Mitochondrial Translocation of Vitamin D Receptor Is Mediated by the Permeability Transition Pore in Human Keratinocyte Cell Line

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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 RXRα 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 complex member StAR. By all means the impairment of pore function led to a reduction of mitochondrial levels of VDR.</p> <h3>Conclusions</h3><p>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.</p> </div

Effect of genetic silencing of StAR on VDR expression.

<p>Subconfluent HaCaT cells were infected with lentiviral StAR shRNA particles to silence the endogenous StAR expression. Mitochondrial fractions from untreated HaCaT (ctrl) and cells infected with shRNA control and StAR were analysed by western blotting for StAR and VDR expression. VDR levels were also evaluated in total lysates. VDAC was used as internal control for protein loading.</p

VDR expression and subcellular distribution in HaCaT cells.

<p>Cell were incubated for 24 hours alone (control, C) or with 1 or 100 nM 1,25D3 (D1 or D100) and harvested. (A) After subcellular fractionation procedures 50 µg of proteins from total extracts (TOT) and soluble fraction (SOL), and 10 µg of proteins from mitochondria (MIT) and nuclear extracts (N) were separated by SDS-PAGE and analysed by western blotting for VDR expression. Equal loading and quality of samples was confirmed by reprobing the membranes with antibodies anti actin, VDAC (mitochondrial marker) and PARP (nuclear marker). (B) Same amount of total extracts and mitochondrial fractions were analysed by western blotting for RXRα expression. (C) 10 µg of proteins from mitochondria (MIT) and nuclear extracts (N) of untreated cells (ctrl) or cells infected with shRNA control and shRNA anti-VDR were analysed by western blotting for VDR expression and afterwards for loading uniformity.</p

Analysis of mitochondrial translocation of VDR in presence of cyclosporin A.

<p>HaCaT cells were treated with cyclosporin A (CsA), cycloheximide (CHX) or 100 nM 1,25D3 (Vit.D) as indicated and 30 µg of mitochondrial proteins were analysed by western blotting for VDR, RXRα and p53 expression. Bands were quantified, normalized for loading as a ratio to VDAC expression and data plotted on graph as percentage of control. Data represent the mean ± S.D of three independent experiments. *<i>p</i><0.05 and **<i>p</i><0.001 compared to control. # <i>p</i><0.05 vs CHX CsA 18 h+vit.D 24 h.</p

VDR association with RXRα and PTP proteins.

<p>(A) Mitochondrial extracts from untreated HaCaT cells were immunoprecipitated with anti-VDR and anti-RXRα rabbit antibody and detection by western blotting was performed with anti-VDR or anti-RXRα biotinylated antibodies and with anti-VDAC rabbit antibody. (B) The interaction between VDR, RXRα and StAR was investigated in mitochondrial fraction by immunoprecipitation with anti-StAR rabbit antibody followed by western blotting and detection with anti-VDR and anti-RXRα biotinylated antibodies. In every assay a 10% input was used as a positive control and immunoprecipitation with normal IgG as negative control.</p

Western blot analysis of the expression of VDR and StAR upon dexamethasone treatment.

<p>30 µg of mitochondrial proteins (A) or whole lysates (B) from untreated HaCaT cells (ctrl) and cells treated for 72 h with dexamethasone (Dex) were analysed by western blotting using an antibody anti-VDR, followed by immunostaining with anti-StAR and finally with anti-VDAC or anti-actin antibody for loading control. The blots are representative of a set of three independent experiments.</p

The Vitamin D Receptor Inhibits the Respiratory Chain, Contributing to the Metabolic Switch that Is Essential for Cancer Cell Proliferation

