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Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
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Editorial
Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013
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The 16th Vitamin D Workshop met in San Francisco for the three days prior to the Endocrine Society meeting. Although this Workshop is a stand alone meeting, the timing and location in proximity to the Endocrine Society meeting represents a decision by leaders in both the Vitamin D Workshop and Endocrine Society to align our efforts in recognition of the mutual and overlapping interests each group shares with the other. During this Workshop we had 24 invited speakers, 24 shorter presentations that were selected from the submitted abstracts, and 109 presentations presented in the form of posters. All presenters were encouraged to submit manuscripts of the work presented which could be in the form of reviews by the invited speakers or original publications by the abstract presenters. These manuscripts were reviewed by at least two members of the Executive Committee, Program Advisory Committee, or other individuals with expertise in the subject matter. Those accepted for publication are found in this special edition of the Journal of Steroid Biochemistry and Molecular Biology. Drs. Daniel Bikle and JoEllen Welsh served as the Special Editors for this publication. We thank the editor of JSBMB, Dr. Jerzy Adamski, his staff, and Elsevier for their interest and support in the publication of the proceedings of the 16th Vitamin D Workshop. What follows is a synopsis of the meeting, focusing on the oral presentations.
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1. D-Code ENCODE
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The leadoff session “D-code ENCODE” focused on the astonishing developments coming out of the ENCODE project and how those results could be utilized by investigators in the Vitamin D field. Our plenary speaker was John Stamatoyannopoulos, one of the principal investigators in the ENCODE project. He made the point that although there were only about 20,000 coding genes, there were around 30,000 non coding genes and at least 4 million regulatory elements. Not surprisingly not all genes and regulatory elements are active in every cell at any moment. Rather there is remarkable cell specificity in what parts of the chromosome are active with approximately 100,000–250,000 DNA hypersensitive sites in a given cell (sites undergoing active transcription), and these sites vary from cell to cell. Although the initial efforts by the ENCODE project were performed with a limited number of cell lines, the project is expanding into additional cell lines and primary cells. The next two speakers in the session, Wes Pike and Carsten Carlberg, then described their efforts to apply these genome-wide techniques and ENCODE data to understand signaling by 1,25(OH)2 D and its
receptor VDR. Their results demonstrate that 1,25(OH)2 D/VDR regulates thousands of genes through a variety of response elements located nearly anywhere in the gene. Of interest as shown by Pike with an osteoblast cell line that differentiates into osteocytes, there is a marked reduction in the number of genes regulated by 1,25(OH)2 D, and only modest overlap with those regulated in the osteoblast indicating the cell and differentiation specificity of genomic regulation by 1,25(OH)2 D. Closely linked to the ENCODE session was the following session on epigenetics and transcriptional regulation. Mark Meyer demonstrated the epigenetic changes that accompanied the transdifferentiation from bone to fat and its reverse. Prashant Singh and T. Seth were more focused on the epigenetic regulation of coregulator complexes in their respective talks on VDR target genes in prostate cancer progression and 24 hydroxylase transcriptional regulation. 2. Vitamin D metabolism Vitamin D metabolism was the major theme for the next three sessions. Michael Collins discussed his studies in tumor induced osteomalacia, a condition in which excess FGF23 from the tumor decreases CYP27B1 while increasing CYP24A1 in the kidney. He left us with the question as to why the 1,25(OH)2 D levels overshoot when the tumor is removed despite little change in PTH, calcium, and phosphate. In a separate session Myles Wolf reviewed the role of FGF23 in inhibiting renal phosphate reabsorption, 1,25(OH)2 D production, and PTH secretion. He pointed out recent data that the fall in 1,25(OH)2 D production in early kidney failure could be reversed with an antibody to FGF23. The reason FGF23 increases in the early stages of renal failure remains unclear. Wenhan Chang presented his fascinating observations that when CYP27B1 (the enzyme that produces 1,25(OH)2 D) is selectively deleted in the parathyroid gland, serum levels of 1,25(OH)2 D and calcium fall despite the large increase in PTH. When the VDR is deleted in the parathyroid gland the rise in PTH is more modest, and serum 1,25(OH)2 D and calcium increase as expected. The results clearly indicate that the parathyroid CYP27B1 is doing more than providing the ligand for the parathyroid VDR, and Chang suggests it may be an important source for circulating 1,25(OH)2 D. John Adams then reviewed the studies of CYP27B1 regulation in macrophages, focusing on the cytokines responsible for this regulation. The report by Alissa Verone suggested the promising role of high dose vitamin D supplementation in the treatment of non small cell lung cancer
http://dx.doi.org/10.1016/j.jsbmb.2014.04.016 0960-0760/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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patients whose tumors have a VDRhigh CYP24A1low genotype. Ken Thummel turned our attention to the role of drug induced CYP3A4 in liver and intestine, and its potential role in drug induced osteomalacia by metabolizing 25OHD to non active metabolites. This can be an important problem in patients with tuberculosis treated with rifampin, an inducer of CYP3A4, as these patients often have low levels of 25OHD to begin with, and as shown previously by the Modlin group fail in their innate immune response to organisms like m. tuberculosis. Andrzej Slominski followed with his discussion of the role of CYP11A1 in vitamin D regulation of epidermal function. CYP11A1, the key regulatory enzyme in cortisol production, also appears to use vitamin D as a substrate with hydroxylations in the side chain at carbons 17, 20, 22, and 23. Some of these metabolites such as 20OHD appear to induce genes that also targets of 1,25(OH)2 D in a VDR dependent fashion. Perhaps these observations can explain why the phenotype of the VDR null mouse differs from that of the CYP27B1 null mouse at least in the skin (the former develops alopecia, the latter does not).
