Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice *.

Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling. INTRODUCTION: The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption. MATERIALS AND METHODS: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test. RESULTS: Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k, the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D(28k), PMCA1b, and klotho, as well as protein levels of calbindin-D28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice. CONCLUSIONS: Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.     

Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model.

CaBP-9k may be involved in the active calcium absorption and embryo implantation. Although we generated CaBP-9k KO mice to explore its function, no distinct phenotypes were observed in these KO mice. It can be hypothesized that TRPV5 and 6 and plasma membrane calcium ATPase 1b may play a role in the regulation of calcium transport to compensate CaBP-9k deficiency in its KO model. INTRODUCTION: Active calcium transport in the duodenum and kidney is carried in three steps: calcium entry through epithelial Ca2+ channels (TRPV5 and TRPV6), buffering and/or transport by calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), and extrusion through the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger 1. Although the molecular mechanism of calcium absorption has been studied using knockouts (KOs) of the vitamin D receptor and CaBP-28k in animals, the process is not fully understood. MATERIALS AND METHODS: We generated CaBP-9k KO mice and assessed the phenotypic characterization and the molecular regulation of active calcium transporting genes when the mice were fed different calcium diets during growth. RESULTS: General phenotypes showed no distinct abnormalities. Thus, the active calcium transport of CaBP-9k-null mice proceeded normally in this study. Therefore, the compensatory molecular regulation of this mechanism was elucidated. Duodenal TRPV6 and CaBP-9k mRNA of wildtype (WT) mice increased gradually during preweaning. CaBP-9k is supposed to be an important factor in active calcium transport, but its role is probably compensated for by other calcium transporter genes (i.e., intestinal TRPV6 and PMCA1b) during preweaning and renal calcium transporters in adult mice. CONCLUSIONS: Depletion of the CaBP-9k gene in a KO mouse model had little phenotypic effect, suggesting that its depletion may be compensated for by calcium transporter genes in the intestine of young mice and in the kidney of adult mice.     

Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mRNAs for avian intestinal calbindin-D28k and the 1,25-dihydroxyvitamin D3 receptor.

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.     

Compensatory Changes in Calcium Metabolism Accompany the Loss of Vitamin D Receptor (VDR) From the Distal Intestine and Kidney of Mice

1,25 Dihydroxyvitamin D₃ (1,25(OH)₂D) increases intestinal Ca absorption when dietary Ca intake is low by inducing gene expression through the vitamin D receptor (VDR). 1,25(OH)₂D‐regulated Ca absorption has been studied extensively in the small intestine, but VDR is also present in the large intestine. Our goal was to determine the impact of large intestinal VDR deletion on Ca and bone metabolism. We used transgenic mice expressing Cre‐recombinase driven by the 9.5‐kb human caudal type homeobox 2 (CDX2) promoter to delete floxed VDR alleles from the caudal region of the mouse (CDX2‐KO). Weanling CDX2‐KO mice and control littermates were fed low (0.25%) or normal (0.5%) Ca diets for 7 weeks. Serum and urinary Ca, vitamin D metabolites, bone parameters, and gene expression were analyzed. Loss of the VDR in CDX2‐KO was confirmed in colon and kidney. Unexpectedly, CDX2‐KO had lower serum PTH (–65% of controls, p < 0.001) but normal serum 1,25(OH)₂D and Ca levels. Despite elevated urinary Ca loss (eightfold higher in CDX2‐KO) and reduced colonic target genes TRPV6 (–90%) and CaBPD_9k (–80%) mRNA levels, CDX2‐KO mice had only modestly lower femoral bone density. Interestingly, duodenal TRPV6 and CaBPD_9k mRNA expression was fourfold and threefold higher, respectively, and there was a trend toward increased duodenal Ca absorption (+19%, p = 0.076) in the CDX2‐KO mice. The major finding of this study is that large intestine VDR significantly contributes to whole‐body Ca metabolism but that duodenal compensation may prevent the consequences of VDR deletion from large intestine and kidney in growing mice. © 2015 American Society for Bone and Mineral Research.     

Regulation of calbindin-D28K gene expression in the chick intestine: effects of serum calcium status and 1,25-dihydroxyvitamin D3.

