Down-regulation of the phosphatidylinositol 3-kinase/Akt pathway is involved in retinoic acid-induced phosphorylation, degradation, and transcriptional activity of retinoic acid receptor gamma 2.

Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARgamma2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARgamma2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARgamma2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.

and -␥) and the retinoid X receptors (RXR␣, -␤, and -␥), which are ligand-dependent transcriptional regulators functioning as RAR/RXR heterodimers both in vivo and in vitro (1)(2)(3). A ligand-independent activation domain called AF-1, which is present in the N-terminal A/B region of RARs, contains serine residues (see Fig. 2A) that are constitutively (i.e. in the absence of ligand) phosphorylated by the Cdk7 subunit of the general transcription factor TFIIH (4,5). We recently demonstrated that phosphorylation of RAR␥2 at these residues is markedly increased in response to RA through activation of p38MAPK. 2 This RA-induced phosphorylation is important for both RAR␥2mediated transcription of RA target genes and degradation of the receptor by the ubiquitin-proteasome pathway. The aim of the present study was to investigate how p38MAPK is activated in response to RA.
Activation of p38MAPK has been traditionally associated with stress responses through a cascade of phosphorylation reactions involving upstream kinases (MAPKKK, MAPKK, and MAPK) (Refs. 6 -9 and references therein). However, it has been recently reported that p38MAPK activity could be regulated through cross-talks with the PI3K/Akt pathway (10 -13). We show here that the RA-induced activation of p38MAPK and therefore the subsequent increase in RAR␥2 phosphorylation resulted from the inhibition of the PI3K/Akt pathway. This down-regulation of the PI3K/Akt pathway was crucial for RAinduced degradation and transactivation activity of RAR␥2, indicating that it is a key step in retinoid signaling.

EXPERIMENTAL PROCEDURES
Reagents, Plasmids, and Chemicals-The pSG5-based expression vectors for mouse (m) RAR␥2, mRAR␥2S66A/S68A, and the DR5-tk-CAT reporter gene were described previously (5,14). All-trans-retinoic acid, LY294002, and wortmannin were from Sigma-Aldrich. STI571 was a gift from Dr. Barbara Willi (Novartis Pharma AG). The vectors for dominant active and negative Akt containing a Myc tag were purchased from Upstate Biotechnology Inc. The cDNA for p38MAPK was provided by P. Cohen (Dundee, UK) and cloned into the pSG5 expression vector.
Polyclonal antibodies specific to RAR␥2 phosphorylated at Ser-66 or Ser-68 were prepared by immunizing rabbits with synthetic phosphopeptides followed by column chromatography with SulfoLink gel columns (Pierce) coupled to the corresponding immunizing phosphorylated peptide. After elution, the antibodies reacting with unphosphorylated RAR␥ were depleted by chromatography on a column coupled to the unphosphorylated peptide.
Cell Lines, Transfections, and CAT Assays-F9 cells were cultured and treated with 10 Ϫ7 M RA as described previously (16). COS-1 cells were grown and transiently transfected in six-well plates using the * This work was supported by funds from the CNRS, the INSERM, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, and Bristol-Myers Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 2 Giannì, M., Bauer, A., Garattini, E., Chambon, P., and Rochette-Egly, C. (2002) EMBO J., in press. DMRIE-C reagent according to the manufacturer's protocol (Invitrogen). All transfections contained 2.5 g of DNA including the DR5-tk-CAT reporter gene (1 g), the RAR␥2 pSG5-based expression vector (0.05 g), the ␤-galactosidase expression vector pCH110 (0.5 g) to correct for variations in transfection efficiency, and Bluescript as a carrier. After a 16-h incubation with DNA, cells were washed and incubated for a further 48 h in medium with or without RA (10 Ϫ6 M). CAT assays were performed using the enzyme-linked immunosorbent assay method (CAT ELISA, Roche Molecular Biochemicals). All assays were normalized to equal ␤-galactosidase activity, and the results were expressed as pg of CAT/unit of ␤-galactosidase.
Extracts, Immunoprecipitations, and Immunoblotting-Whole cell extracts (WCEs) were prepared as described previously (4) and, when required, immunoprecipitated with protein A-Sepharose beads crosslinked with the indicated antibodies. For the detection of the phosphorylated forms of RAR␥2, p38MAPK, Akt, or c-Abl, WCEs were prepared in phosphorylation lysis buffer (17). Proteins, with or without prior immunoprecipitation, were resolved by SDS-10% PAGE, electrotransferred to nitrocellulose membranes, and immunoprobed. All antibodies were diluted in phosphate-buffered saline, 0.05% Tween containing 5% nonfat milk except antibodies against phosphorylated proteins, which were diluted in Tris-buffered saline, 0.05% Tween containing 2% bovine serum albumin. The protein-antibody complexes were detected by chemiluminescence according to the Amersham Biosciences protocol.