3. Vitamin D and malignancy The role of vitamin D regulated pathways in malignancy was the focus of the next session. Susan Gonzalo introduced us to a novel pathway by which 1,25(OH)2 D could act to suppress tumor development, in this case breast cancer. Her group found that 1,25(OH)2 D stabilized 53BP1, a DNA repair enzyme, that when lost contributes to the resistance of triple negative, BRCA1-deficient breast cancers to inhibitors of poly(ADP-ribose) polymerase. This type of breast cancer may be a good target for 1,25(OH)2 D when other agents fail. JoEllen Welsh focused on the role of 1,25(OH)2 D in suppressing the hyaluronic acid (HA)/CD44 pathway in breast cancer cells as a means by which these tumors can be treated. HA promotes survival, invasion, and metastasis of these tumors via its receptor CD44, and by inhibiting the hyaluronan synthase-2, 1,25(OH)2 D can block these events. John White explored the role of the E3 ubiquitin ligase FBw7 in regulating the ratio of cMYC and its antagonist MXD1. 1,25(OH)2 D increases the turnover of cMYC and reduces that of MXD1, and appears to induce this change by regulating the binding to and presumably the catabolism of these factors by FBw7. Johan Lundqvist examined the role of 1,25(OH)2 D suppression of CYP19A1, the aromatase enzyme catalyzing the production of estradiol. This enzyme is the target of therapy for ER + breast cancers as well as ER itself. He demonstrated that at least for these genes the Williams syndrome transcription factor WSTF bound to the promoters of CYP19A1 and ER␣, apparently activating them, and that 1,25(OH)2 D displaced its binding. Of interest is that WSTF has been implicated as part of the suppression by 1,25(OH)2 D in genes such as CYP27B1 suggesting that it plays different roles for different genes, again consistent with our earlier discussion of the cell and gene specificity of 1,25(OH)2 D regulation. In a poster by Yong-Sik Bong et al., the role of VDR deletion in the predisposition to carcinogen induced colon cancer was presented. No alteration in catenin levels or distribution were detected in the VDR null animals, but increased transcriptional activity of -catenin was implied by the increase in cyclin D1 expression. Relevant to these findings in the colon are the results presented by Dan Bikle who noted in keratinocytes that VDR and -catenin have reciprocal roles. VDR blocks -catenin transcriptional activity on genes like cyclin D1 that have only TCF/LEF response elements and are involved primarily in proliferation. On the other hand VDR and -catenin promote the expression of genes like PADI1 and S100a3 that have both VDR and TCF/LEF response elements in proximity and are involved in epidermal and hair follicle differentiation. The epidermis, like the intestinal epithelia, is predisposed to cancer in the absence of VDR.
4. Vitamin D regulation of non coding RNAs Non coding RNAs and their regulation of 1,25(OH)2 D was discussed by a number of speakers. Wenlong Bai reviewed his studies demonstrating that 1,25(OH)2 D inhibited ovarian cancer cell growth by inducing miR-498 that resulted in destabilization of the mRNA for the telomerase catalytic substrate hTERT. Of interest is that the regulation of miR-498 appeared to be unique to ovarian cancer cells, a result that reflects back on the earlier discussion in the ENCODE session of the cell specificity of these regulatory actions of 1,25(OH)2 D. Yan Chun Li explored the role of miR-155 in 1,25(OH)2 D regulation of the innate immune response. MiR-155 inhibits the expression of SOCS1, an inhibitor of the inflammatory response. LPS dramatically increases miR-155 in macrophages, but this is suppressed by 1,25(OH)2 D. In VDR null cells the inflammatory response to LPS is exaggerated due to enhanced miR-155 production and suppression of SOCS1. Yan Jiang examined the regulation of long non coding RNAs (lncRNA) in keratinocytes in vivo and in vitro. She showed in either case a consistent upregulation in VDR null cells of lncRNAs associated with oncogenic activity and a decrease in levels of lncRNAs associated with tumor suppression. She postulated that this pattern of lncRNA expression was consistent with the predisposition of VDR null animals to develop skin cancer when exposed to UVB. Wei-Lin Wang explored the role of the miR-17/92 cluster that they had previously shown was down regulated by 1,25(OH)2 D and androgens in prostate cancer. As PPAR␣ is a predicted target of miR-17/92 and 1,25(OH)2 D and androgens upregulate PPAR␣, she linked the role of 1,25(OH)2 D and androgens on lipid metabolism in prostate cells to their role in regulating miR-17/92. She speculated that by shifting energy toward lipogenesis and away from oxidative phosphorylation required to power proliferation and metastasis 1,25(OH)2 D could block prostate cancer growth. Surprisingly Dicer, the key ribonuclease involved in miRNA production, turns out to be a target of 1,25(OH)2 D as shown in a poster by RJ Gonzalez-Duarte et al.
