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.

BackgroundVitamin 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 FindingsIn 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.ConclusionsThe 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.

Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

Social Impact Assessments (SIAs) can be developed at any stage in a project to propose measures to mitigate potential adverse social impacts and enhance positive social impacts. This paper describes guidance developed on how to make the best use of SIAs in oil and gas projects. Social Impacts of Oil and Gas Projects Potential social impacts of an oil and gas project can be both positive and negative. Adverse impacts such as resource use and the movement of people are often well publicized. However these are often balanced by positive impacts such as the promotion of employment, socio-economic development and improvements in infrastructure. Some typical impacts are described in the table below. SIA is a tool utilized in the oil gas industry to manage social impacts, enhancing positive impacts and avoiding or mitigating adverse ones. SIA provides coompanies an effective risk management tool and communities the opportunity to give input on a project. What is Social Impact Assessment? The Oil and Gas Industry has been aware of these and other impacts for many years and has been developing methods for managing social impacts. In 1997, the International Association of Oil and Gas Producers (OGP) published a report entitled " Principles for Impact Assessment: The Environmental and Social Dimension"1. This report set out principles for the environmental and social dimension of impact assessment for oil and gas exploration and production projects. It was intended for oil and gas companies and their contractors, and highlights the importance of social and environmental assessment in all aspects of project planning and implementation. The document was subsequently endorsed by the International Petroleum Industry Environmental Conservation Association (IPIECA). The document was viewed as a stepping-stone to a more comprehensive guidance document for industry practitioners. In 2002, OGP and IPIECA took the next step and developed ‘Lists of Questions’2 that may need to be asked and answered in considering the social dimension of oil and gas projects. These questions give an extensive insight into the range and diversity of the potential social impacts that an oil and gas project may need to manage. However, these lists of questions were not intended as check lists or as detailed guidance for conducting social impact assessment.

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