Figure 9. Retinoic acid and axonal contact mimickers regulate RXR-c protein levels. 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 and 60 mg/ml BPE, and the protein levels of RXR-c were analyzed by western blot. The upper panel depicts the quantitation of the blots. The data are presented as the mean value 6 SEM referred as the percentage over the controls. Significance was assessed by unpaired Student’s t-test. *: p,0.05; ***: p,0.005. doi:10.1371/journal.pone.0017023.g009 Interestingly, RXR-c signaling has just been shown to be involved in oligodendrocyte differentiation and to accelerate CNS remye- lination [57]. RXR belongs to the Class II subfamily of nuclear receptors and, besides being able to act by itself as a ligand-dependent References 1. Maden M (2007) Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat Rev Neurosci 8: 755–765. 2. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995) Embryonic stem cells express neuronal properties in vitro. Dev Biol 168: 342–357. 3. Corcoran J, Maden M (1999) Nerve growth factor acts via retinoic acid synthesis to stimulate neurite outgrowth. Nat Neurosci 2: 307–308. 4. Haussler M, Sidell N, Kelly M, Donaldson C, Altman A, et al. (1983) Specific high-affinity binding and biologic action of retinoic acid in human neuroblas- toma cell lines. Proc Natl Acad Sci USA 80: 5525–5529. 5. Ca n˜o´n E, Cosgaya JM, Scsucova S, Aranda A (2004) Rapid effects of retinoic acid on CREB and ERK phosphorylation in neuronal cells. Mol Biol Cell 15: 5583–5592. 6. Cosgaya JM, Garcia-Villalba P, Perona R, Aranda A (1996) Comparison of the effects of retinoic acid and nerve growth factor on PC12 cell proliferation, differentiation, and gene expression. J Neurochem 66: 89–98. 7. Cosgaya JM, Perona R, Aranda A (1997) Retinoic acid induces secretion of transforming growth factors by PC12 pheochromocytoma cells. Oncogene 14: 579–587. 8. Scheibe RJ, Ginty DD, Wagner JA (1991) Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase. J Cell Biol 113: 1173–1182. 9. Zhelyaznik N, Schrage K, McCaffery P, Mey J (2003) Activation of retinoic acid signalling after sciatic nerve injury: up-regulation of cellular retinoid binding proteins. Eur J Neurosci 18: 1033–1040. 10. Zhelyaznik N, Mey J (2006) Regulation of retinoic acid receptors a, b and retinoid X receptor a after sciatic nerve injury. Neuroscience 141: 1761–1774. 11. Mey J, Schrage K, Wessels I, Vollpracht-Crijns I (2007) Effects of inflammatory cytokines IL-1b, IL-6, and TNFa on the intracellular localization of retinoid receptors in Schwann cells. Glia 55: 152–164. Regulation of RAR-b and RXR-c in Schwann Cells transcription factor, its major role is as the heterodimerizing partner of the Class I subfamily of nuclear receptors, which is formed by RAR, as well as the vitamin D3 (VDR), the thyroid hormone (TR) or the peroxisome proliferator activated receptors (PPAR) [21]. This central role as a required partner in nuclear receptor signaling implies that the regulation of RXR levels will be relevant not only for its own signaling, but also for regulating the ability of SCs to respond to a vast array of hormones, some of which are likely to play an important role in SC physiology. Further studies need to be conducted in order to determine whether RXR-dependent regulation of myelination is mediated by homodimers or if it is acting just as a heterodimerizing partner for other nuclear receptors. Regarding this, only RXR-a null-mice are not viable and show a clear phenotype. In fact, even the triple compound RXR-a+/2/RXR-b2/2/RXR-c2/2 mutant mice are viable and almost indistinguishable from RXR-a+/2 mice [58], suggesting that all three isoforms are basically interchangeable, making more difficult to study the involvement of RXR on myelin formation in vivo. Animal models of forced overexpression after birth, as well as conditional knock-outs of the different RXR isoforms on Schwann cells should provide further mechanistic insights into the role of RXRs on Schwann cell differentiation and subsequent peripheral myelin formation. Supporting Information Table S1 (DOC) Q-RT-PCR primer list. Acknowledgments We want to thank M. Ituero and A. Moran-Gonzalez for their help with Schwann and DRGN cell cultures. Author Contributions Conceived and designed the experiments: MJL JMC. Performed the experiments: MJL JMC. Analyzed the data: MJL JMC. Wrote the paper: MJL JMC. 12. Kinameri E, Matsuoka I (2003) Autocrine action of BMP2 regulates expression of GDNF-mRNA in sciatic Schwann cells. Brain Res Mol Brain Res 117: 221–227. 13. Johann V, Jeliaznik N, Schrage K, Mey J (2003) Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells. Neurosci Lett 339: 13–16. 14. Dimitroulakos J, Squire J, Pawlin G, Yeger H (1994) NUB-7: a stable I-type human neuroblastoma cell line inducible along N- and S-type cell lineages. Cell Growth Differ 5: 373–384. 15. Tong L, Ji L, Wang Z, Tong X, Zhang L, et al. (2010) Differentiation of neural stem cells into Schwann-like cells in vitro. Biochem Biophys Res Commun 401: 592–597. 16. Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H (2001) Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone- marrow stromal cells. Eur J Neurosci 14: 1771–1776. 17. Jiang L, Zhu J-K, Liu X-L, Xiang P, Hu J, et al. (2008) Differentiation of rat adipose tissue-derived stem cells into Schwann-like cells in vitro. Neuroreport 19: 1015–1019. 18. Matsuse D, Kitada M, Kohama M, Nishikawa K, Makinoshima H, et al. (2010) Human umbilical cord-derived mesenchymal stromal cells differentiate into functional Schwann cells that sustain peripheral nerve regeneration. J Neuropathol Exp Neurol 69: 973–985. 19. Kazakova N, Li H, Mora A, Jessen KR, Mirsky R, et al. (2006) A screen for mutations in zebrafish that affect myelin gene expression in Schwann cells and oligodendrocytes. Developmental Biology 297: 1–13. 20. Latasa M-J, Ituero M, Moran-Gonzalez A, Aranda A, Cosgaya JM (2010) Retinoic acid regulates myelin formation in the peripheral nervous system. Glia 58: 1451–1464. 21. Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev 81: 1269–1304. PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e17023