Calbindin-D28K is a member of a superfamily of calcium binding proteins that share a common avidity for the divalent calcium ion. The ambient concentration of calcium in the blood circulation is thought to orchestrate the release of parathyroid hormone and calcitonin and to govern the activity of renal 1-hydroxylase and thereby synthesis of 1,25-dihydroxyvitamin D3. We report here the results of experiments designed to assess the possible contribution of dietary calcium status upon calbindin-D28K gene expression in the intestine of vitamin D-deficient chicks. The actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and dietary calcium intake upon intestinal calbindin-D28K and calbindin-D28K mRNA were monitored by ELISA and dot-blot hybridization analyses, respectively. Vitamin D3-deficient chicks were fed either a calcium-supplemented diet (3% w/w) or a diet containing low calcium (0.4% w/w). These dietary manipulations evoked a highly significant change in serum calcium status. However, the levels of calbindin-D28K protein and its corresponding mRNA were unaffected. Administration of 1,25-(OH)2D3 (1-16 nmol per animal) to both "normocalcemic" and hypocalcemic vitamin D-deficient chicks resulted in an equivalent stimulation of duodenal calbindin-D28K accumulation of calbindin-D28K mRNA. Intestinal calbindin-D28K was stimulated 20- to 28-fold (above control levels) by 6-8 nmol 1,25-(OH)2D3 in both dietary treatment groups when measured 48 h after the single injection. Hence, despite the existence of a relatively large difference in serum calcium levels, the molecular actions of 1,25-(OH)2D3 in the vitamin D-deficient animal are apparently well insulated from serum calcium chemistry. These observations support the notion that, in the absence of vitamin D3, the calcium ion per se is unable to modulate the calbindin-D28K gene in vivo.     

Vitamin D Receptor Signaling Enhances Locomotive Ability in Mice

Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of wild‐type (WT) mice by 1.6‐fold, whereas exercise did not enhance locomotive ability of VDR knockout (KO) mice. Compared with WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED‐71, ELD), an analog of 1,25(OH)₂D₃, administered to rotarod‐trained C57BL/6 mice enhanced locomotor performance compared with vehicle‐treated nontrained mice. The area of AChR cluster on the extensor digitorum longus was greater in ELD‐treated mice than in vehicle‐treated mice. ELD and 1,25(OH)₂D₃ enhanced expression of IGF‐1, myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability after physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D₃ signaling in locomotive ability. © 2014 American Society for Bone and Mineral Research.     

VDR Haploinsufficiency Impacts Body Composition and Skeletal Acquisition in a Gender-Specific Manner

The vitamin D receptor (VDR) is crucial for virtually all of vitamin D’s actions and is thought to be ubiquitously expressed. We hypothesized that disruption of one allele of the VDR gene would impact bone development and would have metabolic consequences. Body composition and bone mass (BMD) in VDR heterozygous (VDR HET) mice were compared to those obtained in male and female VDR KO and WT mice at 8 weeks of age. Male mice were also evaluated at 16 weeks, and bone marrow mesenchymal stem cell (MSC) differentiation was evaluated in VDR female mice. Additionally, female VDR HET and WT mice received intermittent PTH treatment or vehicle (VH) for 4 weeks. BMD was determined at baseline and after treatment. MRI was done in vivo at the end of treatment; μCT and bone histomorphometry were performed after killing the animals. VDR HET male mice had normal skeletal development until 16 weeks of age but showed significantly less gain in fat mass than WT mice. In contrast, female VDR HET mice showed decreased total-body BMD at age 8 weeks but had a normal skeletal response to PTH. MSC differentiation was also impaired in VDR HET female mice. Thus, female VDR HET mice show early impairment in bone acquisition, while male VDR HET mice exhibit a lean phenotype. Our results indicate that the VDR HET mouse is a useful model for studying the metabolic and skeletal impact of decreased vitamin D sensitivity.     

