Nestin and Steroid Receptor Figure 10. The non-activated GR is retained on the cytoskeleton. (A) A non-phospho specific GR antibody detects nuclear and cytoplasmic GR in Triton X-100 extracted cells. (B) GR-Ser226 specific antibodies recognize cytoskeleton bound GR in extracted cells. (C) GR-Ser211 specific GR antibodies primarily recognize nuclear bound GR. (D–F) Incubation of C6D8 cells with a GR specific ligand (dexamethasone) abolishes cytoplasmic staining with the GR-Ser226 antibody (F) and strengthens the nuclear staining with GR-Ser211 specific GR antibodies (E). (G) Immunoprecipitation (IP) of GR with phosphospecific antibodies indicates reaction with GR subfractions, differing in electrophoretic mobility. GR precipitated from anchoring deficient C6D10 cells primarily reacts with the GR-Ser211 antibody. Bars in A–F = 5 mm. doi:10.1371/journal.pone.0006084.g010 anchoring is of relevance for tissue development. It could be a tool to provide high amounts of receptor molecules by downregulating nestin expression and to activate GR mediated growth suppression during differentiation in a precise time and tissue specific manner. GR is known to play a critical role in regulating proliferative activity during embryonic development. Ligand activated GR was reported to impair proliferation and differentiation of neuronal progenitor cells in vivo and in vitro [11,62,63], to induce a G1 cell cycle arrest or apoptosis in immature thymocytes [64], and to impair proliferation of fibroblasts [65] and undifferentiated mammary epithelial cells [66,46]. Accumulation of GR in undifferentiated cells reaches the highest level just prior to terminal differentiation [67,68,69]. The GR knock-out mouse shows several defects and dies at birth from respiratory dysfunction at a time when brain development is not complete. The lungs display increased cell proliferation rates, suggesting that GR is essential to regulate proliferation during tissue development [70]. The outcome of a conditional GR knock-out in the developing mouse mamma gave evidence that GR loss does not globally affect proliferation of the targeted cell type, but does so at a certain stage in development and differentiation [71]. Regarding the function of glucocorticoids in the developing brain, the interpreta- tion of in vivo data is complicated by the fact that basal levels of glucocorticoids are essential for neuronal development, plasticity and survival, whereas stress levels of glucocorticoids produce neuronal loss [72]. Our data obtained with populations of naive human blood cells suggest that nestin/vimentin dependent anchoring of GR appears very early in the differentiation pathway. Anchoring was typical of cells which were CD133 positive, but CD34 negative. The CD133+/CD 342 phenotype characterizes very primitive periph- eral cells, that are capable of generating differentiated cells of the neuroectoderm, as well as liver, lung, brain, heart, gut, and striated muscle cells [28,73]. These blood cells have in vitro mesenchymal potential and a high hematopoietic activity [74]. In summary, we hypothesize that cytoplasmic anchoring of GR to nestin/vimentin filaments endows undifferentiated cells with significant amounts of unliganded GR that are used to activate GR functions in dependence of the developmental stage. Regulatory elements in the nestin promoter, which specify its tissue specific expression [75,76] could be of primary importance in this control mechanism. Materials and Methods Cell culture C6D10 and C6D8 cells were subcloned from the C6 rat glioma cell line (ATCC (CLL107) [32]. C6K23 cells express an exogenous mouse vimentin protein that contains the Vim3B4-epitope [33]. B8 VimMT3B4 cells stably express an exogenous mouse vimentin protein lacking the vimentin tail domain (introduction of a stop codon by base exchange at position 1358/9 of mouse vimentin cDNA). Rat 1 cells and rat 2 cells were kindly provided by W. Ostertag, Heinrich-Pette-Institute. The cells were grown with Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented PLoS ONE | www.plosone.org 12 June 2009 | Volume 4 | Issue 6 | e6084