5. Vitamin D and immunity/inflammation Margherita Cantorna initiated the session on vitamin D regulation of the immune session by focusing on her recent studies in inflammatory bowel disease (IBD). In a small 24 wk study, 5000 IU vitamin D decreased the disease score in patients, but no placebo control was included. VDRKO animals have alterations in the Th1 and Th17 cells with changes in cytokines like IL-17 and IFN-␥ that predispose them to dextran sodium sulfate (DSS) induced IBD. Both VDRKO and CYP27B1KO have alterations in their gut flora that appear to contribute to the inflammatory response. A potential problem in the use of vitamin D or 1,25(OH)2 D in the management of IBD was shown in a poster by Doris Hummel et al. that proinflammatory cytokines increase the expression of CYP24A1, and could lessen the impact of vitamin D treatment. Tatiana Takiishi reported that long term administration of vitamin D starting at weaning reduced the development of diabetes in NOD mice, a model of type 1 diabetes mellitus, in association with a reduction in IFN-␥ + CD8 cells in pancreatic lymph nodes and spleen. Gabriela Ferreira then showed that dendritic cells from NOD mice could be induced to become tolerogenic by 1,25(OH)2 D. Anita Mehrotra discussed her clinical study in patients on hemodialysis in which she demonstrated a reduction in allogenic immunity over time in those receiving 50,000 IU vitamin D per week. Yan Li examined the role of vitamin D in preventing acute inflammation induced lung injury. VDRKO mice are quite susceptible to LPS induced lung injury associated with an increase in the angiopoietiin-2-tie-2 pathway. 1,25(OH)2 D blocked the LPS induced increase in angiopoeitin by blocking NFB activation. Vitamin D may also be useful in the
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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treatment of asthma particularly in steroid sensitive patients by decreasing IL-17A and increasing IL-10 as presented in a poster by Catherine Hawrlyowicz et al. 6. Vitamin D: classic target tissues The classic actions of vitamin D on intestine and bone were the subject of the next session. James Fleet reviewed data from a number of mouse models in which VDR or its effector molecules such as TRPV6 and calbindin have been deleted in the enterocyte. Deletion of these molecules did not eliminate calcium transport. Moreover, he looked at calcium absorption in a number of mouse strains in comparison to their 1,25(OH)2 D levels, and found a poor correlation. The overall conclusion is that there is still much to learn about the mechanism by which intestinal calcium transport is regulated by vitamin D despite the many years of study. Although VDR regulated intestinal calcium transport is thought to be greatest in the duodenum, Puneet Dhawan developed an animal model in which VDR was restricted to the ileum using a cdx2 promoter. These mice were able to correct the skeletal lesions of the VDRKO mouse suggesting that the ileum could compensate for the reduced calcium absorption in the VDR deficient duodenum. In bone deletion of the VDR appears to increase bone volume as reported by Howard Morris primarily by reducing osteoclast activation, but data presented by Jackson Ryan from the same group indicated that these results might be gender specific: female VDRKO mice on the rescue diet increased BV/TV, whereas male VDRKO mice lost bone. The expression of CYP27B1 in both osteoblasts and osteoclasts as presented in posters by Renee Ormsby et al., AG Turner et al., and Masakazu Kogawa et al. and its role in osteoblast and osteoclast differentiation contributes to the complexity of vitamin D signaling in bone. On the other hand in posters presented by Ayako Shiraishi et al. and Toshio Matsumoto et al., the 1,25(OH)2 D analog eldecalcidol improved the strength of cortical bone and reduced fractures. However, eldecalcitol may act as a partial agonist for the VDR displacing 1,25(OH)2 D as shown in a poster by Suguru Harada et al., possibly explaining the apparent paradox of why eldecalcidol is bone anabolic as is VDR deletion. 7. Vitamin D measurements and world wide vitamin D deficiency The final day of the Workshop was devoted to clinic topics following an outstanding lecture by Keith Yamamoto describing the mechanisms by which the glucocorticoid receptor bound to its response elements in DNA. The first clinical sessions of the day focused on vitamin D assessment, public health issues predisposing to vitamin D deficiency, and means to achieve desired levels. Neil Binkley dealt nicely with the challenges that accurate measurement of serum 25OHD levels encounters, and the methods being utilized to address those challenges. The bottom line is that the number reported from the lab using a good assay still has a substantial error of the measurement that must be considered when deciding whom to treat and with how much vitamin D. Martin Hewison reviewed the status of megalin in enabling the kidney to reclaim 25OHD that would otherwise be lost in the urine. In addition he reminded us that nearly all 25OHD and other vitamin D metabolites are bound to vitamin D binding protein (DBP), but that DBP has different isoforms with different affinities for 25OHD that could affect the ratio of free (the presumably biologically active form) to total 25OHD. This point is made clear in a poster by Jennifer Lai et al. that patients with liver disease and low DBP levels have a higher free 25OHD level despite their reduced total 25OHD level than those without liver disease. Likewise in a poster U.K. Moller et al. showed that women on oral contraceptives have higher
