Regulation of RAR-b and RXR-c in Schwann Cells Figure 7. RAR-b and RXR-c regulation is mediated by RAR. (A) Myelinating cocultures were treated for 7 days with 1 mM of the natural retinoids all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9-cis), or the synthetic compound TTNPB, and the relative mRNA levels of RAR-b and RXR-c were determined by RT-Q-PCR. (B) Isolated SCs were treated for 48 hours with different doses of all-trans-retinoic acid (RA), TTNPB or 9-cis-retinoic acid (9-cis), and the relative levels of RAR-b and RXR-c mRNAs were determined by RT-Q-PCR. All values are shown as the mean 6 SD relative to their respective controls. doi:10.1371/journal.pone.0017023.g007 highly upregulated by RA in a similar fashion to what has been described in other cell types, including PC12 cells [6], neuroblas- toma cells [24], human B- and T-cells [23] or in vivo in neural crest-derived cells of chick embryos [25]. Presumably, this is a direct regulation mediated by the RAREs present in the minimal promoter of this gene [49]. The upregulation of RAR-b by RA is one of the transcriptional regulations more conserved throughout evolution and, in fact, even in the amphioxus, the closest living invertebrate relative of the vertebrates, in which a single RAR gene (AmphiRAR) exists, retinoic acid is able to induce its expression in the nerve cord during development [50]. Interestingly, although cAMP has been described to be able to synergize with the induction of RAR-b by RA in embrionary carcinoma cells [51], we cannot observe this synergism in Schwann cells, in which maximum activation by the retinoid is already achieved in the absence of cAMP activators which, if any, have a negative impact on RAR-b expression levels, in a similar manner to what has been described in neuroblastoma cells [24]. Much less is known about the control of RXR expression levels by retinoids. The down-regulation of some RXR-c transcripts by retinoic acid during differentiation of F9 mouse teratocarcinoma cells has already been described [52], although the mechanisms involved are quite different from the effect of RA on Schwann cells. In F9 cells, the effects of the retinoid are not dependent on de novo protein synthesis, while in Schwann cells, the presence of the protein synthesis inhibitor cycloheximide not only abolishes RXR- c regulation but, in fact, it produces a strong decrease in mRNA levels, uncovering the existence of a labile activator of RXR-c expression in Schwann cells. Additionally, this de novo protein synthesis dependence indicates that the effect of the retinoid is not due to the direct binding of RAR on RXR-c promoter region. In line with this, no putative RARE can be found in the promoter sequences upstream of RXR-c rat gene. Unfortunately, neither the upregulation by axonal contact mimickers nor the downreg- ulation by RA can be found in promoter assays with constructs that compass up to 3 Kb upstream of the transcription starting site of rat RXR-c gene (data not shown). PLoS ONE | www.plosone.org 9 February 2011 | Volume 6 | Issue 2 | e17023