RA-induced Activation of p38MAPK
Results from the Downregulation of the PI3K/Akt Pathway-In RAR␥2-transfected COS-1 cells, p38MAPK phosphorylation was induced after 24 h of RA treatment as assessed by WB analysis with specific antibodies recognizing the active phosphorylated form of the kinase (Fig. 1A, P-p38, compare lanes 1 and 2). To investigate whether this increase in p38MAPK activity involves Akt, we activated or inhibited the PI3K/Akt pathway. First, a constitutively active (da) form of Akt was coexpressed with RAR␥2 in COS-1 cells. This markedly decreased the RA-induced activation of p38MAPK (Fig. 1A, lanes 3-6). On the other hand, overexpression of a dominant negative (dn) form of Akt enhanced p38MAPK phosphorylation (Fig. 1A, lanes 7-10). These results were corroborated by using STI571 (18), an inhibitor of the non-receptor tyrosine kinase c-Abl that down-regulates the PI3K/Akt pathway (19,20). As expected, STI571 (10 M) decreased the level of c-Abl tyrosine phosphorylation (Fig. 1B) while it increased the amount of active phosphorylated Akt (Fig. 1B, P-Akt) as assessed by WB analysis with antibodies specific for the phosphorylated form of Akt. STI571 also suppressed the RA-induced increase in phosphorylated p38MAPK (Fig. 1C, compare lanes 2 and 4). The effects of PI3K inhibitors (LY292002 and wortmannin) on p38MAPK phosphorylation were also evaluated. LY292002 (10 M) and wortmannin (100 nM) decreased the amount of constitutively phosphorylated and activated Akt (Fig. 1D, P-Akt) and increased the activation of p38MAPK induced by RA (Fig. 1E, compare lanes 2 and 3).
RA also activated p38MAPK in mouse embryocarcinoma cells (F9 cells) 2 (Fig. 1C, lane 2). Moreover, as in transfected COS-1 cells, STI571 and LY294002 abrogated (Fig. 1C, compare lanes 2 and 4) and increased (Fig. 1E, compare lanes 2 and  3), respectively, the activation of p38MAPK induced by RA in these cells. Altogether these results indicate that the RA-induced activation of p38MAPK involves the inhibition of the PI3K/Akt pathway.
Inhibition of the PI3K/Akt Pathway Increases RA-induced RAR␥2 Phosphorylation-In transfected COS-1 cells, RAR␥2 is constitutively (i.e. in the absence of RA) phosphorylated at

RA-induced RAR␥ Phosphorylation through PI3K/Akt Inhibition 24860
serine residues 66 and 68 located in the N-terminal A/B region ( Fig. 2A) (5). Moreover, 32 P labeling experiments demonstrated that the amount of RAR␥2 phosphorylated at these residues is increased in response to RA. 2 These results were confirmed by WB analysis using antibodies recognizing specifically RAR␥2 phosphorylated at serine 66 (P-RAR␥S1) or at serine 68 (P-RAR␥S2). Both antibodies detected an increase in the amount of phosphorylated RAR␥2 upon RA treatment (Fig. 2B,  lane 2), indicating that RA increases the phosphorylation of both residues. No increase was observed in COS-1 cells expressing a RAR␥2 mutant in which the two serine residues are mutated into alanine (RAR␥S66A/S68A) (Fig. 2B, lane 4).
The RA-induced increase in phosphorylated RAR␥2 detected with the phospho-RAR␥ antibodies was impaired upon incubation of the transfected cells with SB203580 (10 M), a highly specific inhibitor of p38MAPK (21) (Fig. 2C, lane 3), while the MEK1 inhibitor PD98059 (5 M) had no effect (Fig. 2C, lane 4). Additionally, the increase in RAR␥2 phosphorylation was enhanced upon overexpression of p38MAPK (Fig. 2D, compare  lanes 2 and 4) and appeared earlier (at 10 h instead of 24 h). Altogether these results further support the conclusion that the RA-induced increase in RAR␥2 phosphorylation results from the activation of p38MAPK. 2 We then investigated whether, as expected from the above results, modulating the activity of the PI3K/Akt pathway would affect RA-induced RAR␥2 phosphorylation. Overexpression of the da form of Akt inhibited the increase in RAR␥2 phosphorylation (Fig. 3A, lanes 3 and 4), whereas a dn form of Akt enhanced RAR␥2 phosphorylation (Fig. 3A, lanes 5 and 6). Addition of STI571 also blocked the RA-induced increase in RAR␥2 phosphorylation (Fig. 3B, compare lanes 2 and 4), while in contrast, it was enhanced by the PI3K inhibitors LY294002 and wortmannin (Fig. 3C, compare lanes 2 and 4). Similar results were obtained with F9 cells (data not shown). Collectively these results indicate that the RA-induced RAR␥2 phosphorylation results from the activation of p38MAPK through inhibition of the PI3K/Akt pathway.

