Regulation of RAR-b and RXR-c in Schwann Cells Figure 5. RA upregulates RAR-b mRNA and downregulates RXR-c mRNA in isolated SCs. SCs were treated with 1 mM RA for the times indicated and the relative levels of all six retinoid receptors were determined by Q-RT-PCR. All values are shown as the mean 6 SD relative to their respective controls. doi:10.1371/journal.pone.0017023.g005 cyclopamine was not able to mimic the effect of forskolin, and, in the case of RAR-b, even modulate its expression in the opposite direction, indicates that Hh signaling is not participating on the regulation of RAR-b and RXR-c expression by cAMP. Schwann cell behavior depends on the combination of extrinsic growth factors and hormones along with juxtacrine signaling coming from the neurons. A clear example is the induction of Egr2, the major myelinogenic transcription factor, whose expression depends mainly on axonal contact [44,45] but can also be regulated by extrinsic factors like retinoic acid [20]. Interestingly, Egr2 upregulation by RA is mostly dependent on RXR activation, whose levels are also upregulated in Schwann cells by axonal contact. It is tempting to speculate that the same or similar regulatory mechanisms that control Egr2 expression in Schwann cells could also be regulating RXR-c expression. Another intriguing possibility is that Egr2 upregulation by axonal contact could be partially dependent on RXR-c activation, either by retinoids themselves or by other agents capable of activating this receptor, like docosahex- aenoic acid, a long-chain polyunsaturated fatty acid that is highly enriched in the adult mammalian brain [46] as well as in sciatic nerve during myelin formation [47] and has been shown to enhance neurite formation in DRG sensory neurons [48]. On the other hand, retinoids themselves are also potent regulators of their own receptors in Schwann cells. RAR-b is Figure 6. Influence of axonal contact mimickers on RXR-c and RAR-b regulation by RA. RXR-c downregulation is higher in the presence of axonal contact mimickers, while RAR-b upregulation was similar irrespective of the growth conditions. Isolated SCs were adapted for 24 hours to the absence of the axonal contact mimickers BPE and forskolin and subsequently treated for 48 hours with 1 mM RA in the presence or absence of 2 mM forskolin, 60 mg/ml BPE or the combination of both, and the mRNA levels of RAR-b (A) and RXR-c (B) were analyzed by Q-RT-PCR. In all instances, the levels reached after the acute combined treatment were compared to SCs continuously grown in the presence of BPE and Fk (BPE+Fk always). All values are shown as the mean 6 SEM relative to their respective controls. doi:10.1371/journal.pone.0017023.g006 PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17023