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• W2531971806 abstract "•Vitamin D metabolism is conserved between nematodes and mammals•Vitamin D prevents the age-dependent accumulation of SDS-insoluble proteins•Vitamin D enhances lifespan and protein homeostasis via IRE-1, XBP-1, and SKN-1 Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer’s disease, Parkinson’s disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human β-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency. Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer’s disease, Parkinson’s disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human β-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency. Our understanding of the role of vitamin D has grown significantly over the last several years with evidence that low levels of vitamin D can have a profound effect on human health (Hossein-nezhad and Holick, 2013Hossein-nezhad A. Holick M.F. Vitamin D for health: A global perspective.Mayo Clin. Proc. 2013; 88: 720-755Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar). Following the discovery of the vitamin D receptor (VDR), which is expressed in a wide range of tissues, the role of vitamin D in the prevention and treatment of chronic diseases has become an important area of study (Holick, 1992Holick M.F. Evolutionary biology and pathology of vitamin D.J. Nutr. Sci. Vitaminol. (Tokyo). 1992; : 79-83Crossref PubMed Scopus (15) Google Scholar, Kalueff and Tuohimaa, 2007Kalueff A.V. Tuohimaa P. Neurosteroid hormone vitamin D and its utility in clinical nutrition.Curr. Opin. Clin. Nutr. Metab. Care. 2007; 10: 12-19Crossref PubMed Scopus (209) Google Scholar). Vitamin D deficiency has been linked to various health problems, including cognitive decline, depression, cardiovascular disease, hypertension, type 2 diabetes, and cancer (Butler et al., 2011Butler M.W. Burt A. Edwards T.L. Zuchner S. Scott W.K. Martin E.R. Vance J.M. Wang L. Vitamin D receptor gene as a candidate gene for Parkinson disease.Ann. Hum. Genet. 2011; 75: 201-210Crossref PubMed Scopus (90) Google Scholar, Chan, 2011Chan J. The value of vitamin D supplementation in older people.Nutritional Therapy & Metabolism. 2011; 29: 8-21Google Scholar, Holick, 2003Holick M.F. Vitamin D: A millenium perspective.J. Cell. Biochem. 2003; 88: 296-307Crossref PubMed Scopus (1045) Google Scholar, Ingraham et al., 2008Ingraham B.A. Bragdon B. Nohe A. Molecular basis of the potential of vitamin D to prevent cancer.Curr. Med. Res. Opin. 2008; 24: 139-149Crossref PubMed Scopus (151) Google Scholar, Ito et al., 2011Ito S. Ohtsuki S. Nezu Y. Koitabashi Y. Murata S. Terasaki T. 1α,25-Dihydroxyvitamin D3 enhances cerebral clearance of human amyloid-β peptide(1-40) from mouse brain across the blood-brain barrier.Fluids Barriers CNS. 2011; 8: 20Crossref PubMed Scopus (82) Google Scholar, Liu et al., 2013Liu H.X. Han X. Zheng X.P. Li Y.S. Xie A.M. [Association of vitamin D receptor gene polymorphisms with Parkinson disease].Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2013; 30: 13-16PubMed Google Scholar). During aging, the risk for vitamin D deficiency significantly increases due to reduced nutritional intake of vitamin D, increased adiposity, and decreased cutaneous synthesis of vitamin D. This has led to considerable debate regarding vitamin D supplementation in the elderly and whether deficiencies in vitamin D represent an indicator of ill health or increases one’s susceptibility to chronic disease (Kupferschmidt, 2012Kupferschmidt K. Uncertain verdict as vitamin D goes on trial.Science. 2012; 337: 1476-1478Crossref PubMed Scopus (105) Google Scholar). Vitamin D is a member of the superfamily of secosteroid hormones. There are two major forms of vitamin D, vitamin D2 (ergocalciferol; D2), which is produced by the UV radiation of ergosterol, and vitamin D3 (cholecalciferol; D3), which is a photoproduct produced in the skin from 7-dehydrocholesterol (7DHC) (Smith and Holick, 1987Smith E.L. Holick M.F. The skin: The site of vitamin D3 synthesis and a target tissue for its metabolite 1,25-dihydroxyvitamin D3.Steroids. 