Maternal loss of 24-hydroxylase causes increased intestinal calcium absorption and hypercalcemia during pregnancy but reduced skeletal resorption during lactation in mice.

Inactivation of 24-hydroxylase (CYP24A1) causes mild hypercalcemia in humans that becomes severe and life-threatening during pregnancy through unclear mechanisms. We studied Cyp24a1 null mice during pregnancy, lactation, and post-weaning. We hypothesized that Cyp24a1 nulls have a much greater increase in calcitriol during pregnancy and lactation, leading to markedly increased intestinal calcium absorption and reduced lactational bone loss. WT and Cyp24a1 null sisters were mated to Cyp24a1+/- males. Timepoints included baseline (BL), late pregnancy (LP), mid-lactation (ML), late lactation (LL), and weekly x4 weeks of post-weaning recovery (R1-4). Assessments included intestinal calcium absorption (IntCaAbs) by gavage of 45Ca, bone mineral content (BMC) by DXA, microCT of femurs, 3-point bending tests of tibias, serum hormones, serum and urine minerals, milk analysis, and intestinal gene expression. At LP, whole body BMC increased equally by ~12% in null and WT. Calcitriol was 2.5-fold higher in nulls vs WT, accompanied by 3-fold increased IntCaAbs, hypercalcemia, hypercalciuria, and 6.5-fold higher FGF23. PTH was suppressed in both. Twenty percent of null dams died during delivery but their serum calcium at LP did not differ from Cyp24a1 nulls that survived. At ML, calcitriol, IntCaAbs, and FGF23 declined in both genotypes but remained higher than BL values in Cyp24a1 nulls. By LL, nulls were still hypercalcemic vs WT, and had lost less mean whole body BMC (11% vs. 21%, P<.02), but by micro-CT there were no differences from WT in cortical or trabecular bone mass. Lactational losses in BMC, cortical thickness, and trabecular number were restored by R4 in both genotypes. In summary, ablation of Cyp24a1 increased IntCaAbs and caused hypercalcemia during pregnancy and lactation, late gestational mortality in some nulls, and reduced lactational BMC loss. Treating women with gestational hypercalcemia from CYP24A1 mutations should focus on reducing calcitriol or IntCaAbs, since increased bone resorption is not the cause.

24-hydroxylase breaks down calcitriol, the hormonal form of vitamin D. Genetic deficiency of 24-hydroxylase causes mildly increased blood calcium (hypercalcemia) in adult humans, which can become life-threateningly high during pregnancy. We used a genetically engineered mouse model to determine the cause of this severe hypercalcemia. Calcitriol increased 10-fold during pregnancy in 24-hydroxylase deficient mice versus 4-fold in normal pregnant mice. High calcitriol in turn caused a marked increase in intestinal calcium absorption, which explained the hypercalcemia. Some 24-hydroxylase deficient mice died in late pregnancy. Our findings indicate that affected pregnant women need treatments that specifically reduce intestinal calcium absorption.

Loss of 24-hydroxylated catabolism increases calcitriol and fibroblast growth factor 23 and alters calcium and phosphate metabolism in fetal mice.

