Regulation of RAR-b and RXR-c in Schwann Cells Figure 4. RA upregulates RAR-b and downregulates RXR-c in myelinating cocultures. (A) DRGN/SC cocultures were allowed to myelinate for 7 days in the presence or absence of 1 mM RA, and the relative levels of RAR-a, -b and -c, as well as RXR-a, -b and -c were determined by Q-RT-PCR. All values are shown as the mean 6 SD relative to their respective controls (sister cultures treated only with vehicle). (B) Myelinating cocultures were treated with 1 mM RA from the time of ascorbic acid induction and the levels of all six retinoid receptors were determined by Q-RTR-PCR at different times of treatment. All values are shown as the mean 6 SD relative to their respective controls (myelinating cocultures treated only with vehicle for the same length of time). doi:10.1371/journal.pone.0017023.g004 dependent that involves MAG down-regulation, and another one mainly RXR-dependent involving Egr2 upregulation [20]. This phenomenon is accompanied by the transcriptional regulation of the final effectors, the retinoid receptors. Especially interesting is RXR-c, which is highly upregulated in myelination-capable sensory neuron/Schwann cell cocultures during the phase of Schwann cell proliferation and migration. Our results suggest that this increase most probably is dependent on axonal contact and occurs in the glial component of the coculture, because can be equally observed in isolated SCs in the absence of sensory neurons by the combined treatment with forskolin and BPE. Forskolin is an activator of adenyl cyclase that is a frequent component in Schwann cell cultures as cAMP is an important secondary messenger for their growth and maturation that mimics several of the effects induced by axonal contact [31]. A G-protein coupled receptor Gpr126 has been recently identified to regulate an in vivo surge of cAMP in Schwann cells, which allows for Oct 6 activation at the promyelinating stage [34]. On the other hand, BPE, also routinely used in Schwann cell cultures, is a supplement rich in several growth factors such as neuregulins, bFGF and PDGF that are important for promoting the survival of Schwann cell precursors [35]. Together with the elevation of cAMP levels mimicking axonal contact, these growth factors strongly influence SC physiology and have a synergistic effect on Schwann cell proliferation [36]. Several studies show cross-regulations during embryonic development between the RA and Hedgehog signalling pathway [37] while Desert Hedgehog is also known to be secreted by SCs and their precursors regulating the formation of the perineurium and the maintenance of the nerve, including myelinated fibers [38,39]. Forskolin increases cellular cAMP levels by direct stimulation of adenylyl cyclase which in turn results in the activation of protein kinase A (PKA) [40,41]. On the other hand, activated PKA inhibits Hh signaling [42,43], which opens the possibility that the observed RAR-b and RXR-c regulation by forskolin could be dependent on Hh signaling. The fact that blocking the Hh receptor smoothened by the specific inhibitor PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e17023