Molecular insights into mineralotropic hormone inter-regulation.

The regulation of mineral homeostasis involves the three mineralotropic hormones PTH, FGF23 and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Early research efforts focused on PTH and 1,25(OH)2D3 and more recently on FGF23 have revealed that each of these hormones regulates the expression of the other two. Despite early suggestions of transcriptional processes, it has been only recently that research effort have begun to delineate the genomic mechanisms underpinning this regulation for 1,25(OH)2D3 and FGF23; the regulation of PTH by 1,25(OH)2D3, however, remains obscure. We review here our molecular understanding of how PTH induces Cyp27b1 expression, the gene encoding the enzyme responsible for the synthesis of 1,25(OH)2D3. FGF23 and 1,25(OH)2D3, on the other hand, function by suppressing production of 1,25(OH)2D3. PTH stimulates the PKA-induced recruitment of CREB and its coactivator CBP at CREB occupied sites within the kidney-specific regulatory regions of Cyp27b1. PKA activation also promotes the nuclear translocation of SIK bound coactivators such as CRTC2, where it similarly interacts with CREB occupied Cyp27b1 sites. The negative actions of both FGF23 and 1,25(OH)2D3 appear to suppress Cyp27b1 expression by opposing the recruitment of CREB coactivators at this gene. Reciprocal gene actions are seen at Cyp24a1, the gene encoding the enzyme that degrades 1,25(OH)2D3, thereby contributing to the overall regulation of blood levels of 1,25(OH)2D3. Relative to PTH regulation, we summarize what is known of how 1,25(OH)2D3 regulates PTH suppression. These studies suggest that it is not 1,25(OH)2D3 that controls PTH levels in healthy subjects, but rather calcium itself. Finally, we describe current progress using an in vivo approach that furthers our understanding of the regulation of Fgf23 expression by PTH and 1,25(OH)2D3 and provide the first evidence that P may act to induce Fgf23 expression via a complex transcriptional mechanism in bone. It is clear, however, that additional advances will need to be made to further our understanding of the inter-regulation of each of these hormonal genes.

Genome-wide analyses of gene expression profile identify key genes and pathways involved in skeletal response to phosphate and 1,25-dihydroxyvitamin D3 in vivo.

Phosphate (P) is an essential element involved in various biological actions, such as bone integrity, energy production, cell signaling and molecular component. P homeostasis is modulated by 4 main tissues; intestine, kidney, bone, and parathyroid gland, where 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone and fibroblast growth factor 23 (FGF23) are produced and/or have an influence. In bone, serum P level modulates the production of FGF23 which then controls not only P excretion but also vitamin D metabolism in kidney in an endocrine manner. The hormonally active form of vitamin D, 1,25(OH)2D3, also has a significant effect on skeletal cells via its receptor, the vitamin D receptor, to control gene expression which mediates bone metabolism as well as mineral homeostasis. In this study, we adopted RNA-seq analysis to understand genome-wide skeletal gene expression regulation in response to P and 1,25(OH)2D3. We examined lumbar 5 vertebrae from the mice that were fed P deficient diet for a week followed by an acute high P diet for 3, 6, and 24 h as well as mice treated with 1,25(OH)2D3 intraperitoneally for 6 h. Further identification and exploration of the genes regulated by P and 1,25(OH)2D3 showed that P dynamically modulates the expression of skeletal genes involved in various biological processes while 1,25(OH)2D3 regulates genes highly related to bone metabolism. Our in vivo data were then compared with in vitro data that we previously obtained, which suggests that the gene expression profiles presented in this report mainly represent those of osteocytes. Interestingly, it was found that even though the skeletal response to P is distinguished from that to 1,25(OH)2D3, both factors have an effect on Wnt signaling pathway to modulate bone homeostasis. Taken together, this report presents genome-wide data that provide a foundation to understand molecular mechanisms by which skeletal cells respond to P and 1,25(OH)2D3.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

Reproductive performance and sex ratio adjustment of the wild boar (Sus scrofa) in South Korea.

