Regulation of RAR-b and RXR-c in Schwann Cells Figure 3. Protein synthesis inhibition abrogates RXR-c, while increasing RAR-b mRNA steady state mRNA levels. SCs were treated with the axonal mimickers forskolin and BPE for 24 hours in the presence or absence of 10 mg/ml of the protein synthesis inhibitor cycloheximide (CHX), and RXR-c (A) and RAR-b (B) mRNA levels were determined by Q-RT-PCR. All values are shown as the mean 6 SEM relative to their respective controls in the absence of cycloheximide. doi:10.1371/journal.pone.0017023.g003 relevant dose of 25 nM, all three retinoids were able to significantly regulate RAR-b and RXR-c gene expression. RAR-b and RXR-c are regulated at the transcriptional level Next, we sought to find whether the regulation of RAR-b and RXR-c steady-state mRNA levels by RA is due to a transcriptional mechanism or if, alternatively, other mechanisms such as changes in mRNA stability were involved. Hetereogeneous nuclear RNAs or hnRNAs are the precursors of the mature mRNAs still present on the nucleus of the cell. They present very short half-lives, being processed as they are exported to the cytoplasm. Measuring their levels constitutes a good estimation of the transcription rate for a given gene [33]. In order to ascertain if the transcription rates for RAR-b and RXR-c were regulated by retinoic acid, myelinating cocultures were treated with the hormone for 1, 5 or 14 days and the amount of newly synthesized mRNAs was determined by measuring the hnRNA levels. Retinoic acid produced a strong increase in RAR-b hnRNA levels already at day 1 of treatment, an increment that was maintained, although at a lower level, for the two-weeks time point analyzed (Fig. 8A). Conversely, RXR-c hnRNA levels were already reduced after 1-day treatment with the retinoid, reduction that was maintained and even exacerbated during the whole time period analyzed. Similarly, in isolated SCs, treatment with the hormone for 48 h produced an increase in RAR-b hnRNA, whilst down-regulating RXR-c hnRNA levels (Fig. 8B). As retinoids can regulate gene expression both directly, through binding of their receptors to the promoters of their regulated genes, as well as indirectly, by regulating other transcription factors which, in turn, are the final mediators of the retinoid actions, we decided to analyze if RA regulation was dependent on protein synthesis. While RA was equally able to increase RAR-b mRNA in SCs both in the presence or absence of the protein synthesis inhibitor ciycloheximide, the already quite low levels of RXR-c found in the presence of the inhibitor were not further down-regulated by RA (Fig. 8C), indicating that the later required newly synthesized proteins to be effective. Although these results indicate that both genes are regulated by a transcriptional mechanism, they do not rule out the possible existence of an additional layer of regulation at the post- transcriptional level. To discern this, primary Schwann cells cultures were pre-treated with retinoic acid for 24 hours, followed by the addition of the RNA polymerase inhibitor actynomicin D for different times, and the remaining mRNA levels of RAR-b and RXR-c were analyzed by real time RT-Q-PCR. Although mRNA levels were quite different in the presence or absence of retinoic acid (59-fold higher for RAR-b and 2.5-fold lower for RXR-c), their half-lives were identical in both circumstances (Fig. 8D), indicating that RAR-b and RXR-c are regulated by retinoic acid exclusively at the transcriptional level. RXR-c protein levels are regulated by retinoids Finally, we sought to analyze if the changes in RXR-c steady state mRNAs levels by RA and axonal contact translate into increased receptor abundance at the protein level. Similarly to what occurs at the mRNA level, RA treatment of isolated SCs produced a small decrease in RXR-c protein levels in the absence of axonal contact mimickers. The combined treatment with BPE and forskolin produced an increase in RXR-c protein levels, which was prevented by treatment with the retinoid (Fig. 9). Discussion Previous work from our lab and others has shown that retinoids play an important role during Schwann cell development and function. The results shown in this work demonstrate that retinoid receptors are indeed present in Schwann cells and that their expression levels are tightly regulated by retinoic acid itself as well as by agents that mimic axonal contact. It has been described that retinoic acid signaling components are present in the peripheral nervous system and that after sciatic nerve injury there is an upregulation in retinoic acid signaling as measured by a RARE reporter in a transgenic reporter mouse [10]. This increased RA-signaling is accompanied by a transient upregulation of RAR-a, -b and -c as well as RXR-a, but not of RXR-b and -c, in cells morphologically resembling Schwann cells, although the presence of other cell types (i.e., macrophages) that also express high retinoid receptors levels, makes this deduction less conclusive. Similarly, retinoids play an important role during normal Schwann cell development and function, as regulators of myelin formation throughout two distinct mechanisms, one RAR- PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e17023