Calcitriol circulates at low levels in normal human and rodent fetuses, in part due to increased 24-hydroxylation of calcitriol and 25-hydroxyvitamin D by 24-hydroxylase (CYP24A1). Inactivating mutations of CYP24A1 cause high postnatal levels of calcitriol and the human condition of infantile hypercalcemia type 1, but whether the fetus is disturbed by the loss of CYP24A1 is unknown. We hypothesized that loss of Cyp24a1 in fetal mice will cause high calcitriol, hypercalcemia, and increased placental calcium transport. The Cyp24a1+/- mice were mated to create pregnancies with wildtype, Cyp24a1+/-, and Cyp24a1 null fetuses. The null fetuses were hypercalcemic, modestly hypophosphatemic (compared to Cyp24a1+/- fetuses only), with 3.5-fold increased calcitriol, 4-fold increased fibroblast growth factor 23 (FGF23), and unchanged parathyroid hormone. The quantitative RT-PCR confirmed the absence of Cyp24a1 and 2-fold increases in S100g, sodium-calcium exchanger type 1, and calcium-sensing receptor in null placentas but not in fetal kidneys; these changes predicted an increase in placental calcium transport. However, placental 45Ca and 32P transport were unchanged in null fetuses. Fetal ash weight and mineral content, placental weight, crown-rump length, and skeletal morphology did not differ among the genotypes. Serum procollagen 1 intact N-terminal propeptide and bone expression of sclerostin and Blgap were reduced while calcitonin receptor was increased in nulls. In conclusion, loss of Cyp24a1 in fetal mice causes hypercalcemia, modest hypophosphatemia, and increased FGF23, but no alteration in skeletal development. Reduced incorporation of calcium into bone may contribute to the hypercalcemia without causing a detectable decrease in the skeletal mineral content. The results predict that human fetuses bearing homozygous or compound heterozygous inactivating mutations of CYP24A1 will also be hypercalcemic in utero but with normal skeletal development.

Loss of maternal calcitriol reversibly alters early offspring growth and skeletal development in mice.

Ablation of Cyp27b1 eliminates calcitriol but does not disturb fetal mineral homeostasis or skeletal development. However, independent of fetal genotypes, maternal loss of Cyp27b1 altered fetal mineral and hormonal levels compared to offspring of WT dams. We hypothesized that these maternal influences would alter postnatal skeletal development. Cyp27b1 null and WT females were mated to bear only Cyp27b1+/- offspring. Forty-eight hours after birth, pups were cross-fostered to dams of the same or opposite genotype that bore them. Maternal and offspring samples were collected on days 21 (weaning) and 42. Offspring measurements included minerals and hormones, BMC by DXA, ash weight and mineral content, gene expression, 3-point bending tests, and microCT. Maternal lactational behavior was evaluated. Milk was analyzed for nutritional content. At day 21, offspring fostered by nulls, independent of birth dam, had ~20% lower weight, BMC, ash weight, and ash calcium than pups fostered by WT dams. Adjustment for body weight accounted for the lower BMC but not the lower ash weight and ash calcium. Hormones and serum/urine minerals did not differ across offspring groups. Offspring fostered by nulls had shorter femurs and lower cortical thickness, mean polar moment of inertia, cortical area, trabecular bone volume, and trabecular number. Dam lactational behaviors and milk nutritional content did not differ between groups. At day 42, body weight, ash weight, lengths, BMC, and tibial bone strength were no longer different between pups fostered by null vs WT dams. In summary, pups fostered by Cyp27b1 nulls, regardless of birth dam, have proportionately smaller skeletons at 21 d, impaired microstructure, but normal mineral homeostasis. The skeletal effects are largely recovered by day 42 (3 wk after weaning). In conclusion, maternal loss of calcitriol impairs early postnatal cortical bone growth and trabecular bone mass, but affected offspring catch up after weaning.

USP53 Regulates Bone Homeostasis by Controlling Rankl Expression in Osteoblasts and Bone Marrow Adipocytes.