The wild boar (Sus scrofa), a polygynous species, rapidly expanded its geographical range and increased its population size in South Korea following the extinction of large carnivores and changes to rural environments. Understanding wild boar reproductive traits and strategies is essential for their effective management; however, studies in this area are lacking. Using samples collected from hunting bags, the relationships between 1) litter size and female weight and 2) fetal sex ratio and female body condition were examined to understand wild boar life-history strategies. Wild boars showed a seasonal breeding pattern that maximized reproduction. Litter size (mean = 5.7 ± 1.7) was correlated with female weight, whereas fetal sex ratio was not explained by female body condition. However, the heaviest ranked fetuses within the litters were male-biased. Wild boars aged three years or less accounted for 90% of the total population, and sexual dimorphism developed from two years of age. Considering that their reproductive strategy is more effective (i.e., early gestation and large litter size) than that of other polygynous species, the Trivers-Willard model was not supported for the wild boars in this study. Instead, females adjusted the sex of the heaviest fetus in the litter to maximize lifetime reproductive success.

Rapid genomic changes by mineralotropic hormones and kinase SIK inhibition drive coordinated renal Cyp27b1 and Cyp24a1 expression via CREB modules.

Vitamin D metabolism centers on kidney regulation of Cyp27b1 by mineralotropic hormones, including induction by parathyroid hormone (PTH), suppression by fibroblast growth factor 23 (FGF23) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and reciprocal regulations for Cyp24a1. This coordinated genomic regulation results in production of endocrine 1,25(OH)2D3, which, together with PTH and FGF23, controls mineral homeostasis. However, how these events are coordinated is unclear. Here, using in vivo chromatin immunoprecipitation sequencing in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of phosphorylated (p-133) CREB (pCREB) and its coactivators, CBP (CREB-binding protein) and CRTC2 (CREB-regulated transcription coactivator 2), to previously defined kidney-specific M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with decreased genomic activity and reduced transcripts. Treatment of mice with salt-inducible kinase inhibitors (YKL-05-099 and SK-124) yields rapid genomic recruitment of CRTC2 to Cyp27b1, limited interaction of CBP, and a transcriptional response for both Cyp27b1 and Cyp24a1 that mirrors the actions of PTH. Surprisingly, we find that 1,25(OH)2D3 suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and 1,25(OH)2D3 action. Suppressive actions of 1,25(OH)2D3 and FGF23 at the Cyp27b1 gene are associated with reduced CBP recruitment at these CREB-module enhancers that disrupts full PTH induction. Our findings show that CRTC2 contributes to transcription of both Cyp27b1 and Cyp24a1, demonstrate salt-inducible kinase inhibition as a key modulator of vitamin D metabolism, and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)2D3 in the kidney that regulate mineral homeostasis.

Genomic Mechanisms Governing Mineral Homeostasis and the Regulation and Maintenance of Vitamin D Metabolism.

Our recent genomic studies identified a complex kidney-specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1, as well as suppression by FGF23 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The tissue specificity for this regulatory module appears to be localized exclusively to renal proximal tubules. Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both the M1 and M21 submodules together almost completely eliminates basal Cyp27b1 expression in the kidney, creating a renal specific pseudo-null mouse, resulting in a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse caused by high levels of both 25-hydroxyvitamin D3 [25(OH)D3] and PTH and depletion of 1,25(OH)2D3. Cyp24a1 levels in the double KO mouse also decrease because of compensatory downregulation of the gene by elevated PTH and reduced FGF23 that is mediated by an intergenic module located downstream of the Cyp24a1 gene. Outside of the kidney in nonrenal target cells (NRTCs), expression of Cyp27b1 in these mutant mice was unaffected. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues the aberrant phenotype of this mouse and normalizes the skeleton. In addition, both the high levels of 25(OH)D3 were reduced and the low levels of 1,25(OH)2D3 were fully eliminated in these mutant mice as a result of the rescue-induced normalization of renal Cyp24a1. Thus, these hormone-regulated enhancers for both Cyp27b1 and Cyp24a1 in the kidney are responsible for the circulating levels of 1,25(OH)2D3 in the blood. The retention of Cyp27b1 and Cyp24a1 expression in NRTCs of these endocrine 1,25(OH)2D3-deficient mice suggests that this Cyp27b1 pseudo-null mouse will provide a model for the future exploration of the role of NRTC-produced 1,25(OH)2D3 in the hormone's diverse noncalcemic actions in both health and disease. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

An Overview of Systematic Reviews: Complementary Therapies for Cancer Patients.