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levels of DBP and total vitamin D metabolites, but their calculated free 25OHD and 1,25(OH)2 D were not significantly different from controls nor were parameters of calcium homeostasis including calcium, calcitonin and PTH. Thus measurement of total 25OHD may be misleading in conditions in which DBP levels and affinity for vitamin D metabolites are not taken into account. Elina Hypponen reviewed her GWAS study showing that polymorphisms in the DBP, CYP2R1 (microsomal 25 hydroxylase), DHRC7 (enzyme converting 7-dehydrocholesterol to cholesterol), and CYP24A1 are associated with 25OHD levels, but this accounts for only a few percent of the variation of 25OHD levels in the population. However, the poster by Alena Didriksen et al. found that polymorphisms in DBP and CYP2R1 were associated not only with basal levels of 25OHD, but to the magnitude of the response of serum 25OHD to vitamin D supplementation. Surprisingly the polymorphisms in these genes associated with low 25OHD levels appeared to be protective against aggressive prostate cancer in a study presented as a poster by Alison Mondol et al. Antony Young reported that if you want to increase your 25OHD level go skiing or visit the Canary Islands. You can protect your skin from burning and still increase your vitamin D levels with a good sunscreen. Ingestion of vitamin D is another way to go. A. Burild pointed out that the ingestion of 25OHD increases the serum levels of 25OHD three fold higher than equivalent doses of vitamin D itself. S. Nguyen et al. provided reassuring data in a poster that levels of 25OHD up to 100 ng/ml were not associated with an increased risk of kidney stones. On the other hand Cristina Palacios presented depressing data showing the extent of vitamin D deficiency around the world. This theme was continued in the presentation by Paul Lips who further indicated that 25OHD clearance is accelerated in subjects ingesting a low calcium diet. So diets low in vitamin D and calcium are particularly detrimental to skeletal health. Zulf Mughal in the final talk of the session discussed the cultural problems that underlay vitamin D deficiency and its treatment even in sun rich countries, and reported on efforts by various countries to combat this problem. Most efforts target the pregnant mother and infants using supplements.
8. Vitamin D and the metabolic syndrome The final session examined the relationship between vitamin D and aspects of the metabolic syndrome. Anastassios Pittas reviewed the evidence from a meta-analysis of several longitudinal studies indicating a reciprocal relationship between 25OHD levels and the development of type 2 diabetes mellitus. Pittas was critical of the data from interventional trials as being underpowered to conclusively demonstrate that vitamin D prevents diabetes. Rolf Jorde followed up by reporting that data from the Tromso cohort did not support a relationship between vitamin D administration (20,000–40,000 IU/wk) and an effect on markers of glucose intolerance (Hgb A1c, lipids, CRP) at least in subjects who were not vitamin D deficient at the start of the study. Moreover, in a poster presented by Mirjam Oosterwerff et al. vitamin D supplementation of vitamin D deficient prediabetic non-western immigrants in the Netherlands did not improve either insulin resistance or -cell function after 16 wks relative to placebo controls. Ravi Thadhani turned our attention to the potential role of vitamin D in the prevention/treatment of hypertension. Although animal studies have been suggestive of a positive effect on blood pressure by reducing the renin/angiotensin system, clinical studies have provided mixed results, with little information from large randomized control trials. The VALIDATE-D study presented in a poster by Jenifer Brown is an ongoing clinical trial to address some of these issues in diabetics. A more positive role for vitamin D in preventing atherosclerosis was presented by Carlos Bernal-Mizrachi. He found that macrophages from vitamin D deficient mice take up more cholesterol, predisposing to
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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atherosclerosis. Inhibitors of ER stress could reverse this. Moreover, 1,25(OH)2 D suppressed cholesterol uptake by macrophages from diabetic subjects. Whether these animal and in vitro human cell studies can be translated into preventing atherosclerosis in humans remains to be seen. The author apologizes to all the presenters whose excellent work presented at the Workshop was not summarized in this synopsis. Limitations of space and an effort to adhere to the main themes of the oral sessions precluded this.
