Bram C. J. van der Eerden

Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5

Ca2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca2+ flux to the extracellular Ca2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca2+ channel in maintaining the Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca2+ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca2+ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca2+ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca2+ reabsorption and its essential role in the Ca2+ homeostasis.

Hypervitaminosis D Mediates Compensatory Ca2+ Hyperabsorption in TRPV5 Knockout Mice

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

Phenotypic Dissection of Bone Mineral Density Reveals Skeletal Site Specificity and Facilitates the Identification of Novel Loci in the Genetic Regulation of Bone Mass Attainment

Heritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg) and residual (re) correlations between BMD measured at the upper limbs (UL-BMD), lower limbs (LL-BMD) and skull (SK-BMD), using total-body DXA scans of ∼4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC). Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78) between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43). Likewise, the residual correlation between BMD at appendicular sites (re = 0.55) was higher than the residual correlation between SK-BMD and BMD at appendicular sites (re = 0.20–0.24). To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n∼9,395), combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites). In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01×10−37), whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31×10−14). In addition, we report a novel association between RIN3 (previously associated with Pagets disease) and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4×10−10). Our results suggest that BMD at different skeletal sites is under a mixture of shared and specific genetic and environmental influences. Allowing for these differences by performing genome-wide association at different skeletal sites may help uncover new genetic influences on BMD.

GHRELIN AND BONE

A consequence of gastrectomy is loss of bone mass. Several mechanisms have been proposed, such as malabsorption of vitamins and minerals. Additionally, a peptide hormone produced in the stomach has been shown to mediate a calcitropic effect on bone. The identity of this peptide has not been elucidated, but ghrelin, produced by A-like cells in the fundus of the stomach, could be a good candidate. Ghrelin stimulates growth hormone (GH) secretion both in vivo and in vitro, and could by this means have a positive effect on bone. There is also evidence for direct effects of ghrelin on bone. We discuss here the role that ghrelin may play in bone metabolism, based on the most recent literature.

Unraveling the human bone microenvironment beyond the classical extracellular matrix proteins: a human bone protein library.

A characteristic feature of bone, differentiating it from other connective tissues, is the mineralized extracellular matrix (ECM). Mineral accounts for the majority of the bone tissue volume, being the remainder organic material mostly derived from collagen. This, and the fact that only a limited number of noncollagenous ECM proteins are described, provides a limited view of the bone tissue composition and bone metabolism, the more so considering the increasing understanding of ECM significance for cellular form and function. For this reason, we set out to analyze and extensively characterize the human bone proteome using large-scale mass spectrometry-based methods. Bone samples of four individuals were analyzed identifying 3038 unique proteins. A total of 1213 of these were present in at least 3 out of 4 bone samples. For quantification purposes, we were limited to noncollagenous proteins (NCPs) and we could quantify 1051 NCPs. Most classical bone matrix proteins mentioned in literature were detected but were not among the highly abundant ones. Gene ontology analyses identified high-abundance groups of proteins with a functional link to mineralization and mineral metabolism such as transporters, pyrophosphatase activity, and Ca(2+)-dependent phospholipid binding proteins. ECM proteins were as well overrepresented together with nucleosome and antioxidant activity proteins, which have not been extensively characterized as being important for bone. In conclusion, our data clearly demonstrates that human bone tissue is a reservoir of a wide variety of proteins. In addition to the classical osteoblast-derived ECM, we have identified many proteins from different sources and of unknown function in bone. Thus, this study represents an informative library of bone proteins forming a source for novel bone formation modulators as well as biomarkers for bone diseases such as osteoporosis.

Murine TNFΔARE Crohn's disease model displays diminished expression of intestinal Ca2+ transporters

Background: Patients suffering from Crohns disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU‐rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF‐&agr; levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca2+ handling problem, this study analyzed the Ca2+ homeostasis in heterozygous TNF&Dgr;ARE mice (TNF&Dgr;ARE/+) in comparison to wildtype littermates. Methods: Beside serum Ca2+ and vitamin D levels, the expression of Ca2+ transporters was analyzed in intestine, kidney and bone using quantitative real‐time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. Results: Ca2+ transporters in duodenum (TRPV6, calbindin‐D9K, PMCA1b) and kidney (TRPV5, calbindin‐D28K, NCX1) showed significantly reduced mRNA expression levels in TNP&Dgr;ARE/+ mice, except for renal TRPV5. In bone, only calbindin‐D9K mRNA displayed a significant down‐regulation. These findings were supported by declined duodenal calbindin‐D9K and renal calbindin‐D28K protein values. Likely, this down‐regulation of Ca2+ transporters in TNP&Dgr;ARE/+ mice is mediated by the 58 ± 9% reduction in serum 1,25(OH)2D3 levels. Diminished expression of Ca2+ transporters combined with unchanged serum Ca2+ levels assumes Ca2+ loss from bone to compensate for the bodys overall Ca2+ shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF&Dgr;ARE/+ mice. This finding is further supported by increased total deoxypyridinoline in serum. Conclusions: Our results imply that TNF&Dgr;ARE/+ mice have a disturbed Ca2+ homeostasis characterized by reduced duodenal and renal Ca2+ transporters, diminished 1,25(OH)2D3 levels, and increased bone resorption associated with profound bone abnormalities.