In the skeleton, osteoblasts and osteoclasts synchronize their activities to maintain bone homeostasis and integrity. Investigating the molecular mechanisms governing bone remodeling is critical and helps understand the underlying biology of bone disorders. Initially, we have identified the ubiquitin-specific peptidase gene (Usp53) as a target of the parathyroid hormone in osteoblasts and a regulator of mesenchymal stem cell differentiation. Mutations in USP53 have been linked to a constellation of developmental pathologies. However, the role of Usp53 in bone has never been visited. Here we show that Usp53 null mice have a low bone mass phenotype in vivo. Usp53 null mice exhibit a pronounced decrease in trabecular bone indices including trabecular bone volume (36%) and trabecular number (26%) along with an increase in trabecular separation (13%). Cortical bone parameters are also impacted, showing a reduction in cortical bone volume (12%) and cortical bone thickness (15%). As a result, the strength and mechanical bone properties of Usp53 null mice have been compromised. At the cellular level, the ablation of Usp53 perturbs bone remodeling, augments osteoblast-dependent osteoclastogenesis, and increases osteoclast numbers. Bone marrow adipose tissue volume increased significantly with age in Usp53-deficient mice. Usp53 null mice displayed increased serum receptor activator of NF-κB ligand (RANKL) levels, and Usp53-deficient osteoblasts and bone marrow adipocytes have increased expression of Rankl. Mechanistically, USP53 regulates Rankl expression by enhancing the interaction between VDR and SMAD3. This is the first report describing the function of Usp53 during skeletal development. Our results put Usp53 in display as a novel regulator of osteoblast-osteoclast coupling and open the door for investigating the involvement of USP53 in pathologies. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Vitamin D and Diseases of Mineral Homeostasis: A Cyp24a1 R396W Humanized Preclinical Model of Infantile Hypercalcemia Type 1.

Infantile hypercalcemia type 1 (HCINF1), previously known as idiopathic infantile hypercalcemia, is caused by mutations in the 25-hydroxyvitamin D 24-hydroxylase gene, CYP24A1. The R396W loss-of-function mutation in CYP24A1 is the second most frequent mutated allele observed in affected HCINF1 patients. We have introduced the site-specific R396W mutation within the murine Cyp24a1 gene in knock-in mice to generate a humanized model of HCINF1. On the C57Bl6 inbred background, homozygous mutant mice exhibited high perinatal lethality with 17% survival past weaning. This was corrected by crossbreeding to the CD1 outbred background. Mutant animals had hypercalcemia in the first week of life, developed nephrolithiasis, and had a very high 25(OH)D3 to 24,25(OH)2D3 ratio which is a diagnostic hallmark of the HCINF1 condition. Expression of the mutant Cyp24a1 allele was highly elevated while Cyp27b1 expression was abrogated. Impaired bone fracture healing was detected in CD1-R396w/w mutant animals. The augmented lethality of the C57Bl6-R396W strain suggests an influence of distinct genetic backgrounds. Our data point to the utility of unique knock-in mice to probe the physiological ramifications of CYP24A1 variants in isolation from other biological and environmental factors.

NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH.

PTH induces phosphorylation of the transcriptional coregulator NACA on serine 99 through Gαs and PKA. This leads to nuclear translocation of NACA and expression of the target gene Lrp6, encoding a coreceptor of the PTH receptor (PTH1R) necessary for full anabolic response to intermittent PTH (iPTH) treatment. We hypothesized that maintaining enough functional PTH1R/LRP6 coreceptor complexes at the plasma membrane through NACA-dependent Lrp6 transcription is important to ensure maximal response to iPTH. To test this model, we generated compound heterozygous mice in which one allele each of Naca and Lrp6 is inactivated in osteoblasts and osteocytes, using a knock-in strain with a Naca99 Ser-to-Ala mutation and an Lrp6 floxed strain (test genotype: Naca99S/A; Lrp6+/fl;OCN-Cre). Four-month-old females were injected with vehicle or 100 µg/kg PTH(1-34) once daily, 5 days a week for 4 weeks. Control mice showed significant increases in vertebral trabecular bone mass and biomechanical properties that were abolished in compound heterozygotes. Lrp6 expression was reduced in compound heterozygotes vs. controls. The iPTH treatment increased Alpl and Col1a1 mRNA levels in the control but not in the test group. These results confirm that NACA and LRP6 form part of a common genetic pathway that is necessary for the full anabolic effect of iPTH.

Expression and Role of Ubiquitin-Specific Peptidases in Osteoblasts.