1,25-dihydroxyvitamin D(3)-independent stimulatory effect of estrogen on the expression of ECaC1 in the kidney

Estrogen deficiency results in a negative Ca(2+) balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca(2+) handling and regulation. The epithelial Ca(2+) channel ECaC1 (or TRPV5) is the entry channel involved in active Ca(2+) transport. Ca(2+) entry is followed by cytosolic diffusion, facilitated by calbindin-D(28K) and/or calbindin-D(9k), and active extrusion across the basolateral membrane by the Na(+)/Ca(2+)-exchanger (NCX1) and plasma membrane Ca(2+)-ATPase (PMCA1b). In this transcellular Ca(2+) transport, ECaC1 probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17beta-estradiol (17beta-E(2)) is involved in Ca(2+) reabsorption via regulation of the expression of ECaC1. The ovariectomized rat model was used to investigate the regulation of ECaC1, at the mRNA and protein levels, by 17beta-E(2) replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17beta-E(2) treatment at pharmacologic doses increased renal mRNA levels of ECaC1, calbindin-D(28K), NCX1, and PMCA1b and increased the protein abundance of ECaC1. Furthermore, the involvement of 1,25-dihydroxyvitamin D(3) in the effects of 17beta-E(2) was examined in 25-hydroxyvitamin D(3)-1alpha-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D(9K), calbindin-D(28K), NCX1, and PMCA1b was not significantly altered after 17beta-E(2) treatment. In contrast, ECaC1 mRNA and protein levels were both significantly upregulated. Moreover, 17beta-E(2) treatment partially restored serum Ca(2+) levels, from 1.63 +/- 0.06 to 2.03 +/- 0.12 mM. In conclusion, this study suggests that 17beta-E(2) is positively involved in renal Ca(2+) reabsorption via the upregulation of ECaC1, an effect independent of 1,25-dihydroxyvitamin D(3).     

Villin promoter-mediated transgenic expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) increases intestinal calcium absorption in wild-type and vitamin D receptor knockout mice

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D–regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild‐type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium–specific expression of a 3X Flag‐tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG‐VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG‐VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three‐fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to <10% of WT but small intestine calbindin‐D_9k expression was elevated >15 times in TG mice. TG‐VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D_9K mRNA and protein levels were elevated in TG‐VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR‐independent upregulation of intestinal calbindin D_9k in TG‐VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research.     

Adenovirus‐delivered microRNA targeting the vitamin D receptor reduces intracellular Ca2+ concentrations by regulating the expression of Ca2+‐transport proteins in renal epithelial cells

What’s known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone‐forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²⁺ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed.

OBJECTIVE

? To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects.

MATERIALS AND METHODS

? The adenovirus vector‐delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR‐dependent epithelial Ca²⁺ transport proteins were detected using real‐time polymerase chain reaction and Western blot assays, respectively. ? Fluorescent Ca²⁺ indicator Fluo‐4 NW (Fluo‐4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500‐IX71, Japan) were used to detect the cytosolic free Ca²⁺ concentration at different time points after infection.

RESULTS

? The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin‐D_28k and plasma membrane Ca²⁺‐ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. ? There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²⁺‐exchanger (NCX1). ? Furthermore, VDR knockdown results in a decrease in intracellular Ca²⁺ concentration ([Ca²⁺]i) in NRK cell lines.

CONCLUSIONS

? Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²⁺]i in NRK cell lines. ? The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.     

The ClC-5 knockout mouse model of Dent's disease has renal hypercalciuria and increased bone turnover.

Dent's disease is a nephrolithiasis disorder associated with hypercalciuria and low molecular weight proteinuria that is caused by mutations in the voltage-gated chloride channel ClC-5. Because the exact cause of hypercalciuria in this disease is unknown and could come from a renal, intestinal, or bone origin, we have investigated overall calcium handling in the ClC-5 knockout mouse (ClC-5 KO). On a high calcium diet, ClC-5 KO mice had elevated serum 1alpha,25-dihydroxyvitamin D3 (1alpha,25D3), alkaline phosphatase (AP), osteocalcin (OC), and urinary deoxypyridinoline (DPD), but serum parathyroid hormone (PTH), calcium, and intestinal calcium uptake was similar to that of wild-type (WT) mice. A 30-fold decrease in dietary calcium intake caused elevation of serum PTH and urinary cyclic adenosine monophosphate in ClC-5 KO mice and decreased the renal calcium excretion, which still remained 2-fold above that of WT mice. On this low calcium diet, both groups of mice had the same serum 1alpha,25D3, with similar increments in intestinal calcium absorption, serum AP, OC, and urinary DPD. These data indicate that the hypercalciuria in the ClC-5 KO mice on low and high calcium diets is of bone and renal origin and is not caused by increased intestinal calcium absorption, despite an elevated serum 1alpha,25D3. These mice data suggest that young patients with this disease may have a propensity for altered bone homeostasis that should be monitored clinically.     