Introduction: This article critically examines the systematic reviews (SR) and meta-analysis (MA) of complementary therapies for cancer patients to appraise the evidence level, and offers suggestions for future research and practice. Methods: The Cochrane Library and MEDLINE were searched from their inception to January 2018, to identify SR and MA of complementary therapies available for cancer patients. Final selected SR and MA were methodologically evaluated for their quality by applying the Assessing the Methodological Quality of Systematic Reviews 2 (AMSTAR2) instrument. Data extraction and risk of quality assessments were performed by 2 independent reviewers. Results: A total of 104 studies were included in the analysis. The majority of the individual clinical trials included in the SR and MA were performed in China (48%) and the United States (26.9%). Breast cancer was the most studied cancer type (25%), and acupuncture was the most studied intervention (21%). Side effects of cancer such as pain, depression, and fatigue were effectively managed with complementary therapies. The methodologically problematic items included not listing the excluded studies and lack of protocol or protocol registration. Conclusions: With increasing interest in research, complementary therapies appear to be beneficial in reducing side effects and raising the quality of life of cancer patients. Complementary therapies have generally been studied for all cancers, with acupuncture being the most researched, regardless of the cancer type. Since AMSTAR2 is a stricter assessment tool than before, future studies need to consider the risk of methodological bias with caution and discuss appropriate overall quality assessment tools.

Diet of the wild boar (Sus scrofa): implications for management in forest-agricultural and urban environments in South Korea.

The wild boar is one of the most widely distributed in the world. In South Korea, the wild boar population has rapidly increased and their habitat use has expanded from forests to urban environments. This expansion has led to increased conflicts with humans, such as the severe damaging of crops and the attacking of people in urban areas. We assessed the stomach contents of wild boar killed by hunters in two different environments in Geochang and Seoul, South Korea, from 2012 to 2017. We compared the feeding habits between sites and between seasons and explored the relationship between the number of earthworms and the main diet. The diet of wild boars inhabiting the two environments were found to differ and vary seasonally. Wild boar in Geochang preferred crops, when available, to natural food resources. Although wild boar in Seoul also preferred crops, they had a higher composition of natural food in their diets because of a low availability of crops. The preference of crops and discarded food waste in urban areas is expected to have accelerated the appearance of wild boar in urban areas. The consumption of earthworms did not differ between the two study sites, but it did differ seasonally due to availability. The number of earthworms was significantly negatively correlated with crop availability in both sites. Effective management plans that involve targeted hunting by baiting with food in Seoul and direct hunting in the fall in Geochang should be implemented to resolve the human-wild boar conflicts in these areas.

Recent Advances in the Discovery of Novel Antiprotozoal Agents.

Parasitic diseases have serious health, social, and economic impacts, especially in the tropical regions of the world. Diseases caused by protozoan parasites are responsible for considerable mortality and morbidity, affecting more than 500 million people worldwide. Globally, the burden of protozoan diseases is increasing and is been exacerbated because of a lack of effective medication due to the drug resistance and toxicity of current antiprotozoal agents. These limitations have prompted many researchers to search for new drugs against protozoan parasites. In this review, we have compiled the latest information (2012-2017) on the structures and pharmacological activities of newly developed organic compounds against five major protozoan diseases, giardiasis, leishmaniasis, malaria, trichomoniasis, and trypanosomiasis, with the aim of showing recent advances in the discovery of new antiprotozoal drugs.

A Control Region Near the Fibroblast Growth Factor 23 Gene Mediates Response to Phosphate, 1,25(OH)2D3, and LPS In Vivo.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene's regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.

A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues.

Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3 Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element-binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1-deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2-deleted strain did not affect 1,25(OH)2D3 induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.

Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse.

Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

Fabrication Process of Bilayer RGO/PEDOT:PSS Film for Flexible Transparent Conductive Electrode.