1987; 49: 103-131Crossref PubMed Scopus (23) Google Scholar). The vitamin D photoproduct is biologically inert, requiring two separate hydroxylation steps by cytochrome P450 enzymes to produce the biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)2D3) (Figure 1A). As the concentration of 1,25-(OH)2D3 increases, VDRs throughout the body become activated, resulting in extensive alterations in gene expression and numerous physiological alterations. C. elegans is an excellent model for longevity studies and investigating aspects of chronic disease pathology. Many of the classical signaling pathways and transcription factors that modulate stress response and aging have been identified in the nematode. Enhancing the activity of the FOXO transcription factor DAF-16, which functions in the insulin/IGF-1 signaling pathway, significantly increases lifespan (Kenyon, 2005Kenyon C. The plasticity of aging: Insights from long-lived mutants.Cell. 2005; 120: 449-460Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar). Additionally, the activity of the heat shock transcription factor, HSF-1, and the Nrf2-like xenobiotic and oxidative stress-response factor, SKN-1, also affect normal aging in the worm (Tullet et al., 2008Tullet J.M. Hertweck M. An J.H. Baker J. Hwang J.Y. Liu S. Oliveira R.P. Baumeister R. Blackwell T.K. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.Cell. 2008; 132: 1025-1038Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). These stress response transcription factors up- or downregulate a diverse range of target genes. Protein homeostasis plays an important role in aging and age-related disease. Normal aging in C. elegans is associated with a loss in protein homeostasis and an accumulation of insoluble protein (David et al., 2010David D.C. Ollikainen N. Trinidad J.C. Cary M.P. Burlingame A.L. Kenyon C. Widespread protein aggregation as an inherent part of aging in C. elegans.PLoS Biol. 2010; 8: e1000450Crossref PubMed Scopus (431) Google Scholar, Reis-Rodrigues et al., 2012Reis-Rodrigues P. Czerwieniec G. Peters T.W. Evani U.S. Alavez S. Gaman E.A. Vantipalli M. Mooney S.D. Gibson B.W. Lithgow G.J. Hughes R.E. Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan.Aging Cell. 2012; 11: 120-127Crossref PubMed Scopus (116) Google Scholar, Walther et al., 2015Walther D.M. Kasturi P. Zheng M. Pinkert S. Vecchi G. Ciryam P. Morimoto R.I. Dobson C.M. Vendruscolo M. Mann M. Hartl F.U. Widespread proteome remodeling and aggregation in aging C. elegans.Cell. 2015; 161: 919-932Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Neurological diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) share common cellular and molecular features including protein aggregation and inclusion body formation. Neurotoxic aggregated forms of endogenous proteins, such as amyloid-β (AD), α-synuclein (PD), huntingtin (HD), and TAR DNA-binding protein 43 kDa (TDP-43; ALS) underlie the pathogenesis of these diseases. Analogous to their effects on longevity, DAF-16, HSF-1, and SKN-1 all contribute to maintenance of protein homeostasis in C. elegans (Alavez et al., 2011Alavez S. Vantipalli M.C. Zucker D.J. Klang I.M. Lithgow G.J. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan.Nature. 2011; 472: 226-229Crossref PubMed Scopus (295) Google Scholar, Dostal et al., 2010Dostal V. Roberts C.M. Link C.D. Genetic mechanisms of coffee extract protection in a Caenorhabditis elegans model of β-amyloid peptide toxicity.Genetics. 2010; 186: 857-866Crossref PubMed Scopus (95) Google Scholar). In C. elegans, DAF-16 and HSF-1 both regulate the formation of age-induced polyglutamine-repeat protein aggregates, similar to those found in HD (Hsu et al., 2003Hsu A.L. Murphy C.T. Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor.Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1121) Google Scholar). Deficiency in either DAF-16 or HSF-1 correlates with premature accumulation of age-associated insoluble protein (Walther et al., 2015Walther D.M. Kasturi P. Zheng M. Pinkert S. Vecchi G. Ciryam P. Morimoto R.I. Dobson C.M. Vendruscolo M. Mann M. Hartl F.U. Widespread proteome remodeling and aggregation in aging C. elegans.Cell. 2015; 161: 919-932Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). SKN-1 has also been shown to be required for the maintenance of protein homeostasis (Alavez et al., 2011Alavez S. Vantipalli M.C. Zucker D.J. Klang I.M. Lithgow G.J. Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan.Nature. 2011; 472: 226-229Crossref PubMed Scopus (295) Google Scholar). Additionally, SKN-1 activity is associated with another mechanism previously shown to be important in lifespan extension, the endoplasmic reticulum unfolded protein response (ER-UPR) (Glover-Cutter et al., 2013Glover-Cutter K.M. Lin S. Blackwell T.K. Integration of the unfolded protein and oxidative stress responses through SKN-1/Nrf.PLoS Genet. 2013; 9: e1003701Crossref PubMed Scopus (110) Google Scholar), which is induced in response to proteotoxic stress in the ER to suppress the accumulation of unfolded or misfolded proteins (Zhang and Kaufman, 2006Zhang K. Kaufman R.J. The unfolded protein response: A stress signaling pathway critical for health and disease.Neurology. 2006; 66: S102-S109Crossref PubMed Scopus (510) Google Scholar). In C. elegans, not only does SKN-1 play a prominent role in the transcriptional regulation of the ER-UPR, but specific ER-UPR regulators are also, in turn, important for SKN-1 target gene expression (Glover-Cutter et al., 2013Glover-Cutter K.M. Lin S. Blackwell T.K. Integration of the unfolded protein and oxidative stress responses through SKN-1/Nrf.PLoS Genet. 2013; 9: e1003701Crossref PubMed Scopus (110) Google Scholar). Consistent with the hypothesis that impaired protein homeostasis can drive aging, we and others have shown that normal aging is associated with insoluble protein accumulation, and genes encoding these insoluble proteins are enriched for those that determine lifespan (David et al., 2010David D.C. Ollikainen N. Trinidad J.C. Cary M.P. Burlingame A.L. Kenyon C. Widespread protein aggregation as an inherent part of aging in C. elegans.PLoS Biol. 2010; 8: e1000450Crossref PubMed Scopus (431) Google Scholar, Reis-Rodrigues et al., 2012Reis-Rodrigues P. Czerwieniec G. Peters T.W. Evani U.S. Alavez S. Gaman E.A. Vantipalli M. Mooney S.D. Gibson B.W. Lithgow G.J. Hughes R.E. Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan.Aging Cell. 2012; 11: 120-127Crossref PubMed Scopus (116) Google Scholar). Vitamin D has been shown to extend lifespan in C. elegans (Messing et al., 2013Messing J.A. Heuberger R. Schisa J.A. Effect of vitamin D3 on lifespan in Caenorhabditis elegans.Curr. Aging Sci. 2013; 6: 220-224Crossref PubMed Scopus (8) Google Scholar). Moreover, short-term treatment with vitamin D reduces amyloid-β (Aβ) peptide aggregation and improves cognition in mouse models of AD (Durk et al., 2014Durk M.R. Han K. Chow E.C. Ahrens R. Henderson J.T. Fraser P.E. Pang K.S. 1α,25-Dihydroxyvitamin D3 reduces cerebral amyloid-β accumulation and improves cognition in mouse models of Alzheimer’s disease.J. Neurosci. 