The ubiquitin-proteasome system regulates biological processes in normal and diseased states. Recent investigations have focused on ubiquitin-dependent modifications and their impacts on cellular function, commitment, and differentiation. Ubiquitination is reversed by deubiquitinases, including ubiquitin-specific peptidases (USPs), whose roles have been widely investigated. In this review, we explore recent findings highlighting the regulatory functions of USPs in osteoblasts and providing insight into the molecular mechanisms governing their actions during bone formation. We also give a brief overview of our work on USP53, a target of PTH in osteoblasts and a regulator of mesenchymal cell lineage fate decisions. Emerging evidence addresses questions pertaining to the complex layers of regulation exerted by USPs on osteoblast signaling. We provide a short overview of our and others' understanding of how USPs modulate osteoblastogenesis. However, further studies using knockout mouse models are needed to fully understand the mechanisms underpinning USPs actions.

Ubiquitin specific peptidase Usp53 regulates osteoblast versus adipocyte lineage commitment.

We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (- 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

Vitamin D Regulates CXCL12/CXCR4 and Epithelial-to-Mesenchymal Transition in a Model of Breast Cancer Metastasis to Lung.

Vitamin D deficiency is associated with poor cancer outcome in humans, and administration of vitamin D or its analogs decreases tumor progression and metastasis in animal models. Using the mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of mammary cancer, we previously demonstrated a significant acceleration of carcinogenesis in animals on a low vitamin D diet and a reduction in spontaneous lung metastases when mice received vitamin D through perfusion. We investigate here the action mechanism for vitamin D in lung metastasis in the same non-immunodeficient model and demonstrate that it involves the control of epithelial to mesenchymal transition as well as interactions between chemokine C-X-C motif chemokine 12 (CXCL12) and its receptor C-X-C chemokine receptor type 4 (CXCR4). In vitro, 10-9M vitamin D treatment modifies the phenotype of MMTV-PyMT primary mammary tumor cells and significantly decreases their invasiveness and mammosphere formation capacity by 40% and 50%, respectively. Vitamin D treatment also inhibits phospho-signal transducer and activator of transcription 3 (p-STAT3), zinc finger E-box-binding homeobox 1 (Zeb1), and vimentin by 52%, 75%, and 77%, respectively, and increases E-cadherin by 87%. In vivo, dietary vitamin D deficiency maintains high levels of Zeb1 and p-STAT3 in cells from primary mammary tumors and increases CXCL12 expression in lung stroma by 64%. In lung metastases, vitamin D deficiency increases CXCL12/CXCR4 co-localization by a factor of 2.5. These findings indicate an involvement of vitamin D in mammary cancer "seed" (primary tumor cell) and "soil" (metastatic site) and link vitamin D deficiency to epithelial-to-mesenchymal transition (EMT), CXCL12/CXCR4 signaling, and accelerated metastasis, suggesting vitamin D repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing spread to distant organs.

The ER protein TLC domain 3B2 and its enzymatic product lactosylceramide enhance chondrocyte maturation.

Purpose/Aim of study: We previously cloned Tlcd3b2 (Tram-Lag1-CLN8 domain 3B2, formerly Fam57b2) from bone fracture repair callus tissue of Cyp24a1 knockout mice and showed that it synthesizes lactosylceramide (LacCer) under allosteric control of the vitamin D metabolite, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Tlcd3b2 was mainly detected in chondrocytes and the 24,25(OH)2D3-TLCD3B2-LacCer signaling cascade was shown to be important for optimal bone fracture repair, suggesting a role for TLCD3B2 in chondrocyte differentiation or maturation. We report the subcellular localization of TLCD3B2 and its effect on chondrocyte differentiation. Materials and Methods: Immunofluorescence detection of epitope-tagged mutants was used to assess localization. ATDC5 chondrogenic cells were transfected with Tlcd3b2 expression vectors to examine effects on chondrocyte differentiation. Results and Conclusions: TLCD3B2 localized to the endoplasmic reticulum, with both the N- and C-termini facing the cytosolic compartment. Chondrogenic ATDC5 cells stably overexpressing Tlcd3b2 showed elevated type 2 (Col2a1) and type 10 (Col10a1) collagen gene expression and increased proteoglycan synthesis, and the effect on Col2a1 was enhanced by treatment with 24,25(OH)2D3. LacCer treatment of ATDC5 cells potentiated Col10a1 expression. Our results show that TLCD3B2 is an ER protein and implicate its expression and enzymatic product in chondrocyte maturation.

