 ml of TEDK containing 15% sucrose and laid on top of a 30–45% sucrose step gradient. Gradients were run at 76,000 x g for 3 h and the interface between 15–30% sucrose, containing purified plasma membranes, was picked up and diluted in TEDK, and the membranes were repelleted at 100,000 x g for 1 h. The purified plasma membrane was resuspended in TEDK buffer containing inhibitors.

Nuclear fraction was obtained by resuspending cells in 60 mM KCl, 15 mM NaCl, 20 mM Tris-HCl (pH 8), 0.15 mM spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA. Cell suspension was mixed with one volume of the same buffer containing 0.5% Nonidet P-40 and incubated for 5 min on ice. Nuclei were pelleted by centrifugation at 1500 x g for 10 min at 4 C, and washed with the same buffer without detergent. The nuclei were repelleted and lysed with RIPA buffer containing 0.5% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate.

Detergent-resistant membrane microdomains (lipid rafts) from NIH-3T3 cells overexpressing RAR{alpha} and Myr-RAR{alpha} were fractionated in sucrose step gradients as previously described (63, 64), with modifications. About 15 x 106 cells were suspended in 500 µl of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 0.25% Triton X-100) plus inhibitors (1 mM PMSF, 40 µg/ml aprotinin, 40 µg/ml leupeptin, 1 mM sodium ortovanadate, 1 mM NaF) and solubilized for 30 min at 4 C. Sucrose concentration of the lysates was adjusted to 41% before they were overlaid with 2.7 ml of 35% sucrose and 0.8 ml of 16% sucrose prepared in 10 mM Tris-HCl, pH 7.4. Sucrose gradients were ultracentrifuged (40,000 rpm in a SW60 rotor, 18 h, 4 C), and ten 0.4-ml fractions were collected from each gradient (from the top to the bottom, fractions 1–10; fraction 10 included the pellet), precipitated with 6.5% trichloroacetic acid in the presence of 0.05% sodium deoxycholate, washed with 80% cold acetone, dissolved, and boiled in 2x Laemmli sample buffer. Fractions were analyzed by SDS-PAGE followed by Western blotting.


   ACKNOWLEDGMENTS
 
We thank J. M. Escamilla and A. Llopis for excellent technical assistance; Dr. R. Alvarez and Dr. M. J. Vega for preparing ALRT1550 and LG100567; G. Sarrió and M. Tatay for help with plasmid cloning; Dr. H. Gronemeyer for the gift of the MEF(RAR{alpha}ß{gamma})L–/L– cells; Dr. A. Aranda for providing the GST-RAR{alpha} construct; and V. Andrés, A. Díez-Juan, A. C. Carrera, P. Crespo, T. Espert, C. Gallego, T. Iglesias, B. Jiménez, A. Muñoz, F. Palau, V. Rubio, and P. Sanz for suggestions, protocols, gifts of materials, and use of equipment.


   FOOTNOTES
 
S.M. was the recipient of a predoctoral fellowship (Formación de Personal Investigador) from the Ministry of Science and Technology and a contract from Programa de contratación de Técnicos, Ministry of Education and Science. This work was supported by grants of the Spanish former Ministry of Science and Technology and Ministry of Education and Science [SAF2003-00311 and SAF2006-00647 (to D.B.)]; SAF2004-07131 (to A.R.d.L.), European Commission [QLK3-2002-02029 "Anticancer Retinoids" (to A.R.d.L.)], Generalitat Valenciana [GRUPOS 03/15 and ACOMP 06/212 (to D.B.)] and Fondo Europeo de Desarrollo Regional.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2007

Abbreviations: AMD, Actinomycin D; CHX, cycloheximide; DDT, dithiothreitol; ECL, enhanced chemiluminescence; ER, estrogen receptor; GFP, green fluorescent protein; GST, glutathione-S-transferase; 4-HPR, N-(4-hydroxyphenyl)-retinamide; hsp90, heat shock protein 90; MEF, mouse embryo fibroblast; PI3K, phosphatidylinositol-3-kinase; PMSF, phenylmethylsulfonyl fluoride; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoic X receptor; TTNPB, 4[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)-1-propenyl] benzoic acid.

Received for publication January 31, 2007. Accepted for publication June 18, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  RARβ  |  RARγ
Coregulators:   CAV1
Ligands:   Am 580  |  all-trans-Retinoic acid  |  9-cis-Retinoic acid  |  Arotinoid acid



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