Daniel Bikle (MD, PhD) Q1 University of California San Francisco, Veterans Affairs Medical Center, 4150 Clement St (111N), San Q2 Francisco, CA 94121, United States E-mail address: [email protected] Available online xxx
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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SBMB 4197 1–4
Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
1
2
3
Editorial
Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013
4
26
The 16th Vitamin D Workshop met in San Francisco for the three days prior to the Endocrine Society meeting. Although this Workshop is a stand alone meeting, the timing and location in proximity to the Endocrine Society meeting represents a decision by leaders in both the Vitamin D Workshop and Endocrine Society to align our efforts in recognition of the mutual and overlapping interests each group shares with the other. During this Workshop we had 24 invited speakers, 24 shorter presentations that were selected from the submitted abstracts, and 109 presentations presented in the form of posters. All presenters were encouraged to submit manuscripts of the work presented which could be in the form of reviews by the invited speakers or original publications by the abstract presenters. These manuscripts were reviewed by at least two members of the Executive Committee, Program Advisory Committee, or other individuals with expertise in the subject matter. Those accepted for publication are found in this special edition of the Journal of Steroid Biochemistry and Molecular Biology. Drs. Daniel Bikle and JoEllen Welsh served as the Special Editors for this publication. We thank the editor of JSBMB, Dr. Jerzy Adamski, his staff, and Elsevier for their interest and support in the publication of the proceedings of the 16th Vitamin D Workshop. What follows is a synopsis of the meeting, focusing on the oral presentations.
27
1. D-Code ENCODE
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
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The leadoff session “D-code ENCODE” focused on the astonishing developments coming out of the ENCODE project and how those results could be utilized by investigators in the Vitamin D field. Our plenary speaker was John Stamatoyannopoulos, one of the principal investigators in the ENCODE project. He made the point that although there were only about 20,000 coding genes, there were around 30,000 non coding genes and at least 4 million regulatory elements. Not surprisingly not all genes and regulatory elements are active in every cell at any moment. Rather there is remarkable cell specificity in what parts of the chromosome are active with approximately 100,000–250,000 DNA hypersensitive sites in a given cell (sites undergoing active transcription), and these sites vary from cell to cell. Although the initial efforts by the ENCODE project were performed with a limited number of cell lines, the project is expanding into additional cell lines and primary cells. The next two speakers in the session, Wes Pike and Carsten Carlberg, then described their efforts to apply these genome-wide techniques and ENCODE data to understand signaling by 1,25(OH)2 D and its
receptor VDR. Their results demonstrate that 1,25(OH)2 D/VDR regulates thousands of genes through a variety of response elements located nearly anywhere in the gene. Of interest as shown by Pike with an osteoblast cell line that differentiates into osteocytes, there is a marked reduction in the number of genes regulated by 1,25(OH)2 D, and only modest overlap with those regulated in the osteoblast indicating the cell and differentiation specificity of genomic regulation by 1,25(OH)2 D. Closely linked to the ENCODE session was the following session on epigenetics and transcriptional regulation. Mark Meyer demonstrated the epigenetic changes that accompanied the transdifferentiation from bone to fat and its reverse. Prashant Singh and T. Seth were more focused on the epigenetic regulation of coregulator complexes in their respective talks on VDR target genes in prostate cancer progression and 24 hydroxylase transcriptional regulation. 2. Vitamin D metabolism Vitamin D metabolism was the major theme for the next three sessions. Michael Collins discussed his studies in tumor induced osteomalacia, a condition in which excess FGF23 from the tumor decreases CYP27B1 while increasing CYP24A1 in the kidney. He left us with the question as to why the 1,25(OH)2 D levels overshoot when the tumor is removed despite little change in PTH, calcium, and phosphate. In a separate session Myles Wolf reviewed the role of FGF23 in inhibiting renal phosphate reabsorption, 1,25(OH)2 D production, and PTH secretion. He pointed out recent data that the fall in 1,25(OH)2 D production in early kidney failure could be reversed with an antibody to FGF23. The reason FGF23 increases in the early stages of renal failure remains unclear. Wenhan Chang presented his fascinating observations that when CYP27B1 (the enzyme that produces 1,25(OH)2 D) is selectively deleted in the parathyroid gland, serum levels of 1,25(OH)2 D and calcium fall despite the large increase in PTH. When the VDR is deleted in the parathyroid gland the rise in PTH is more modest, and serum 1,25(OH)2 D and calcium increase as expected. The results clearly indicate that the parathyroid CYP27B1 is doing more than providing the ligand for the parathyroid VDR, and Chang suggests it may be an important source for circulating 1,25(OH)2 D. John Adams then reviewed the studies of CYP27B1 regulation in macrophages, focusing on the cytokines responsible for this regulation. The report by Alissa Verone suggested the promising role of high dose vitamin D supplementation in the treatment of non small cell lung cancer
http://dx.doi.org/10.1016/j.jsbmb.2014.04.016 0960-0760/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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patients whose tumors have a VDRhigh CYP24A1low genotype. Ken Thummel turned our attention to the role of drug induced CYP3A4 in liver and intestine, and its potential role in drug induced osteomalacia by metabolizing 25OHD to non active metabolites. This can be an important problem in patients with tuberculosis treated with rifampin, an inducer of CYP3A4, as these patients often have low levels of 25OHD to begin with, and as shown previously by the Modlin group fail in their innate immune response to organisms like m. tuberculosis. Andrzej Slominski followed with his discussion of the role of CYP11A1 in vitamin D regulation of epidermal function. CYP11A1, the key regulatory enzyme in cortisol production, also appears to use vitamin D as a substrate with hydroxylations in the side chain at carbons 17, 20, 22, and 23. Some of these metabolites such as 20OHD appear to induce genes that also targets of 1,25(OH)2 D in a VDR dependent fashion. Perhaps these observations can explain why the phenotype of the VDR null mouse differs from that of the CYP27B1 null mouse at least in the skin (the former develops alopecia, the latter does not).