Simultaneous Exposure of Excess Fluoride and Calcium Deficiency Alters VDR,CaR, and Calbindin D 9 k mRNA Levels in Rat Duodenal Mucosa

Fluoride ingestion reduces intestinal calcium absorption; its molecular basis has not been studied. We studied the mRNA expression of calcium-sensing receptor (CaR), vitamin D receptor (VDR) and calbindin D 9 k (D 9 k) by northern blot analysis in the duodenal mucosa of rats. Weanling pups fed with chow diet containing adequate calcium (0.5% w/w) and drinking water (NaF < 1 ppm) served as controls (Group I) and were studied at 9 and 15 weeks. The pups, born to rats fed with a calcium-deficient diet (0.03%) and excess fluoride water (NaF 50 ppm), were continued on the same diet and water (Group II) until 9 weeks of age. Subsequently, Group II rats were divided into 4 subgroups; 3 subgroups with fluoride free water [II-A adequate calcium, II-B excess calcium (Ca 2%) and II-D calcium deficient], whereas II-C received fluorinated water and adequate calcium diet until 15 weeks. At 9 weeks, as compared to group-I, group-II had decreased VDR (P < 0.001) and D 9 k mRNA (P < 0.001), whereas CaR mRNA levels increased (P < 0.05). At 15 weeks, as compared to group-I, VDR mRNA further reduced in group II-D (P < 0.001) and II-C (P < 0.001), whereas it increased in group II-A. Removal of fluoride ingestion and calcium replenishment increased D 9 k mRNA expression, maximally in adequate calcium group (P < 0.001), while it was further reduced in group II-C (P < 0.001). CaR expression decreased significantly in all the groups. We conclude that excess fluoride reduces the mRNA levels of VDR and D 9 k in the duodenal mucosa of rats, thereby possibly reducing calcium absorption. Calcium supplementation with simultaneous fluoride removal improves their expression.     

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats.

The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats. INTRODUCTION: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known. MATERIALS AND METHODS: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots. RESULTS AND CONCLUSIONS: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.     

补肾中药对骨质疏松大鼠CaBp─D9k基因及表达的影响

本实验应用分子生物学基因重组及杂交技术，对地塞米松诱发的骨质疏松大鼠肠道钙结合蛋白Ｄ９ｋ（ＣａＢｐ─Ｄ９ｋ）基因及其ｍＲＮＡ水平及补肾中药和维生素Ｄ３（Ｖｉｔ─Ｄ３）治疗后的变化进行了研究。结果发现，地塞米松、补肾中药和Ｖｉｔ─Ｄ３均不能影响肠道ＣａＢｐ─Ｄ９ｋ基因，但对其转录却可产生不同影响，补肾中药可能是通过增加肠道ＣａＢｐ─Ｄ９ｋｍＲＮＡ水平，进而增加ｃａＢｐ─Ｄ９ｋ的含量而促进肠道钙的吸收，通过肠道（４５）Ｃａ的吸收实验进一步证明了这一点。     

Increased mandibular condylar growth in mice with estrogen receptor beta deficiency

Temporomandibular joint (TMJ) disorders predominantly afflict women of childbearing age, suggesting a role for female hormones in the disease process. In long bones, estrogen acting via estrogen receptor beta (ERβ) inhibits axial skeletal growth in female mice. However, the role of ERβ in the mandibular condyle is largely unknown. We hypothesize that female ERβ‐deficient mice will have increased mandibular condylar growth compared to wild‐type (WT) female mice. This study examined female 7‐day‐old, 49‐day‐old, and 120‐day‐old WT and ERβ knockout (KO) mice. There was a significant increase in mandibular condylar cartilage thickness as a result of an increased number of cells, in the 49‐day‐old and 120‐day‐old female ERβ KO compared with WT controls. Analysis in 49‐day‐old female ERβ KO mice revealed a significant increase in collagen type X, parathyroid hormone–related protein (Pthrp), and osteoprotegerin gene expression and a significant decrease in receptor activator for nuclear factor κ B ligand (Rankl) and Indian hedgehog (Ihh) gene expression, compared with WT controls. Subchondral bone analysis revealed a significant increase in total condylar volume and a decrease in the number of osteoclasts in the 49‐day‐old ERβ KO compared with WT female mice. There was no difference in cell proliferation in condylar cartilage between the genotypes. However, there were differences in the expression of proteins that regulate the cell cycle; we found a decrease in the expression of Tieg1 and p57 in the mandibular condylar cartilage from ERβ KO mice compared with WT mice. Taken together, our results suggest that ERβ deficiency increases condylar growth in female mice by inhibiting the turnover of fibrocartilage. © 2013 American Society for Bone and Mineral Research.     