Inhibition of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) stimulates osteoblastogenesis by potentiating bone morphogenetic protein 2 (BMP2) responses.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA-PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA-PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast-specific conditional Prkdc-knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA-PKcs negatively regulates osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.

Vitamin D supplementation improves bone mineralisation independent of dietary phosphate in male X-linked hypophosphatemic (Hyp) mice.

The disorder of X-linked hypophosphatemia (XLH), results in the supressed renal production of active 1α,25-dihydroxyvitamin D (1,25(OH)2D) due to elevated fibroblast growth factor-23 (FGF23) levels. While adequate 25(OH)D levels are generally associated with improved mineralisation of the skeleton independent of circulating 1,25(OH)2D levels, it is unclear whether raising 25(OH)D to sufficiently high levels through dietary vitamin D3 administration contributes to improving bone mineralisation in the murine homolog for XLH, Hyp mice. Three-week-old male Hyp mice were fed one of four diets containing either 1000 IU (C) or 20,000 IU (D) vitamin D3/kg diet with either 0.35% phosphate or 1.25% phosphate (P) until 12 weeks of age (n = 12/group). When compared to C-fed mice, D-fed mice significantly elevated serum 25(OH)D levels to 72.8 ± 4.9 nmol/L (2-fold, p < 0.001) and increased both cortical bone mineral density (15%, p < 0.01), and vertebral trabecular BV/TV% (80%, p < 0.001), despite persistent hypophosphatemia and normocalcemia. The increase in bone volume was associated with improved Tb.Th (12%, p < 0.01) and Tb.N (63%, p < 0.001). Unlike with D-diet, P-fed mice resulted in increased femoral (15%, p < 0.001) and vertebral (12%, p < 0.001) length, and a 34% increase in vertebral trabecular BV/TV% when compared to control fed animals (p < 0.001). However, the addition of the high P diet to the high D diet did not result in additive effects on bone mineralisation when compared to the effects of D diet alone, despite serum 25(OH)D levels elevated to 118.8 ± 8.6 nmol/L. In D-fed mice, the increase in bone mineral density and volume was associated with reduced osteoid volume, reduced ObS/BS, and a trend for reduced serum PTH levels, suggesting reduced bone turnover in these animals. Thus, elevating serum 25(OH)D levels independently improves bone mineralisation in Hyp mice without causing hypercalcemia, suggesting that further studies are required in XLH patients to establish the role of increasing 25(OH)D levels in improving bone mineralisation.

Nfil3, a target of the NACA transcriptional coregulator, affects osteoblast and osteocyte gene expression differentially.

Intermittent administration of PTH(1-34) has a profound osteoanabolic effect on the skeleton. At the cellular level, osteoblasts and osteocytes are two crucial cell types that respond to PTH stimulation in bone. The transcriptional cofactor Nascent polypeptide Associated Complex and coregulator alpha (NACA) is a downstream target of the PTH-Gαs-PKA axis in osteoblasts. NACA functions as a transcriptional cofactor affecting bZIP factor-mediated transcription of target promoters in osteoblasts, such as Osteocalcin (Bglap2). Here, we used RNA-Seq and ChIP-Seq against NACA in PTH-treated MC3T3-E1 osteoblastic cells to identify novel targets of the PTH-activated NACA. Our approach identified Nuclear factor interleukin-3-regulated (Nfil3) as a target promoter of this pathway. Knockdown of Naca reduced the response of Nfil3 to PTH(1-34) stimulation. In silico analysis of the Nfil3 promoter revealed potential binding sites for NACA (located within the ChIP fragment) and CREB. We show that following PTH stimulation, phosphorylated-CREB binds the proximal promoter of Nfil3 in osteoblasts. The activity of the Nfil3 promoter (-818/+182 bp) is regulated by CREB and this activation relies on the presence of NACA. In addition, we show that knockdown of Nfil3 enhances the expression of osteoblastic differentiation markers in MC3T3-E1 cells while it represses osteocytic marker gene expression in IDG-SW3 cells. These results show that the PTH-induced NACA axis regulates Nfil3 expression and suggest that NFIL3 acts as a transcriptional repressor in osteoblasts while it exhibits differential activity as an activator in osteocytes.