2014; 34: 7091-7101Crossref PubMed Scopus (107) Google Scholar). These observations prompted us to investigate whether vitamin D promotes widespread cellular protein homeostasis and consequently influences aging. We found that D3 feeding suppressed the toxicity induced by human β-amyloid (Aβ3-42) aggregation and rescued paralysis of worms expressing a metastable perlecan protein. Critically, we found that vitamin D3 treatment slowed proteome-wide, age-related protein insolubility. We examined the mechanism by which vitamin D influences protein homeostasis and longevity and found that the beneficial effects of vitamin D3 require the stress response pathway genes SKN-1, IRE-1, and XBP-1. The role for this secosteroid hormone in suppressing age-related proteotoxic stress provides an explanation for the observed elevated risk for neurological disease associated with human vitamin D deficiency. To test the suitability of C. elegans as a model for investigating vitamin D mechanisms, we asked whether worms fed vitamin D3 have the ability to produce the bioactive form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), which is required for VDR activity. We grew large populations of synchronously aging N2 wild-type hermaphrodite worms at 25°C on either vitamin-D3- or control-treated nematode growth media (NGM) plates and prepared lipid extracts on the second day of adulthood (5-day-old worms). We tested the worm lipid extracts for biological activity in a one-hybrid human cell-based VDR activity assay and found that lipid extracts made from D3-fed worms showed enhanced human VDR transcriptional activity, as evidenced by increased luciferase activity, compared to control-treated worms (Figure 1B). Addition of vitamin D3 alone to the VDR expressing cells had no effect on VDR transcriptional activity (data not shown). This demonstrated that C. elegans worms are able to metabolize vitamin D3 into a ligand that activates human VDR. To test whether worms metabolized vitamin D3 to the known active ligand, 1,25-(OH)2D3, lipid extracts made from vitamin D3-fed worms were subjected to liquid chromatography/mass spectroscopy (LC-MS). A signal identical to the 1,25-(OH)2D3 standard was present in the D3-fed lipid extracts, but not in extracts from control-treated worms (Figure 1C). Quantification of the amount of 1,25-(OH)2D3 in the lipid extracts derived from D3-fed worms revealed approximately 5.95E-03 pg/worm. By comparison, in humans, plasma 1,25-(OH)2D3 levels range from 10 to 70 pg/mL (Bikle et al., 1984Bikle D.D. Gee E. Halloran B. Haddad J.G. Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease.J. Clin. Invest. 1984; 74: 1966-1971Crossref PubMed Scopus (269) Google Scholar). Since C. elegans are grown on a live Escherichia coli (E. coli) food source, we tested whether exposure of E. coli to D3 would result in 1,25-(OH)2D3 production but found that the bacteria alone did not make this active form of vitamin D (data not shown). Collectively these data demonstrated that C. elegans are capable of synthesizing 1,25-(OH)2D3, and that lipid extracts derived from these worms can activate human VDR, confirming that this critical component of vitamin D metabolism is conserved between nematodes and mammals. We confirmed that vitamin D3 extended C. elegans lifespan (Messing et al., 2013Messing J.A. Heuberger R. Schisa J.A. Effect of vitamin D3 on lifespan in Caenorhabditis elegans.Curr. Aging Sci. 2013; 6: 220-224Crossref PubMed Scopus (8) Google Scholar). Feeding vitamin D3 throughout adulthood extended lifespan in a dose-dependent manner, and was not toxic even at the highest concentration (250 μM) tested (Figure 2A; Table S1). Given that normal aging is modulated by a network of transcription factors (Hsu et al., 2003Hsu A.L. Murphy C.T. Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor.Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1121) Google Scholar, Tullet et al., 2008Tullet J.M. Hertweck M. An J.H. Baker J. Hwang J.Y. Liu S. Oliveira R.P. Baumeister R. Blackwell T.K. