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

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

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

shRNA-mediated VDR knock down in HaCaT cells nearly abolishes VDR expression and drastically reduces cell growth.

<p>Subconfluent HaCaT cells were infected with lentiviral VDR shRNA 3 and shRNA control particles. Seven days after infection and puromycin selection, VDR expression was evaluated in the cellular extracts. (<b>A</b>) mRNA expression levels were quantified using real-time analysis of VDR transcripts, and the values are expressed as fold changes in the silenced cells compared to the controls. (<b>B</b>) VDR expression in the mitochondrial fractions (left panels) and total lysates (right panels) from shRNA-transfected control and shRNA-transfected VDR cells was analyzed using western blotting. Tubulin detected in total extracts and VDAC levels in mitochondrial fractions were used as internal controls for protein loading. The effects of VDR silencing on proliferation were evaluated using a crystal violet assay in cells that had been stained at various times after seeding (<b>C</b>), as well as using cell cycle analysis (<b>D</b>) in cells that were harvested at day 7 post-infection. (<b>E</b>) Cell viability was evaluated using the MTT assay at day 7 post-infection and the values are expressed as the percentage of absorbance of the shRNA control. The data are expressed as the means ±SD of three independent experiments. * P<0.05 compared to the control.</p