<div><p>We recently described the mitochondrial localization and import of the vitamin D receptor (VDR) in actively proliferating HaCaT cells for the first time, but its role in the organelle remains unknown. Many metabolic intermediates that support cell growth are provided by the mitochondria; consequently, the identification of proteins that regulate mitochondrial metabolic pathways is of great interest, and we sought to understand whether VDR may modulate these pathways. We genetically silenced VDR in HaCaT cells and studied the effects on cell growth, mitochondrial metabolism and biosynthetic pathways. VDR knockdown resulted in robust growth inhibition, with accumulation in the G0G1 phase of the cell cycle and decreased accumulation in the M phase. The effects of VDR silencing on proliferation were confirmed in several human cancer cell lines. Decreased VDR expression was consistently observed in two different models of cell differentiation. The impairment of silenced HaCaT cell growth was accompanied by sharp increases in the mitochondrial membrane potential, which sensitized the cells to oxidative stress. We found that transcription of the subunits II and IV of cytochrome c oxidase was significantly increased upon VDR silencing. Accordingly, treatment of HaCaT cells with vitamin D downregulated both subunits, suggesting that VDR may inhibit the respiratory chain and redirect TCA intermediates toward biosynthesis, thus contributing to the metabolic switch that is typical of cancer cells. In order to explore this hypothesis, we examined various acetyl-CoA-dependent biosynthetic pathways, such as the mevalonate pathway (measured as cholesterol biosynthesis and prenylation of small GTPases), and histone acetylation levels; all of these pathways were inhibited by VDR silencing. These data provide evidence of the role of VDR as a gatekeeper of mitochondrial respiratory chain activity and a facilitator of the diversion of acetyl-CoA from the energy-producing TCA cycle toward biosynthetic pathways that are essential for cellular proliferation.</p></div

VDR silencing inhibits the proliferation of several human cancer cell lines.

<p>(<b>A</b>) The cells were infected with lentiviral VDR shRNA 3 or shRNA control and the silencing efficacy was examined in both the total and mitochondrial extracts using western blotting. Tubulin detected in total extracts and VDAC levels in mitochondrial fractions were used as internal controls for protein loading. (<b>B</b>) Both the silenced and control cells were subjected to proliferation assays seven days after infection and selection. The cells were stained at 72 hours or five days after seeding, and the values for the silenced cells are expressed as the percentage of their respective controls. The data are expressed as the means ±SD of three independent experiments. * P<0.05 compared to the control.</p

Highest affinity VDRE sites in the mtDNA sequence, as detected using <i>in silico</i> analysis.

<p>MATRIX: One of the possible VDRE sites matching the mtDNA sequence (the sequence and matrix are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115816#pone.0115816.s004" target="_blank">S1 table</a>). START SITE: The start site of the sequence referred to in the UCSC database (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115816#s4" target="_blank">Methods</a> section). SCORE: The affinity score is reported as a percentage of the maximum score for each matrix. STRAND: The strand of the sequence. For overlapping sequences, more than one matrix, start site and score are reported. Sites in the D-loop are highlighted in bold text.</p><p>Highest affinity VDRE sites in the mtDNA sequence, as detected using <i>in silico</i> analysis.</p

Proliferating human cells express mitochondrial VDR, whereas differentiated cells display reduced levels of receptor expression.

<p>(<b>A</b>) VDR expression was analyzed in a panel of several human cell lines using western blotting in total lysates (tot VDR) and mitochondrial extracts (mitoc VDR). For RD and MCF7 cells, VDR detection required a longer duration of exposure to ECL. (<b>B</b>) Two models of cellular differentiation were used to assess VDR levels in the total lysates and mitochondrial fractions: Human proliferating HaCaT cells vs. human primary differentiated keratinocytes and differentiation-inducible RD18 cells carrying a doxycycline-inducible miR-206-expressing lentiviral vector in the absence (uninduced: NI) or presence of doxycycline for four (induced: IND4) and six days (induced: IND6). Tubulin detected in total extracts and VDAC levels in mitochondrial fractions were used as internal controls for protein loading. The blots are representative of a set of three independent experiments.</p