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans.Cell. 2008; 132: 1025-1038Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar), we tested whether members of this network were required for the beneficial effects of vitamin D3 on lifespan in C. elegans. First, we tested the requirement of DAF-16 in D3-induced lifespan extension. We found that D3 feeding extended the lifespan of daf-16(mu86) worms, which lack functional DAF-16 protein (Figure 2B; Table S1). Additionally, D3 treatment did not alter the subcellular localization of a DAF-16::GFP fusion protein (TJ356 strain; data not shown). These data suggest that the effect of D3 feeding on lifespan extension is independent of DAF-16. In addition, we found that vitamin-D3-induced lifespan extension did not require DAF-12 (Figures S1A and S1B; Table S1), the proposed ortholog of VDR in C. elegans (Antebi et al., 2000Antebi A. Yeh W.H. Tait D. Hedgecock E.M. Riddle D.L. daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans.Genes Dev. 2000; 14: 1512-1527Crossref PubMed Google Scholar, Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. The nuclear receptor superfamily: The second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6064) Google Scholar). Furthermore, in a cell-based luciferase reporter assay, 1,25-(OH)2D3 did not increase DAF-12 transcriptional activity (data not shown). Taken together, we conclude that vitamin-D3-induced lifespan extension is independent of DAF-12. We next tested the requirement of the HSF-1 in D3-induced lifespan extension. D3 treatment resulted in marginal or no lifespan extension in hsf-1(sy441) mutant worms (Figure 2C; Table S1), suggesting that the effect of D3 on lifespan may partially require the participation of HSF-1-regulated genes. Last, we examined the effect of SKN-1 in D3-induced lifespan extension. We observed no lifespan extension by D3 for skn-1(zu135) mutant worms, demonstrating that SKN-1 is required for the effects of D3 feeding (Figure 2D; Table S1). SKN-1 activity is associated with another mechanism important in lifespan extension, the endoplasmic reticulum unfolded protein response (ER-UPR) (Glover-Cutter et al., 2013Glover-Cutter K.M. Lin S. Blackwell T.K. Integration of the unfolded protein and oxidative stress responses through SKN-1/Nrf.PLoS Genet. 2013; 9: e1003701Crossref PubMed Scopus (110) Google Scholar). Specifically, SKN-1 regulates transcription of the entire core of the ER-UPR and many downstream ER stress defense genes. Moreover, ER stress influences the levels of skn-1 mRNA and SKN-1 protein (Glover-Cutter et al., 2013Glover-Cutter K.M. Lin S. Blackwell T.K. Integration of the unfolded protein and oxidative stress responses through SKN-1/Nrf.PLoS Genet. 2013; 9: e1003701Crossref PubMed Scopus (110) Google Scholar). Proteotoxic stress triggers the ER-UPR by activating the stress sensors ribonuclease inositol requiring protein-1 (IRE-1), PERK kinase homolog (PEK-1), and activating transcription factor-6 (ATF-6) (Calfon et al., 2002Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.Nature. 2002; 415: 92-96Crossref PubMed Scopus (2132) Google Scholar, Shen et al., 2001Shen X. Ellis R.E. Lee K. Liu C.Y. Yang K. Solomon A. Yoshida H. Morimoto R. Kurnit D.M. Mori K. Kaufman R.J. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development.Cell. 2001; 107: 893-903Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, Shen et al., 2005Shen X. Ellis R.E. Sakaki K. Kaufman R.J. Genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in C. elegans.PLoS Genet. 2005; 1: e37Crossref PubMed Scopus (181) Google Scholar). Activation of each sensor produces a transcription factor that activates genes to increase the protein-folding capacity in the ER. Of the three stress responsive ER-UPR pathways, IRE-1 is the most conserved. Upon activation of the UPR, IRE1-dependent splicing of a small intron from the xbp-1 mRNA leads to synthesis of XBP-1 transcription factor, which, in turn, induces expression of hsp-4 and other ER-UPR-associated genes. Given the requirement for SKN-1 in D3-mediated lifespan extension and that SKN-1 and the ER-UPR form a regulatory network, we tested the dependency of each ER-UPR pathway for the lifespan response to D3 feeding. We found that the D3-induced increase on survival was dependent on