We developed a facile method to achieve a homogeneous coating of poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) on a graphene oxide (GO) layer with outstanding sheet resistance. We fabricated a transparent bilayer GO/PEDOT:PSS film as a flexible transparent conductive electrode (TCF). GO layer was coated on flexible PET and PI substrate by dip coating. The coated GO layers were modulated by their sizes and post heat treatment. The GO layers were thermally reduced and over coated with a PEDOT:PSS layer. Compared to the values of PEDOT:PSS, the sheet resistance of the bilayer film decreased by 5.2% and cyclic bending durability increased by 47.4%. The synergetic conductive network between the reduced graphene oxide (RGO) layer and the PEDOT:PSS layer resulted in low sheet resistance; the initial network retained under cyclic bending. The bilayer TCF film can be applied to multifunctional electrical devices for which flexibility and high conductivity are necessary.

Flavin-Based Light-Driven Fluorescent Probe for the Detection of Antioxidant Amino Acids.

We have synthesized a flavin-N(5)-oxide derivative with a p-toluenesulfonyl (Ts-OF) group as a "turn-on" fluorescent probe for the detection of several antioxidant amino acids and biothiols. Oxidized flavin was synthesized by using dithiothreitol as the reducing agent. Ts-OF showed a light-driven fluorescence enhancement in the presence of several amino acids and biothiols such as histidine (His), methionine (Met), cysteine (Cys), glutathione (GSH), and homocysteine (Hcy). The 1H NMR study indicated the reductive elimination of the p-toluenesulfonyl group from Ts-OF in the presence of antioxidants and photo-irradiation.

Effect of ceramic material and resin cement systems on the color stability of laminate veneers after accelerated aging.

STATEMENT OF PROBLEM: Laminate veneers are susceptible to color change during clinical service. Studies that compare the effects of different ceramic and resin cement systems on color stability are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the color stability of laminate veneers after accelerated aging using different ceramic and resin cement systems. MATERIAL AND METHODS: Ceramic specimens (N=168; shade A1; thickness, 0.50 ±0.05 mm; diameter, 10.00 ±0.10 mm) were prepared using nanofluorapatite and lithium disilicate (high translucency [HT] to low translucency [LT]) ceramics. Light-polymerizing (LP) cements were classified by brightness (high or low). Dual-polymerizing cements were classified by composition (base-only [DB] or base-catalyst [DC]) for comparison of color stability on the basis of polymerization type. DB cement was light-polymerizing, whereas DC cement was dual-polymerizing. They were further classified by shade (transparent, white, or yellow [n=7, each]). Color difference (ΔE) values were obtained by spectrophotometric quantification of L* (lightness), a* (green-red), and b* (blue-yellow) values before and after aging. The Kruskal-Wallis, Mann-Whitney U, Wilcoxon signed rank, and Bonferroni post hoc tests were used for statistical analysis. RESULTS: After specimens were subjected to accelerated aging, HT ceramic specimens luted with yellow-shade DC cement exhibited the greatest color change (ΔE=2.11), whereas HT and LT ceramic specimens luted with low-brightness LP cement exhibited the least color change (ΔE=1.37). In HT ceramic specimens, which exhibited the greatest color change of the 3 ceramic types, transparent shade cement exhibited significantly lower ΔE values than the other shades with DB (P<.001) and DC cements (P=.010). High-brightness cement exhibited significantly higher ΔE values than low-brightness cement when used with NF (P=.017), HT (P<.001), and LT (P<.001) ceramics. The ΔE values of DB cement were not always lower than those of DC cement. For all specimens, the aging of laminate veneers decreased the L* values and increased the a* and b* values. CONCLUSIONS: Ceramic and resin-cement systems affected the color stability of laminate veneers. Relative to other ceramics, HT lithium disilicate ceramics exhibited greater color changes upon aging. For HT ceramics, the use of transparent shade resin cement is recommended. The lower the brightness of resin cement, the higher the color stability of veneers. For luting of 0.5-mm-thick laminate veneers with dual-polymerizing cement, light polymerization did not yield better color stability than dual polymerization over time.