3. Vitamin D and malignancy The role of vitamin D regulated pathways in malignancy was the focus of the next session. Susan Gonzalo introduced us to a novel pathway by which 1,25(OH)2 D could act to suppress tumor development, in this case breast cancer. Her group found that 1,25(OH)2 D stabilized 53BP1, a DNA repair enzyme, that when lost contributes to the resistance of triple negative, BRCA1-deficient breast cancers to inhibitors of poly(ADP-ribose) polymerase. This type of breast cancer may be a good target for 1,25(OH)2 D when other agents fail. JoEllen Welsh focused on the role of 1,25(OH)2 D in suppressing the hyaluronic acid (HA)/CD44 pathway in breast cancer cells as a means by which these tumors can be treated. HA promotes survival, invasion, and metastasis of these tumors via its receptor CD44, and by inhibiting the hyaluronan synthase-2, 1,25(OH)2 D can block these events. John White explored the role of the E3 ubiquitin ligase FBw7 in regulating the ratio of cMYC and its antagonist MXD1. 1,25(OH)2 D increases the turnover of cMYC and reduces that of MXD1, and appears to induce this change by regulating the binding to and presumably the catabolism of these factors by FBw7. Johan Lundqvist examined the role of 1,25(OH)2 D suppression of CYP19A1, the aromatase enzyme catalyzing the production of estradiol. This enzyme is the target of therapy for ER + breast cancers as well as ER itself. He demonstrated that at least for these genes the Williams syndrome transcription factor WSTF bound to the promoters of CYP19A1 and ER␣, apparently activating them, and that 1,25(OH)2 D displaced its binding. Of interest is that WSTF has been implicated as part of the suppression by 1,25(OH)2 D in genes such as CYP27B1 suggesting that it plays different roles for different genes, again consistent with our earlier discussion of the cell and gene specificity of 1,25(OH)2 D regulation. In a poster by Yong-Sik Bong et al., the role of VDR deletion in the predisposition to carcinogen induced colon cancer was presented. No alteration in catenin levels or distribution were detected in the VDR null animals, but increased transcriptional activity of -catenin was implied by the increase in cyclin D1 expression. Relevant to these findings in the colon are the results presented by Dan Bikle who noted in keratinocytes that VDR and -catenin have reciprocal roles. VDR blocks -catenin transcriptional activity on genes like cyclin D1 that have only TCF/LEF response elements and are involved primarily in proliferation. On the other hand VDR and -catenin promote the expression of genes like PADI1 and S100a3 that have both VDR and TCF/LEF response elements in proximity and are involved in epidermal and hair follicle differentiation. The epidermis, like the intestinal epithelia, is predisposed to cancer in the absence of VDR.
4. Vitamin D regulation of non coding RNAs Non coding RNAs and their regulation of 1,25(OH)2 D was discussed by a number of speakers. Wenlong Bai reviewed his studies demonstrating that 1,25(OH)2 D inhibited ovarian cancer cell growth by inducing miR-498 that resulted in destabilization of the mRNA for the telomerase catalytic substrate hTERT. Of interest is that the regulation of miR-498 appeared to be unique to ovarian cancer cells, a result that reflects back on the earlier discussion in the ENCODE session of the cell specificity of these regulatory actions of 1,25(OH)2 D. Yan Chun Li explored the role of miR-155 in 1,25(OH)2 D regulation of the innate immune response. MiR-155 inhibits the expression of SOCS1, an inhibitor of the inflammatory response. LPS dramatically increases miR-155 in macrophages, but this is suppressed by 1,25(OH)2 D. In VDR null cells the inflammatory response to LPS is exaggerated due to enhanced miR-155 production and suppression of SOCS1. Yan Jiang examined the regulation of long non coding RNAs (lncRNA) in keratinocytes in vivo and in vitro. She showed in either case a consistent upregulation in VDR null cells of lncRNAs associated with oncogenic activity and a decrease in levels of lncRNAs associated with tumor suppression. She postulated that this pattern of lncRNA expression was consistent with the predisposition of VDR null animals to develop skin cancer when exposed to UVB. Wei-Lin Wang explored the role of the miR-17/92 cluster that they had previously shown was down regulated by 1,25(OH)2 D and androgens in prostate cancer. As PPAR␣ is a predicted target of miR-17/92 and 1,25(OH)2 D and androgens upregulate PPAR␣, she linked the role of 1,25(OH)2 D and androgens on lipid metabolism in prostate cells to their role in regulating miR-17/92. She speculated that by shifting energy toward lipogenesis and away from oxidative phosphorylation required to power proliferation and metastasis 1,25(OH)2 D could block prostate cancer growth. Surprisingly Dicer, the key ribonuclease involved in miRNA production, turns out to be a target of 1,25(OH)2 D as shown in a poster by RJ Gonzalez-Duarte et al.