IRE-1/XBP-1 signaling. Worms carrying the loss-of-function allele, ire-1(v33), showed significantly reduced lifespan with D3 feeding compared to vehicle-treated worms (Figure 2E; Table S1). Lifespan of worms maintaining the loss-of-function allele, xbp-1(zc12), showed no significant change with D3 feeding compared to vehicle-treated worms (Figure 2F; Table S1). Interestingly, ire-1(v33) mutant worms exhibited a shortened lifespan upon D3 feeding. In contrast, D3 feeding significantly increased lifespan in worms maintaining loss-of-function alleles for pek-1 and atf-6 (Table S1; Figure S1C). Collectively, these data specifically implicate the stress response genes SKN-1, IRE-1, and XBP-1 in vitamin-D3-induced lifespan extension. Given that the effect of D3 feeding on lifespan is dependent on a genetic network, we next surveyed the downstream target genes of HSF-1, SKN-1, and IRE-1/XBP-1. We examined the effect of D3 feeding on the expression of an HSF-1 target gene encoding a molecular chaperone, using the transgenic transcriptional reporter strain, phsp-16.2::GFP. D3 treatment had no effect on the expression of this transcriptional reporter (Figures S2A and S2C). We examined other molecular chaperones not under direct regulation by HSF-1, hsp-6 (mitochondrial chaperone) and hsp-4 (ER chaperone). Using the transcriptional reporter stains phsp-6::GFP and phsp-4::GFP, we found that vitamin D3 feeding only increased the levels of hsp-4 expression (Figures S2A, S2B, and S2D). HSP-4 is a direct target of the IRE-1/XBP-1 pathway, and it is upregulated in response to ER stress. Surprisingly, we failed to observe a significant D3-associated upregulation of hsp-4 mRNA levels in wild-type N2 worms as assessed by RNA sequencing (RNA-seq) and quantitative real-time PCR at various time points (data not shown). These results indicate that lifespan extension from D3 requires IRE-1/XBP-1 but does not appear to result in robust constitutive induction of the downstream XBP-1 target gene, hsp-4. We then tested the effects of D3 treatment on the expression of a target of SKN-1 using a transgenic transcriptional reporter strain pgst-4::GFP. GST-4 (glutathione transferase-4) is involved in the phase II oxidative stress response and its expression reports on SKN-1 activity. D3 feeding significantly upregulated pgst-4::GFP compared to control-treated worms (Figure 3A) in a SKN-1-dependent manner (Figure 3B). We confirmed this result by quantitative real-time PCR analysis of endogenous gst-4 mRNA levels (Figure 3C). To gain a more detailed picture of the genomic response to vitamin D3, we undertook a genome-wide analysis of altered mRNA abundance. Specifically, synchronous populations of D3 fed and control L4 stage hermaphrodite worms were processed for RNA-sequencing. We observed 253 significantly upregulated and 78 significantly downregulated genes in response to vitamin D3 treatment (data not shown). Gene ontology (GO) analysis of this dataset revealed several clusters of genes with functional properties that are consistent with previously reported microarray studies of 1,25-(OH)2D3-regulated genes (Heikkinen et al., 2011Heikkinen S. Väisänen S. Pehkonen P. Seuter S. Benes V. Carlberg C. Nuclear hormone 1α,25-dihydroxyvitamin D3 elicits a genome-wide shift in the locations of VDR chromatin occupancy.Nucleic Acids Res. 2011; 39: 9181-9193Crossref PubMed Scopus (187) Google Scholar, Hossein-nezhad et al., 2013Hossein-nezhad A. Spira A. Holick M.F. Influence of vitamin D status and vitamin D3 supplementation on genome wide expression of white blood cells: A randomized double-blind clinical trial.PLoS ONE. 2013; 8: e58725Crossref PubMed Scopus (192) Google Scholar). These included a significant enrichment of genes associated with apoptosis, immune functions, response to stimulus, transport, cellular component organization, development, and metabolism. Given the dependency of HSF-1, SKN-1, and