during sciatic nerve development [20]. This is especially notorious for RXR-c, which was expressed at much higher levels in myelinating SC/DRGN cocultures than in isolated SCs or sensory neurons, in which it was present at very low levels (Fig. 1B). For comparison, RAR-b mRNA levels that were initially higher in DRGN than in SCs, remained relatively unchanged from DRGN cultures in myelination-competent SC/DRGN cocultures (Fig. 1B). These results suggest that cell-to-cell contact upregulates RXR- c expression, either in the SCs, in the DRGNs or in both cell types. As SCs express higher levels of retinoid receptors than DRGNs, and it is well known that neuronal contact strongly influence its physiology, we decided to analyze whether axonal contact could upregulate RXR-c expression in SCs. DRGNs are pseudo-unipolar neurons; they present a bifurcated axon with central and peripheral branches and no dendrites. Axonal contact in SCs can be partially mimicked in vitro by the exposition to cAMP-elevating agents [31] such as the adenylate cyclase activator forskolin, especially in combination with the neuregulin glial growth factor (GGF). In order to ascertain whether, effectively, axonal contact is the responsible for the increased levels of RXR-c mRNA observed in myelination-competent SC/DRGN cocul- tures, isolated SCs were treated with forskolin, both in the presence or absence of a GGF-enriched BPE. Treatment with either BPE or forskolin produced an increase in RXR-c mRNA levels with a maximum effect obtained with the combination of both factors (Fig. 2A). On the other hand, BPE increased the low basal RAR-b mRNA levels, while forskolin produced a small but reproducible down-regulation that was even more apparent with the combination of both factors (Fig. 2B). As expected, myelin- associated glycoprotein (MAG) mRNA levels and Egr2, two markers of differentiated SCs, were highly up-regulated by forskolin, both in the presence or absence of BPE (Fig. 2C–D). Although forskolin is used frequently to mimic axonal contact, it also influences Hedgehog (Hg) signaling. As several studies show cross-regulations during embryonic development between RA and Hg signaling pathways, we decided to analyze if forskolin treatment could be able to influence Hg signaling in SC cultures, which, in turn, might be responsible for changes in RAR-b and RXR-c expression levels and not the mimicked axonal contact effect by forskolin. For this purpose, Hh signaling was blocked by treatment with the specific Hh receptor smoothened inhibitor cyclopamine, and the RAR-b and RXR-c mRNA levels were determined. While treatment with forskolin alone produced an increase in RXR-c and a reduction in RAR-b mRNA levels, cyclopamine treatment failed to mimic these effects (Fig. 2E–F). Cyclopamine did not affected RXR-c mRNA levels whilst, contrary to the effect of forskolin, it did produce a small increase in RAR-b mRNA levels. In the presence of the smoothened inhibitor cyclopamine, forskolin was still able to increase RXR-c mRNA levels and to decrease RAR-b mRNA levels. These results indicate that the Hh signaling is not involved in the effect of the axonal mimicker forskolin on SCs. Next, we analyzed whether de novo protein synthesis was required for RXR-c induction by axonal contact mimickers. Interestingly, the presence of the protein synthesis inhibitor ciycloheximide produced an almost complete suppression of RXR-c steady state mRNA levels (Fig. 3A), which could not be reverted by the addition of BPE and forskolin. This result suggests that SCs express a labile inducer, whose continuous presence is required for maintenance of RXR-c mRNA levels. On the other hand, RAR-b mRNA steady-state levels were increased in the absence of new protein synthesis, while the small reduction achieved by the combined treatment with BPE and forskolin was still visible although at a lesser extent (Fig. 3B). Regulation of RAR-b and RXR-c in Schwann Cells All together, these results suggest that RXR-c and RAR-b steady-state mRNA levels are maintained in normal conditions and regulated by axonal contact mimickers throughout different mechanisms. Retinoic acid differentially regulates RAR-b and RXR-c mRNA levels in SCs The fact that RA can modulate myelin formation in the PNS [20], together with the multiple descriptions of retinoids being able to regulate their own receptors levels in several cell types, led us to clarify whether the same could be true in myelinating Schwann cell/sensory neurons cocultures. Retinoic acid treatment produced a tremendous increase in RAR-b mRNA levels without affecting RAR-a or -c mRNA levels in myelinating Schwann cell/sensory neurons cocultures (Fig. 4A). On the other hand, the retinoid produced a small but reproducible decrease in RXR-a and -b levels, at the same time that RXR-c mRNA was highly down-regulated. This effect could already be seen after 4 days of treatment, a time point in which active myelin formation is taking place, as well as after 10 days of treatment in which myelin formation in control cocultures has already taken place almost completely (Fig. 4B). Although retinoid receptors expression in SCs is generally higher that in isolated DRG neurons, the later also express them, so, at this moment, we could not know whether their regulation by RA in myelinating cocultures is mostly due to the SC component or if the neuronal levels are also changed. To ascertain if the variations seen in myelinating cocultures are due to a direct regulation of their mRNA levels in Schwann cells, we examined if RA could also regulate retinoid receptors in pure isolated primary Schwann cells cultures. As observed in myelinating cocultures, retinoic acid treatment increased RAR-b mRNA levels without affecting RAR-a and -c levels (Fig. 5). Regarding RXRs, retinoic acid practically did not change RXR-a and -b mRNA levels, while RXR-c was highly down-regulated, similarly to what occurred in myelinating cocultures. As axonal contact was able to modulate retinoid receptors levels, and the above experiments depicted in Fig. 5 were performed with isolated SCs in the continuous presence of the axonal contact mimickers forskolin and BPE, we next asked whether RA-regulation of RAR-b and RXR-c in SCs was dependent on the presence of these factors. As expected, RA produced a strong up-regulation of RAR-b mRNA levels irrespective of the presence of forskolin and/or BPE (Fig. 6A). Unexpectedly, RA downregulation of RXR-c was at least partially dependent on the presence of the axonal contact mimickers. While RA was able to inhibit RXR-c in the presence of the axonal mimickers BPE and forskolin, either alone or in combination, the retinoid was only able to slightly down-regulate the already diminished levels found in the absence of axonal mimickers (Fig. 6B). Analyzing the results from a different perspective, in the presence of RA, neither BPE nor forskolin, either alone or in combination, were able to upregulate RXR-c mRNA levels. RAR-b and RXR-c are regulated by a RAR-dependent mechanism As RA actions can be mediated by both RAR and RXR, and both groups of receptors have been shown to be active during myelin formation in Schwann cells [20], we asked which receptor(s) mediate RAR-b and RXR-c regulation. Myelinating cocultures were treated for seven days with the natural retinoid all-trans-retinoic acid, which binds to RAR as well as to RXR although with a lower affinity, as well as with 9-cis-retinoic acid that binds with similar PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17023