5. Vitamin D and immunity/inflammation Margherita Cantorna initiated the session on vitamin D regulation of the immune session by focusing on her recent studies in inflammatory bowel disease (IBD). In a small 24 wk study, 5000 IU vitamin D decreased the disease score in patients, but no placebo control was included. VDRKO animals have alterations in the Th1 and Th17 cells with changes in cytokines like IL-17 and IFN-␥ that predispose them to dextran sodium sulfate (DSS) induced IBD. Both VDRKO and CYP27B1KO have alterations in their gut flora that appear to contribute to the inflammatory response. A potential problem in the use of vitamin D or 1,25(OH)2 D in the management of IBD was shown in a poster by Doris Hummel et al. that proinflammatory cytokines increase the expression of CYP24A1, and could lessen the impact of vitamin D treatment. Tatiana Takiishi reported that long term administration of vitamin D starting at weaning reduced the development of diabetes in NOD mice, a model of type 1 diabetes mellitus, in association with a reduction in IFN-␥ + CD8 cells in pancreatic lymph nodes and spleen. Gabriela Ferreira then showed that dendritic cells from NOD mice could be induced to become tolerogenic by 1,25(OH)2 D. Anita Mehrotra discussed her clinical study in patients on hemodialysis in which she demonstrated a reduction in allogenic immunity over time in those receiving 50,000 IU vitamin D per week. Yan Li examined the role of vitamin D in preventing acute inflammation induced lung injury. VDRKO mice are quite susceptible to LPS induced lung injury associated with an increase in the angiopoietiin-2-tie-2 pathway. 1,25(OH)2 D blocked the LPS induced increase in angiopoeitin by blocking NFB activation. Vitamin D may also be useful in the
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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treatment of asthma particularly in steroid sensitive patients by decreasing IL-17A and increasing IL-10 as presented in a poster by Catherine Hawrlyowicz et al. 6. Vitamin D: classic target tissues The classic actions of vitamin D on intestine and bone were the subject of the next session. James Fleet reviewed data from a number of mouse models in which VDR or its effector molecules such as TRPV6 and calbindin have been deleted in the enterocyte. Deletion of these molecules did not eliminate calcium transport. Moreover, he looked at calcium absorption in a number of mouse strains in comparison to their 1,25(OH)2 D levels, and found a poor correlation. The overall conclusion is that there is still much to learn about the mechanism by which intestinal calcium transport is regulated by vitamin D despite the many years of study. Although VDR regulated intestinal calcium transport is thought to be greatest in the duodenum, Puneet Dhawan developed an animal model in which VDR was restricted to the ileum using a cdx2 promoter. These mice were able to correct the skeletal lesions of the VDRKO mouse suggesting that the ileum could compensate for the reduced calcium absorption in the VDR deficient duodenum. In bone deletion of the VDR appears to increase bone volume as reported by Howard Morris primarily by reducing osteoclast activation, but data presented by Jackson Ryan from the same group indicated that these results might be gender specific: female VDRKO mice on the rescue diet increased BV/TV, whereas male VDRKO mice lost bone. The expression of CYP27B1 in both osteoblasts and osteoclasts as presented in posters by Renee Ormsby et al., AG Turner et al., and Masakazu Kogawa et al. and its role in osteoblast and osteoclast differentiation contributes to the complexity of vitamin D signaling in bone. On the other hand in posters presented by Ayako Shiraishi et al. and Toshio Matsumoto et al., the 1,25(OH)2 D analog eldecalcidol improved the strength of cortical bone and reduced fractures. However, eldecalcitol may act as a partial agonist for the VDR displacing 1,25(OH)2 D as shown in a poster by Suguru Harada et al., possibly explaining the apparent paradox of why eldecalcidol is bone anabolic as is VDR deletion. 7. Vitamin D measurements and world wide vitamin D deficiency The final day of the Workshop was devoted to clinic topics following an outstanding lecture by Keith Yamamoto describing the mechanisms by which the glucocorticoid receptor bound to its response elements in DNA. The first clinical sessions of the day focused on vitamin D assessment, public health issues predisposing to vitamin D deficiency, and means to achieve desired levels. Neil Binkley dealt nicely with the challenges that accurate measurement of serum 25OHD levels encounters, and the methods being utilized to address those challenges. The bottom line is that the number reported from the lab using a good assay still has a substantial error of the measurement that must be considered when deciding whom to treat and with how much vitamin D. Martin Hewison reviewed the status of megalin in enabling the kidney to reclaim 25OHD that would otherwise be lost in the urine. In addition he reminded us that nearly all 25OHD and other vitamin D metabolites are bound to vitamin D binding protein (DBP), but that DBP has different isoforms with different affinities for 25OHD that could affect the ratio of free (the presumably biologically active form) to total 25OHD. This point is made clear in a poster by Jennifer Lai et al. that patients with liver disease and low DBP levels have a higher free 25OHD level despite their reduced total 25OHD level than those without liver disease. Likewise in a poster U.K. Moller et al. showed that women on oral contraceptives have higher