RA-induced Down-regulation of the PI3K/Akt Pathway Is Involved in RAR␥2 Degradation and Transactivation-Agonistic
ligands convert RAR␥2 into a strong transcriptional activator. Concomitantly, RAR␥2 is degraded by the ubiquitin-proteasome pathway (22). We recently demonstrated that the RA-induced increase in RAR␥2 phosphorylation that is mediated through activation of p38MAPK is required for both degradation and transactivation of the receptor. 2 Therefore, we investigated whether modulating the PI3K/Akt pathway would also affect RA-induced RAR␥2 degradation and transcriptional potential.
Expression of da Akt (Fig. 4A, lanes 5 and 6) blocked RAinduced RAR␥2 degradation that occurs at 48 h in transfected COS-1 cells. Addition of STI571 had the same effect in COS-1 cells and F9 cells (Fig. 4C, compare lanes 2 and 4). In contrast, overexpression of a dn form of Akt (Fig. 4A, lanes 3 and 4) as well as the PI3K inhibitors LY294002 and wortmannin (Fig.  4E, compare lanes 4 and 6 to lane 2, and data not shown) increased RAR␥2 degradation.
The transcriptional activity of RAR␥2 was similarly affected. Indeed, in COS-1 cells expressing RAR␥2 and a CAT reporter gene under the control of a DR5-RA response element (DR5-
Collectively these results indicate that the down-regulation of the PI3K/Akt pathway is involved in both RA-induced degradation and transcriptional activity of RAR␥2. DISCUSSION We previously found that the RA-induced increase in RAR␥2 phosphorylation is mediated through activation of p38MAPK. 2 Here we report that this activation implicates the down-regulation of the PI3K/Akt pathway. Indeed, blocking PI3K with wortmannin or LY294002 amplified the observed RA-induced increase in p38MAPK activity and RAR␥2 phosphorylation. Reciprocally, stimulation of the PI3K/Akt pathway upon STI571 treatment or overexpression of da Akt down-regulated these processes.
Our present results are in agreement with recent reports demonstrating that Akt negatively regulates p38MAPK (10 -12) and that disruption of the PI3K/Akt pathway prevents these effects, resulting in the activation of the p38MAPK (13). How RA inhibits the PI3K/Akt pathway was recently elucidated in mouse embryocarcinoma cells (F9 cells) by Bastien et al., 3 who have shown that RA acts at two levels, phosphorylation of the phosphatase PTEN and inhibition of PI3K through its p85␣ subunit, both of them leading to Akt inhibition.
Interestingly our present study has demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targets not only the phosphorylation of RAR␥2 through the activation of the p38MAPK but also its transcriptional activity and its degradation by the proteasome. Thus, RAR␥2 phosphorylation, RAR␥2 turnover, and RAR␥2-mediated transcription of RA target genes are interrelated events resulting from the RAinduced down-regulation of the PI3K/Akt pathway, which therefore plays an important role in RA signaling.
It is interesting to note that Akt is a mediator of cell growth and survival, while RA has pronounced antiproliferative potential that is usually linked to its capacity to induce differentiation. In keeping with this activity, RA is used in the treatment of several cancers (23,24). As a number of tumoral processes have been correlated with constitutively high Akt activity (11,25,26) and therefore to aberrant downstream kinase activities, one can speculate that inhibition of this pathway would improve the efficiency of RA therapy. In that respect it should be noted that STI571, which not only inhibits c-Abl tyrosine kinase but also other receptor tyrosine kinases that are often amplified in carcinoma (27) and lead to increased activation of the PI3K/Akt pathway, is currently used in cancer therapy (28 -31). Moreover it synergizes with retinoids in terms of cytodifferentiation and growth inhibition (32) and is capable of partially reversing the RA resistance of some acute promyelo-cytic leukemia cells (32). Altogether these results strongly suggest that the combination of retinoids with agents that affect the PI3K/Akt pathway could be exploited at the clinical level to improve retinoid therapy and/or reverse RA resistance. In conclusion, this study will provide new insights not only into retinoid signaling but also into retinoid therapy.