IRE-1/XBP-1 in D3-induced lifespan extension, we further examined our RNA-seq dataset to determine whether expression of target genes of any of these transcription factors might be perturbed by vitamin D3 treatment. First, we examined whether our RNA-seq dataset was enriched for heat shock proteins (HSPs), since HSF-1 has been shown to be a major transcriptional regulator of these genes. We observed no significant enrichment for HSPs in the transcriptional effects of D3 feeding. These data are consistent with our previous finding that D3 feeding had no effect on the molecular chaperone transcriptional reporter strain, phsp-16.2::GFP. We next examined SKN-1 gene targets from a previously reported array that examined differential expression between skn-1 knockdown and control worms, profiled at L4 larval stage (Oliveira et al., 2009Oliveira R.P. Porter Abate J. Dilks K. Landis J. Ashraf J. Murphy C.T. Blackwell T.K. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf.Aging Cell. 2009; 8: 524-541Crossref PubMed Scopus (241) Google Scholar). Comparison of our dataset with the subset of genes found to be downregulated in skn-1 knockdown animals revealed a striking enrichment for genes expressed in response to D3 feeding (empirical p = 10−6; Table S2). Genes negatively regulated by SKN-1 were not significantly perturbed by vitamin D (empirical p = 0.48). Since SKN-1 activates the transcription of genes encoding phase II detoxification enzymes in response to oxidative stress (An and Blackwell, 2003An J.H. Blackwell T.K. SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response.Genes Dev. 2003; 17: 1882-1893Crossref PubMed Scopus (515) Google Scholar) and vitamin D induces SKN-1 gene targets, we next evaluated whether D3 induced oxidative stress. To test this, we measured reactive oxygen species (ROS) levels, using the superoxide ROS indicator dihydroethidium (DHE), in synchronously aged N2 worms grown from eggs on either vitamin-D3- or control-treated NGM plates. We found that DHE-derived fluorescent ethidium levels were unchanged between D3-treated and vehicle-treated worms. In contrast, N2 worms treated with paraquat (PQ), a known oxidative stress inducer, had significantly increased ROS levels compared to both vitamin-D3- or control-treated worms (data not shown). We asked whether vitamin-D3-treated worms differed in their resistance to PQ. Treatment with either vitamin D3 or vehicle control during development and subsequent exposure to PQ resulted in no difference in survival between D3 and control worms. These data indicate that vitamin D does not induce oxidative stress nor oxidative stress resistance. We next considered whether ER-UPR gene targets could be affected by vitamin D feeding. To test this, we used three previously reported definitions of the ER-UPR pathway: genes annotated in the ER-UPR according to the Gene Ontology consortium (http://amigo.geneontology.org/amigo); genes dependent on ire-1 and/or xbp-1 for response to the UPR inducer tunicamycin (Shen et al., 2005Shen X. Ellis R.E. Sakaki K. Kaufman R.J. Genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in C. elegans.PLoS Genet. 2005; 1: e37Crossref PubMed Scopus (181) Google Scholar); and genes dependent on pek-1 and/or atf-6 for tunicamycin response. In contrast to our findings with SKN-1, in each cohort, and in a cohort defined as their union, we observed no significant enrichment for the transcriptional effects of D3 feeding (empirical p = 0.17, 0.99, 0.71, and 0.78, respectively), consistent with our single-gene analyses of ER-UPR targets (data not shown). Given the IRE-1/XBP-1 and SKN-1 dependency for D3 lifespan extension, we further examined the crosstalk between these" @default.
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• W2531971806 title "Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1" @default.
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