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levels of DBP and total vitamin D metabolites, but their calculated free 25OHD and 1,25(OH)2 D were not significantly different from controls nor were parameters of calcium homeostasis including calcium, calcitonin and PTH. Thus measurement of total 25OHD may be misleading in conditions in which DBP levels and affinity for vitamin D metabolites are not taken into account. Elina Hypponen reviewed her GWAS study showing that polymorphisms in the DBP, CYP2R1 (microsomal 25 hydroxylase), DHRC7 (enzyme converting 7-dehydrocholesterol to cholesterol), and CYP24A1 are associated with 25OHD levels, but this accounts for only a few percent of the variation of 25OHD levels in the population. However, the poster by Alena Didriksen et al. found that polymorphisms in DBP and CYP2R1 were associated not only with basal levels of 25OHD, but to the magnitude of the response of serum 25OHD to vitamin D supplementation. Surprisingly the polymorphisms in these genes associated with low 25OHD levels appeared to be protective against aggressive prostate cancer in a study presented as a poster by Alison Mondol et al. Antony Young reported that if you want to increase your 25OHD level go skiing or visit the Canary Islands. You can protect your skin from burning and still increase your vitamin D levels with a good sunscreen. Ingestion of vitamin D is another way to go. A. Burild pointed out that the ingestion of 25OHD increases the serum levels of 25OHD three fold higher than equivalent doses of vitamin D itself. S. Nguyen et al. provided reassuring data in a poster that levels of 25OHD up to 100 ng/ml were not associated with an increased risk of kidney stones. On the other hand Cristina Palacios presented depressing data showing the extent of vitamin D deficiency around the world. This theme was continued in the presentation by Paul Lips who further indicated that 25OHD clearance is accelerated in subjects ingesting a low calcium diet. So diets low in vitamin D and calcium are particularly detrimental to skeletal health. Zulf Mughal in the final talk of the session discussed the cultural problems that underlay vitamin D deficiency and its treatment even in sun rich countries, and reported on efforts by various countries to combat this problem. Most efforts target the pregnant mother and infants using supplements.
8. Vitamin D and the metabolic syndrome The final session examined the relationship between vitamin D and aspects of the metabolic syndrome. Anastassios Pittas reviewed the evidence from a meta-analysis of several longitudinal studies indicating a reciprocal relationship between 25OHD levels and the development of type 2 diabetes mellitus. Pittas was critical of the data from interventional trials as being underpowered to conclusively demonstrate that vitamin D prevents diabetes. Rolf Jorde followed up by reporting that data from the Tromso cohort did not support a relationship between vitamin D administration (20,000–40,000 IU/wk) and an effect on markers of glucose intolerance (Hgb A1c, lipids, CRP) at least in subjects who were not vitamin D deficient at the start of the study. Moreover, in a poster presented by Mirjam Oosterwerff et al. vitamin D supplementation of vitamin D deficient prediabetic non-western immigrants in the Netherlands did not improve either insulin resistance or -cell function after 16 wks relative to placebo controls. Ravi Thadhani turned our attention to the potential role of vitamin D in the prevention/treatment of hypertension. Although animal studies have been suggestive of a positive effect on blood pressure by reducing the renin/angiotensin system, clinical studies have provided mixed results, with little information from large randomized control trials. The VALIDATE-D study presented in a poster by Jenifer Brown is an ongoing clinical trial to address some of these issues in diabetics. A more positive role for vitamin D in preventing atherosclerosis was presented by Carlos Bernal-Mizrachi. He found that macrophages from vitamin D deficient mice take up more cholesterol, predisposing to
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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atherosclerosis. Inhibitors of ER stress could reverse this. Moreover, 1,25(OH)2 D suppressed cholesterol uptake by macrophages from diabetic subjects. Whether these animal and in vitro human cell studies can be translated into preventing atherosclerosis in humans remains to be seen. The author apologizes to all the presenters whose excellent work presented at the Workshop was not summarized in this synopsis. Limitations of space and an effort to adhere to the main themes of the oral sessions precluded this.
Daniel Bikle (MD, PhD) Q1 University of California San Francisco, Veterans Affairs Medical Center, 4150 Clement St (111N), San Q2 Francisco, CA 94121, United States E-mail address: [email protected] Available online xxx
Please cite this article in press as: D. Bikle, Highlights from the 16th Vitamin D Workshop, San Francisco, CA, June 11–14, 2013, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.04.016
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