Preclinical safety and efficacy of 24R,25-dihydroxyvitamin D3 or lactosylceramide treatment to enhance fracture repair.

BACKGROUND/OBJECTIVE: Cyp24a1-null mice deficient in 24,25(OH)2D3 display impaired callus formation during the endochondral phase of bone fracture repair. The 24,25(OH)2D3 metabolite acted by binding to the TLC domain containing 3B isoform 2 (TLCD3B2, previously named FAM57B2) effector protein, which then synthesizes lactosylceramide (LacCer). Treatment with 24,25(OH)2D3 or LacCer restored callus size and mechanical properties in Cyp24a1-null mice. METHODS: To assess the safety of these molecules and test their efficacy for bone healing in wild-type, non-genetically modified mice, we treated 12-week-old, osteotomized C57BL/6 female mice with each compound for up to 21 days post-osteotomy. Control cohorts were injected with vehicle. RESULTS: Neither compound was found to exhibit any nephro- nor hepato-toxicity. Calcemia remained stable throughout the experiment and was unaffected by either treatment. Supplementation with 24,25(OH)2D3 increased circulating levels of this metabolite about 8-fold, decreased 1,25(OH)2D3 levels, and significantly increased circulating 1,24,25(OH)3D3 levels, suggesting 1?-hydroxylation of 24,25(OH)2D3. TLCD3B2 was found to be expressed in fracture callus at the surface of unmineralized or pre-mineralized cartilage on day 10 and day 12 post-osteotomy and to progressively recede to become undetectable by day 18. Treatment with 24,25(OH)2D3 or LacCer reduced the number of TLCD3B2-positive cells. Both treatments also significantly increased stiffness and elastic modulus of the healing bone callus. CONCLUSION: Exogenous administration of 24,25(OH)2D3 or LacCer improved the biomechanical properties of repaired bones in wild-type animals without affecting circulating calcium levels or other blood parameters, demonstrating preclinical safety and efficacy. TRANSLATIONAL POTENTIAL: Our data suggest the use of 24R,25-dihydroxyvitamin D3 or lactosylceramide for ameliorating fracture healing in clinical practice.

Vitamin D-regulated osteocytic sclerostin and BMP2 modulate uremic extraskeletal calcification.

We induced chronic kidney disease (CKD) with adenine in WT mice, mice with osteocyte-specific deletion of Cyp27b1, encoding the 25-hydroxyvitamin D 1(OH)ase [Oct-1(OH)ase-/-], and mice with global deletion of Cyp27b1 [global-1α(OH)ase-/-]; we then compared extraskeletal calcification. After adenine treatment, mice displayed increased blood urea nitrogen, decreased serum 1,25(OH)2D, and severe hyperparathyroidism. Skeletal expression of Cyp27b1 and of sclerostin and serum sclerostin all increased in WT mice but not in Oct-1α(OH)ase-/- mice or global-1α(OH)ase-/- mice. In contrast, skeletal expression of BMP2 and serum BMP2 rose in the Oct-1α(OH)ase-/- mice and in the global-1α(OH)ase-/- mice. Extraskeletal calcification occurred in muscle and blood vessels of mice with CKD and was highest in Oct-1α(OH)ase-/-mice. In vitro, recombinant sclerostin (100 ng/mL) significantly suppressed BMP2-induced osteoblastic transdifferentiation of vascular smooth muscle A7r5 cells and diminished BMP2-induced mineralization. Our study provides evidence that local osteocytic production of 1,25(OH)2D stimulates sclerostin and inhibits BMP2 production in murine CKD, thus mitigating osteoblastic transdifferentiation and mineralization of soft tissues. Increased osteocytic 1,25(OH)2D production, triggered by renal malfunction, may represent a "primary defensive response" to protect the organism from ectopic calcification by increasing sclerostin and suppressing BMP2 production.

Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation.

The transcriptional cofactor nascent polypeptide-associated complex and co-regulator α (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA's activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified protein phosphatase 1 catalytic subunit α (PP1A) as a NACA-associated Ser/Thr phosphatase. NACA interacted with multiple components of the PP1A holoenzyme complex: the PPP1CA catalytic subunit and the regulatory subunits PPP1R9B, PPP1R12A and PPP1R18. MS analysis revealed that NACA co-expression with PPP1CA causes dephosphorylation of NACA at Thr-89, Ser-151, and Thr-174. NACA Ser/Thr-to-alanine variants displayed increased nuclear localization, and NACA dephosphorylation was associated with specific recruitment of novel NACA interactants, such as basic transcription factor 3 (BTF3) and its homolog BTF3L4. NACA and PP1A cooperatively potentiated cJUN transcriptional activity of the AP-1-responsive MMP9-luciferase reporter, which was abolished when Thr-89, Ser-151, or Thr-174 were substituted with phosphomimetic aspartate residues. We confirmed the NACA-PP1A interaction in MC3T3-E1 osteoblastic cells and observed that NACA phosphorylation status at PP1A-sensitive sites is important for the regulation of AP-1 pathway genes and for osteogenic differentiation and matrix mineralization. These results suggest that PP1A dephosphorylates NACA at specific residues, impacting cJUN transcriptional activity and osteoblast differentiation and function.

Hepatic posttranscriptional network comprised of CCR4-NOT deadenylase and FGF21 maintains systemic metabolic homeostasis.

Whole-body metabolic homeostasis is tightly controlled by hormone-like factors with systemic or paracrine effects that are derived from nonendocrine organs, including adipose tissue (adipokines) and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is a hormone-like protein, which is emerging as a major regulator of whole-body metabolism and has therapeutic potential for treating metabolic syndrome. However, the mechanisms that control FGF21 levels are not fully understood. Herein, we demonstrate that FGF21 production in the liver is regulated via a posttranscriptional network consisting of the CCR4-NOT deadenylase complex and RNA-binding protein tristetraprolin (TTP). In response to nutrient uptake, CCR4-NOT cooperates with TTP to degrade AU-rich mRNAs that encode pivotal metabolic regulators, including FGF21. Disruption of CCR4-NOT activity in the liver, by deletion of the catalytic subunit CNOT6L, increases serum FGF21 levels, which ameliorates diet-induced metabolic disorders and enhances energy expenditure without disrupting bone homeostasis. Taken together, our study describes a hepatic CCR4-NOT/FGF21 axis as a hitherto unrecognized systemic regulator of metabolism and suggests that hepatic CCR4-NOT may serve as a target for devising therapeutic strategies in metabolic syndrome and related morbidities.

Mineral Homeostasis in Murine Fetuses Is Sensitive to Maternal Calcitriol but Not to Absence of Fetal Calcitriol.

