D or medium alone was added VDR import was similar to control. time course experiment. (C) VDR expression after 72 hours of the same treatments as in (A).(TIF) pone.0054716.s002.tif (299K) GUID:?E2C55BD2-4D46-4F6A-973A-626A477AEE82 Abstract Background Vitamin D receptor (VDR) is a well known transcriptional regulator, active as heterodimer in association with coactivators and corepressors. In addition it has been described the extranuclear distribution of the receptor and in particular the recently reported mitochondrial localization in platelets and megakaryocytes is intriguing because it appears to be a common feature of steroid receptors. Whereas for other members of the steroid receptor family the mitochondrial function has been explored, up to now nothing is known about a mitochondrial form of VDR in human proliferating cells. Methodology/Principal Findings In this study we characterized for the first time the mitochondrial localization of VDR in the human keratinocyte cell line HaCaT. In proliferating HaCaT cells VDR was abundantly expressed in mitochondria in association with its binding partner RXR and the import was ligand-independent. By immunoprecipitation studies we demonstrated the interaction of VDR with proteins of the permeability transition pore (PTP), VDAC and StAR. We then adopted different pharmacological and silencing approaches with the aim of hampering PTP function, either affecting PTP opening or abating the expression of the CHIR-090 complex member StAR. By all means the impairment of pore function led to a reduction of mitochondrial levels of VDR. Conclusions The results reported here demonstrate a ligand-independent mitochondrial import of VDR through the permeability transition pore, and open interesting new perspectives on PTP function as transporter and on VDR role in mitochondria. Introduction Vitamin D (the active form 1,25-dihydroxyvitamin D3, 1,25D3) exerts its antiproliferative and differentiating properties through its receptor (VDR). Like other members of the steroid hormone receptors family, VDR is well known as transcriptional factor and CHIR-090 carries on its function upon nuclear translocation, heterodimerization with RXR and association with coactivators or corepressors to VDRE regions of DNA. VDR activity is controlled by p53, which both induces receptor CHIR-090 expression and synergizes with it at VDRE sites [1]. With this mechanism, for example, p53 and VDR trigger cell cycle arrest through p21 (waf1/cip1) action [2]. Besides their classical nuclear function for many steroid receptors a non genomic signalling pathway has been described, either plasma membrane bound or at mitochondrial level. Steroid receptors such as RXR, thyroid receptor (TR), glucocorticoid receptor (GR), estrogen receptor (ER) show an additional mitochondrial localization which suggests a role either in mitochondrial biogenesis or metabolism [3]C[5]. Many nuclear transcription factors may participate in regulating mitochondrial function through transcriptional regulation of mitochondrial DNA [6]. Also p53 can translocate to mitochondria, where it is involved in apoptosis. The transcription independent pathway of p53 mediated apoptosis has been described as a destabilization of the outer mitochondrial membrane by complexing with the anti-apoptotic proteins Bcl-xL and Bcl2 and by activation of the cytoplasmic proapoptotic protein Bax [7]. Apart from our previous work on human platelets and megakaryocytes, where we described a VDR located in mitochondria [8], there are very few evidences of a mitochondrial localization for VDR, presented as mere observations [9]C[11]. The aim of this work was therefore to characterize VDR subcellular distribution in proliferating human cells. We carried out our study on a cellular model highly expressing VDR, the HaCaT cell line, a spontaneously transformed Col4a4 human epithelial cell line from adult skin which is immortal but maintains full epidermal differentiation capacity [12]. Human keratinocytes not only respond to vitamin D CHIR-090 with changes in proliferation and differentiation but also synthesize vitamin D and its metabolites [13]. HaCaT cells CHIR-090 express nuclear VDR and have been used as a model for the investigation of the vitamin D pathway and its modulation [14]. In this study, we.