The impact of VDR expression and regulation in vivo.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). These actions include orchestration of mineral homeostasis which is coordinated by the kidney, intestine, bone and parathyroid gland wherein the VDR transcriptionally regulates expression of the genes involved in this complex process. Mutations in human VDR (hVDR) cause hereditary vitamin D resistant rickets, a genetic syndrome characterized by hypocalcemia, hyperparathyroidism and rickets resulting from dysregulation of mineral homeostasis. Expression of the VDR is regulated by external stimuli in a tissue-specific manner. However, the mechanisms of this tissue-specificity remain unclear. Studies also suggest that phosphorylation of hVDR at serine 208 impacts the receptor's transcriptional activity. These experiments were conducted in vitro, however, and therefore limited in their conclusions. In this report, we summarize (1) our most recently updated ChIP-seq data from mouse tissues to identify regulatory regions responsible for the tissues-specific regulation of the VDR and (2) our studies to understand the mechanism of hormonal regulation of Vdr expression in bone and kidney in vivo using transgenic mouse strains generated by mouse mini-genes that contain comprehensive genetic information capable of recapitulating endogenous Vdr gene regulation and expression. We also defined the functional human VDR gene locus in vivo by using a human mini-gene comparable to that in the mouse to generate a humanized VDR mouse strain in which the receptor is expressed at normal levels (normal expressor). The present report also shows that a humanized mouse model in which the VDR is expressed at levels about 10-fold lower than the normal expressor mouse rescued the VDR-null phenotype despite its reduced transcriptional activity relative to wildtype expression. We also generated an additional humanized mouse model expressing hVDR bearing a mutation converting serine 208 to alanine (hVDR-S208A). In spite of the mutation, target gene expression induced by the ligand was unchanged relative to a mouse strain expressing comparable levels of wildtype hVDR. Further characterization also showed that serum calcium and parathyroid hormone levels were normal and alopecia was not observed in this hVDR-S208A mouse strain as well. Taken together, our in vivo studies using ChIP-seq analyses and the mini-gene transgenic mice improve our understanding of the tissue-specific regulatory mechanisms of controlling VDR expression and the mechanisms of action of the VDR.

Polyion complex micelle formed from tetraphenylethene containing block copolymer.

BACKGROUND: Polymeric micelles attract great attention in drug delivery and therapeutics. Various types of block copolymers have been designed for the application in biomedical fields. If we can introduce additional functional groups to the block copolymers, we can achieve advanced applications. In this regards, we tried to introduce aggregation induced emission enhancement (AIE) unit in the block copolymer. METHODS: The formation of polyion complex micelle was confirmed by dynamic light scattering and transmission electron microscopy. HeLa cells were incubated with polyion complex micelle and broad-band visible light using a halogen lamp (150 W) was irradiated to evaluate photocytotoxicity of polyion complex (PIC) micelle. RESULTS: For the design of functional polymeric micelle, aggregation induced emission enhancement unit was introduced in the middle of block copolymer. We newly synthesized a new type block copolymer (PEG-TPE-PEI) possessing tetraphenylethene (TPE) group, as an AIE unit, in the middle of polymeric segments of PEG and PEI, which successfully formed PIC micelle with DP. The formation of PIC micelle was confirmed by dynamic light scattering, ζ potential measurement and transmission electron microscopy. CONCLUSIONS: PEG-TPE-PEI successfully formed PIC micelle by mixing with negatively charged dendrimer porphyrin. The PIC micelle exhibited photocytotoxicity upon illumination of broadband visible light.

A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation.

The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease.

The vitamin D receptor: contemporary genomic approaches reveal new basic and translational insights.

The vitamin D receptor (VDR) is the single known regulatory mediator of hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in higher vertebrates. It acts in the nucleus of vitamin D target cells to regulate the expression of genes whose products control diverse, cell type-specific biological functions that include mineral homeostasis. In this Review we describe progress that has been made in defining new cellular sites of action of this receptor, the mechanisms through which this mediator controls the expression of genes, the biology that ensues, and the translational impact of this receptor on human health and disease. We conclude with a brief discussion of what comes next in understanding vitamin D biology and the mechanisms that underlie its actions.