The transient receptor potential channel TRPV6 is dynamically expressed in bone cells but is not crucial for bone mineralization in mice

Bone is the major store for Ca2+ in the body and plays an important role in Ca2+ homeostasis. During bone formation and resorption Ca2+ must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca2+ transport machinery in these bone cells. In this study, we examined the epithelial Ca2+ channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast‐like cells as well as in peripheral blood mononuclear cell‐derived human osteoclasts and murine tibial bone marrow‐derived osteoclasts. Also other transcellular Ca2+ transport genes, calbindin‐D9k and/or ‐D28K, Na+/Ca2+ exchanger 1, and plasma membrane Ca2+ ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone‐facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) did not change TRPV6 expression in both mineralizing and non‐mineralizing SV‐HFO cultures. Lentiviral transduction‐mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6D541A/D541A mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non‐functional. In summary, TRPV6 and other proteins involved in transcellular Ca2+ transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice. J. Cell. Physiol. 227: 1951–1959, 2012.

Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses

This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β-glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine into lipid vesicle-containing adipocytes within 1 to 2 weeks. Phenotypic changes during those highly dynamic differentiation processes can be detected by biochemical and histological assays and gene expression analyses of differentiation markers. In addition, this unit describes an electroporation method that allows the transient genetic perturbation of hMSCs.

Ghrelin and bone

Ghrelin is a gut‐derived peptide hormone, first isolated from the stomach. Ghrelin was initially characterized as a growth hormone (GH) secretagogue, but it plays a more important role as a potent orexigen and modulator of whole‐body energy homeostasis. Ghrelin itself is closely regulated by metabolic status. Bone remodeling constantly renews the skeleton in a highly energy‐dependent fashion. Accordingly, bone metabolism is tightly coupled to energy metabolism through the integration of peripheral and central mechanisms, involving the sympathetic nervous system and factors such as leptin. Ghrelin has been shown to modulate osteoblast differentiation and function, both directly and perhaps also through regulation of the GH–insulin‐like growth factor axis. However, recently it has also been shown that ghrelin interacts with leptin in modulating bone structure, constituting a new mechanism that couples bone metabolism with energy homeostasis. In this review, we discuss the role that ghrelin plays modulating bone cell function, and its integrative role in coupling bone metabolism with energy metabolism.

Bone Resorption Inhibitor Alendronate Normalizes the Reduced Bone Thickness of TRPV5−/− Mice†

TRPV5 is a Ca2+‐selective channel involved in transcellular Ca2+ absorption expressed in kidney and in the ruffled border of osteoclasts. Studies in hypercalciuric TRPV5 knockout (TRPV5−/−) mice, which display significantly increased vitamin D levels, showed that TRPV5 ablation increases number and size of osteoclasts but impairs osteoclast‐mediated bone resorption. The latter is not in line with the observed decreased bone thickness in TRPV5−/− mice. Bisphosphonates also inhibit osteoclast‐mediated bone resorption. The aim of this study was to evaluate the effect of alendronate on the expression of the Ca2+ transporters in bone, kidney, and duodenum and, importantly, the bone phenotype in TRPV5−/− mice. Wildtype (TRPV5+/+) and TRPV5−/− mice were treated during 10 wk with 2 mg/kg alendronate or vehicle weekly and housed in metabolic cages at the end of treatment. Urine and blood samples were taken for biochemical analysis, and duodenum, kidney, and femur were sampled. Expression of Ca2+ transporters and osteoclast ruffled border transporters in bone and cultured osteoclasts was determined by QPCR analysis. Femurs were scanned using μCT, and resorption pit assays were performed in bone marrow cultures isolated from TRPV5+/+ and TRPV5−/− mice. Alendronate treatment enhanced bone thickness in TRPV5+/+ mice but also normalized the disturbed bone morphometry parameters in TRPV5−/− mice. Bone TRPV5 expression was specifically enhanced by alendronate, whereas the expression of Ca2+ transporters in kidney and intestine was not altered. The expression of the osteoclast ruffled border membrane proteins chloride channel 7 (CLC‐7) and the vacuolar H+‐ATPase did not differ between both genotypes, but alendronate significantly enhanced the expression and PTH levels in TRPV5−/− mice. The expression of TRPV5, CLC‐7, and H+‐ATPase in osteoclast cultures was not affected by alendronate. The number of resorption pits was reduced in TRPV5−/− bone marrow cultures, but the response to vitamin D was similar to that in TRPV5+/+ cultures. The alendronate‐induced upregulation of TRPV5 in bone together with the decreased resorptive capacity of TRPV5−/− osteoclasts in vitro suggests that TRPV5 has an important role in osteoclast function. However, our data indicate that significant bone resorption still occurs in TRPV5−/− mice, because alendronate treatment normalized bone thickness in these mice. Thus, TRPV5−/− mice are able to rescue the resulting defect in osteoclast‐mediated bone resorption, possibly mediated by the long‐term hypervitaminosis D or other (non)hormonal compensatory mechanisms.