Both in vitro by using a myelinating DRGN/SC coculture system, as well as in vivo during the development of the sciatic nerve, RA is a strong inhibitor of PNS myelin formation through two different mechanisms, one involving Egr2 (also known as Krox20) upregulation, which is dependent on retinoid X receptor (RXR) activation, and another one that implies MAG downregulation through binding of the retinoid to retinoic acid receptor (RAR). The members of the nuclear receptor superfamily act as ligand- dependent transcription factors that regulate the transcription of the genes that contain specific hormone response elements (HREs) in their promoters. The retinoids exert their actions through their binding to two groups of nuclear receptors. All-trans-RA binds with high affinity to RARs and, with a lower affinity, to RXRs, each one being codified by three separate genes (-a, -b and -c). Another retinoid, 9-cis-RA presents a similar affinity in its binding to both receptors, while the so called rexinoids are able to differentially activate RXRs. RARs function only as heterodimers with RXRs, regulating the transcription of the genes that present retinoic acid response elements (RAREs) in their promoters. Gene regulation by rexinoids through RXR can be mediated by retinoid X response elements (RXREs) present in their promoters to which they bind as RXR-RXR homodimers, as well as by acting as heterodimers of RXR in combination with several members of the nuclear receptor superfamily [21]. In addition to the concentration of retinoids and rexinoids, the ability of a particular cell type to respond to these hormones also depends on the relative expression levels of the different receptors present at this particular cell type. The pleiotropic effects of retinoids in development can partially be explained by the patterns of expression of the various RAR and RXR genes [22]. Nuclear receptor expression is under the control of several factors, and, among others, the retinoids are one of the most important. For instance, RA is a potent regulator of RAR-b expression in different cell types, both of neural and non-neural origin [6,23,24,25], although there are also descriptions of regulation of RAR-a by estrogen [26], RAR-b and RXR-b by interleukin-1 [27], RAR-b by nerve growth factor [28], as well as RAR-a and -c by b- carotene [29]. As SC differentiation is under the control of retinoids, we sought to investigate if retinoid receptors levels were also regulated in this cell type during its differentiation, as well as by the retinoid itself. Using both purified SC cultures, as well as myelinating cocultures of SCs with sensory DRGNs, we have been able to show that RXR-c is transcriptionally upregulated by axonal contact, while RAR-b, as well as RXR-c levels are differentially regulated by RA in SCs. RA produces a strong upregulation of RAR-b, while inhibiting RXR-c expression. Materials and Methods Ethics Statement All animal work was done in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/ 2005). All experimental protocols were approved by the Ethics Committee of the Consejo Superior de Investigaciones Cientificas (SAF2003-02528; SAF2004-05343; SAF2007-61693). Rat SC cultures SCs were isolated and purified as previously indicated [30]. After cytosine arabinoside and Thy-1.1 purification, SCs cultures were plated in poly-L-Lysine-coated plates and expanded in 10% heat-inactivated fetal bovine serum (FBS)-containing Dulbecco’s modified Eagle’s medium (DMEM), in the continuous presence of 2 mM forskolin (Calbiochem) and 60 mg/ml bovine pituitary Regulation of RAR-b and RXR-c in Schwann Cells extract (BPE; Gibco). Cultures were always used within less than four passages and all experiments were repeated with SCs from different preparations. For experiments, freshly seeded SCs were allowed to attach O.N. in regular media, changed to DMEM containing 10% of newborn bovine serum (NBS) depleted of retinoids (achieved by sequential incubations with AG1-X8 resin and charcoal), in the presence of 2 mM forskolin and 60 mg/ml BPE. When the effect of BPE and/or forskolin was to be analyzed, these factors were removed form the cell culture media already at the time of seeding and the cultures were allowed to adapt to the new conditions for at least 24 hours before adding the different treatments. In any case, controls were always treated with an equivalent amount of vehicle (0.01% ethanol for a typical 1 mM RA treatment) for the same time. The data depicted in Figure 5 is a reverse time-course in with all the samples were processed at the same time, having been treated with the retinoid for the time indicated in the X-axis. Control cultures were treated with vehicle (0.01% ethanol) for the longest time point analyzed (48 hours). Rat DRGN/SC Cocultures Purified DRGN and SC cultures were prepared by using methods previously described [30]. In short, neuronal cultures were established from DRGs obtained from Wistar rat embryos at 15 days of gestation from our animal facility. DRGNs were dissociated and plated onto collagen coated coverslips or plastic 6- well plates. Nonneuronal cells were eliminated by cycling (three 2- day cycles) with a fluorodeoxyuridine-containing medium (10 mM). Nerve growth factor (NGF)-dependent neurons were then maintained for 1 week in medium consisting of 10% FBS in high-glucose minimum essential medium (MEM) and 100 ng/ml of NGF. SCs were isolated from the sciatic nerves of 4-day-old rat pups as previously described [30]. SCs were purified by using cytosine arabinoside and Thy-1.1 antibody (Sigma) mediated lysis of the fibroblasts. Approximately 100,000 purified SCs were then seeded onto 3-weeks-old purified neuronal cultures of <50,000 cells. On contact with the axons, SCs proliferated rapidly until the axons were fully populated. When proliferation ceased, SCs began to elongate and ensheath the axons (premyelination stage). During this stage, media was progressively changed to media containing 10% NBS depleted of retinoids. At this time (<7 days after seeding), once the SCs have ceased to proliferate because they have completely populated the cultures and already have established a one-to-one relationship with the axons, the cocultures were induced to myelinate (myelination stage) by the addition of ascorbic acid (50 mg/ml), which is necessary for the formation of the basal lamina, an absolute requirement for myelin formation. This allows us to discriminate between the proliferation/ premyelination stages and the properly called myelination stage. Treatments with the different retinoids (all at 1 mM unless otherwise indicated) were initiated at the time of ascorbic acid addition (considered always as day 0 in all the experiments), and replenished with every feeding, together with new ascorbic acid, every 2 to 3 days. Sister control cultures received an equivalent amount of vehicle (0.01% ethanol for the 1 mM RA treatments). Western Blot Analysis Samples were prepared for Western blot analysis by homoge- nization in radioimmunoprecipitation assay (RIPA) buffer [PBS with 1% Nonidet P-40/0.5% deoxycholate/0.1% sodium dodecyl sulfate (SDS)/1 mM PMSF/Complete protease inhibitor tablets (Roche)] followed by high-speed centrifugation. Protein determi- nation was made by using the BCATM Protein Assay Kit (Pierce). Equivalent amounts of total protein extract from each sample were PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e17023