Effects of global or targeted deletion of the EP4 receptor on the response of osteoblasts to prostaglandin in vitro and on bone histomorphometry in aged mice

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre–LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E₂ (PGE₂) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE₂ in cell culture but may not be essential for maintenance of bone remodeling in vivo.     

Coordinated control of renal Ca2+ handling

Ca2+ homeostasis is an important factor, which is underlined by the numerous clinical symptoms that involve Ca2+ deficiencies. The overall Ca2+ balance is maintained by the concerted action of Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone, which are all under the control of the calciotropic hormones that are released upon a demand for Ca2+. In the kidney, these calciotropic hormones affect active Ca2+ reabsorption, which consists of TRPV5 as the apical entry gate for Ca2+ influx, calbindin-D28K as an intracellular ferry for Ca2+ and, NCX1 and PMCA1b for extrusion of Ca2+ across the basolateral membrane. This review highlights the action of hormones on renal Ca2+ handling and focuses on the coordinated control of the renal Ca2+ transport proteins. Parathyroid hormone stimulates renal Ca2+ handling by regulating active Ca2+ reabsorption on both the genomic and non-genomic level. Estrogens harbor calciotropic hormone characteristics positively regulating the expression of TRPV5, independently of vitamin D. Besides having a strong regulatory effect on the expression of the intestinal Ca2+ transport proteins, vitamin D contributes to the overall Ca2+ balance by enhancing the expression of the Ca2+ transport machinery in the kidney. Dietary Ca2+ is involved in regulating its own handling by controlling the expression of the renal Ca2+ transport proteins. Thus, the magnitude of Ca2+ entry via TRPV5 controls the expression of the other Ca2+ transport proteins underlining the gatekeeper function of this Ca2+ channel in the renal Ca2+ handling.     

The surface antigen CD45R identifies a population of estrogen-regulated murine marrow cells that contain osteoclast precursors

We examined the osteoclastogenic potential of murine bone marrow cells that were fractionated according to their expression of the surface antigen CD45R. Osteoclast-like cells (OCL) with many authentic osteoclast characteristics readily formed in purified CD45R⁺ murine bone marrow cell cultures after treatment with receptor activator of nuclear factor κB ligand (RANKL) and M-CSF. Ovariectomy (Ovx) caused a 1.5- to 2-fold increase in OCL number in unfractionated and CD45R⁺ murine bone marrow cell cultures without affecting OCL formation in CD45R⁻ marrow cells. Limiting dilution assays confirmed that Ovx caused an increase in osteoclast precursor cell number in CD45R⁺ but not CD45R⁻ cells. Mice deficient in the type 1 IL-1 receptor (IL-1R1 KO) do not lose bone mass after Ovx. We found that unfractionated, CD45R⁺, and CD45R⁻ bone marrow cells from IL-1R1 KO mice showed no increase in OCL formation in vitro after Ovx. In both the wild-type (WT) and the IL-1R1 KO mice Ovx was associated with a 2-fold increase in pre-B-lymphocytes. About 1.3–3.5% of murine marrow cells expressed surface RANK (the receptor for RANKL) while about 11.9–15% of murine bone marrow cells expressed c-Fms (the receptor for M-CSF). There was little effect of Ovx on cells expressing either RANK or c-Fms. These results demonstrate that CD45R expression identifies a subset of murine bone marrow cells whose ability to form OCL in vivo is regulated by estrogen in WT but not IL-1R1 KO cells. The effects of estrogen on bone mass may be related to these responses.