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

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

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

Extremely low frequency electromagnetic fields affect proliferation and mitochondrial activity of human cancer cell lines

<div><p></p><p><i>Purpose</i>: To date, the effects of electromagnetic fields on cell metabolism have been overlooked. The objective of the present study was to investigate the influence of extremely low frequency electromagnetic fields (ELF-EMF) over mitochondrial metabolism and the consequent impact on cancer cell growth.</p><p><i>Materials and methods</i>: The effects of ELF-EMF on cancer growth were investigated in several human cancer cell lines by crystal violet assay. The modulation of mitochondrial activity was assessed by cytofluorimetric evaluation of membrane potential and by real-time quantification of mitochondrial transcription. Moreover the expression of several mitochondrial proteins and their levels in the organelle were evaluated.</p><p><i>Results</i>: The long-term exposure to ELF-EMF reduced the proliferation of several cancer cell lines and the effect was associated to an increased mitochondrial activity without evident changes in ATP levels. The results of our experiments excluded a transcriptional modulation of mitochondrial respiratory complexes, rather suggesting that ELF-EMF increased the energy demand. The altered mitochondrial metabolism led to changes in mitochondrial protein profile. In fact we found a downregulated expression of mitochondrial phospho-ERK, p53 and cytochrome c.</p><p><i>Conclusion</i>: The results of the present study indicate that ELF-EMF can negatively modulate cancer cell growth increasing respiratory activity of cells and altering mitochondrial protein expression.</p></div

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

Effects of VDR silencing on mitochondrial activity.

<p>HaCaT cells were infected with shRNA control or VDR shRNA 3 and the mitochondrial membrane potential was examined using JC-1 cytofluorimetric evaluation, in the presence or absence of two different stressors: (<b>A</b>) Control and silenced cells were treated with either 10 mM H₂O₂ or (<b>B</b>) 0.5 M sorbitol. In both figures, a representative image from the cytofluorimetric analysis is shown in the top panel, whereas the results from three separate experiments are plotted in the graph in the lower panel. The FL-2/FL-1 ratio was calculated and the values are expressed as a percentage of the untreated shRNA control. * P<0.05 compared to the untreated shRNA control, <b>^$</b> P<0.05 compared to the treated shRNA control. (<b>C</b>) Real time analysis of COX II (COX II) and IV (COX IV) subunit transcript expression in control and silenced cells. Fold changes are plotted on the graphs as the means ±SD of three independent experiments. * P<0.05 and ** P<0.01 compared to the shRNA control. (<b>D</b>) HaCaT cells were grown in the presence or absence of 10 nM vitamin D and COX II and COX IV transcript expression were evaluated using real-time analysis after 24 and 48 hours of treatment. The values plotted on the graphs represent the fold change in transcript expression in treated versus untreated cells and are displayed as the means ±SD of three independent experiments. <b>^§</b> P<0.05 and <b>^§§</b> P<0.01 compared to the untreated cells.</p