Vitamin D receptor (VDR) null fetuses have normal serum minerals, parathyroid hormone (PTH), skeletal morphology, and mineralization but increased serum calcitriol, placental calcium transport, and placental expression of Pthrp, Trpv6, and (as reported in this study) Pdia3. We examined Cyp27b1 null fetal mice, which do not make calcitriol, to determine if loss of calcitriol has the same consequences as loss of VDR. Cyp27b1 null and wild-type (WT) females were mated to Cyp27b1+/- males, which generated Cyp27b1 null and Cyp27b1+/- fetuses from Cyp27b1 null mothers, and Cyp27b1+/- and WT fetuses from WT mothers. Cyp27b1 null fetuses had undetectable calcitriol but normal serum calcium and phosphorus, PTH, fibroblast growth factor 23 (FGF23), skeletal mineral content, tibial lengths and morphology, placental calcium transport, and expression of Trpv6 and Pthrp; conversely, placental Pdia3 was downregulated. However, although Cyp27b1+/- and null fetuses of Cyp27b1 null mothers were indistinguishable, they had higher serum and amniotic fluid calcium, lower amniotic fluid phosphorus, lower FGF23, and higher 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D than in WT and Cyp27b1+/- fetuses of WT mothers. In summary, loss of fetal calcitriol did not alter mineral or bone homeostasis, but Cyp27b1 null mothers altered mineral homeostasis in their fetuses independent of fetal genotype. Cyp27b1 null fetuses differ from Vdr null fetuses, possibly through high levels of calcitriol acting on Pdia3 in Vdr nulls to upregulate placental calcium transport and expression of Trpv6 and Pthrp. In conclusion, maternal calcitriol influences fetal mineral metabolism, whereas loss of fetal calcitriol does not. © 2018 American Society for Bone and Mineral Research.

Calcioic acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D3 and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D3.

Calcitroic acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3) proceeds via analogous steps culminating in calcioic acid; however this C23-truncated acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2D3 was found to restore normal fracture repair in Cyp24a1-/- mice devoid of 24,25-(OH)2D3. We set out to study the multi-step catabolism of D3 metabolites in vivo using LC-MS/MS methods in vehicle or 24,25-(OH)2D3-treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2D3 (7 ng/mL) and 25-OH-D3-26,23-lactone (4 ng/mL). We also detected 24-oxo-25-OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1-/- mice. In 24,25-(OH)2D3-treated Cyp24a1+/- mice, serum 24,25-(OH)2D3 rose to 200 ng/mL while 25-OH-D3-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2D3 Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)2D3-treated Cyp24a1+/- mice. In 24,25-(OH)2D3-treated Cyp24a1-/- mice, serum 24,25-(OH)2D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2D3 dose. Serum 1,25-(OH)2D3 levels were suppressed in 24,25-(OH)2D3-treated Cyp24a1+/- and Cyp24a1-/- mice. Circulating 1,24,25-(OH)3D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2D3. While undetectable in vehicle-treated Cyp24a1-/- mice, 1,24,25-(OH)3D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2D3-treated nulls suggesting conversion of 24,25-(OH)2D3 to 1,24,25-(OH)3D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.

Optimal bone fracture repair requires 24R,25-dihydroxyvitamin D3 and its effector molecule FAM57B2.

The biological activity of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] remains controversial, but it has been suggested that it contributes to fracture healing. Cyp24a1-/- mice, synthesizing no 24R,25(OH)2D3, show suboptimal endochondral ossification during fracture repair, with smaller callus and reduced stiffness. These defects were corrected by 24R,25(OH)2D3 treatment, but not by 1,25-dihydroxyvitamin D3. Microarrays with Cyp24a1-/- callus mRNA identified FAM57B2 as a mediator of the 24R,25(OH)2D3 effect. FAM57B2 produced lactosylceramide (LacCer) upon specific binding of 24R,25(OH)2D3. Fam57b inactivation in chondrocytes (Col2-Cre Fam57bfl/fl) phenocopied the callus formation defect of Cyp24a1-/- mice. LacCer or 24R,25(OH)2D3 injections restored callus volume, stiffness, and mineralized cartilage area in Cyp24a1-null mice, but only LacCer rescued Col2-Cre Fam57bfl/fl mice. Gene expression in callus tissue suggested that the 24R,25(OH)2D3/FAM57B2 cascade affects cartilage maturation. We describe a previously unrecognized pathway influencing endochondral ossification during bone repair through LacCer production upon binding of 24R,25(OH)2D3 to FAM57B2. Our results identify potential new approaches to ameliorate fracture healing.