Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring Rudolph Reimer1., Heike Helmbold1., Beata Szalay1, Christian Hagel2, Heinrich Hohenberg1, Wolfgang Deppert1, Wolfgang Bohn 1 * 1 Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, Hamburg, Germany, 2 Institute of Neuropathology, Universita¨tsklinikum Hamburg-Eppendorf, Hamburg, Germany Abstract Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner. Citation: Reimer R, Helmbold H, Szalay B, Hagel C, Hohenberg H, et al. (2009) Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring. PLoS ONE 4(6): e6084. doi:10.1371/journal.pone.0006084 Editor: Nils Cordes, Dresden University of Technology, Germany Received August 22, 2008; Accepted May 27, 2009; Published June 29, 2009 Copyright: 2009 Reimer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from Deutsche Krebshilfe to W.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wolfgang.bohn@hpi.uni-hamburg.de . These authors contributed equally to this work. Introduction The class IV IF-protein nestin [1] is a well known marker of embryonic stem cell derived progenitors that have the potential to develop into neuroectodermal, endodermal and mesodermal lineages [2]. In the developing brain nestin is found coexpressed with vimentin from neural tube closure until the end of gliogenesis. Nestin is not only present in early developmental stages, it reappears transiently in adult tissue after injury to muscle [3], brain [4,5] and liver [6], situations requiring high proliferative activity of undifferentiated cells to repair the damaged tissue. Thus, nestin is also a major marker of regenerating tissue. The close relationship between nestin expression and prolifer- ative activity in tissue suggests that functions of nestin exceed those of increasing the mechanical stability of the cell. Indeed, phosphorylation and structural organization of nestin are highly dynamic throughout the cell cycle [7]. Nestin promotes phos- phorylation-dependent disassembly of vimentin IFs during mitosis [8], and it interacts with Cdk5/p35 [9], a kinase regulating differentiation of neuronal and muscle cells. Downregulation of nestin in neuronal cells activates Cdk5/p35 dependent apoptosis, suggesting that nestin is a survival determinant, protecting neuronal progenitors from stress induced cell death [10]. In intial studies on the distribution of nestin in mouse embryo tissue we detected a coincident strong accumulation of GR in nestin positive cells. Therefore, we asked whether this may indicate a structural or even functional relationship. GR is the cellular mediator of hormone regulated stress in the body and a key regulator of growth and differentiation in embryonic tissue, notably the brain, the immune system, bone, cartilage, and muscle [11,12,13,14]. Some studies emphasized a role of microtubules in intracellular transport of activated GR into the nucleus [15,16]. Purified tubulin showed direct binding of receptor molecules [17,18,19]. FKBP 52, a chaperone which associates with GR, has an affinity for dynein, a microtubule dependent cytoplasmic motor protein [20,21]. Thus, there is evidence that microtubules control intracellular trafficking of the hormone activated receptor. But, neither disruption of microtubules nor of microfilaments was sufficient to induce nuclear localization of the unliganded receptor, nor did it impair nuclear transport of the liganded receptor [22,23]. Thus, microtubules are involved in nuclear transport of GR, but not necessarily control retention of GR in the cytoplasm. Particulate receptor heterocomplexes PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e6084