B.C.J. van der Eerden

Klotho Prevents Renal Calcium Loss

Disturbed calcium (Ca(2+)) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca(2+) absorption is primary, which causes increased urinary Ca(2+) excretion, leading to elevated 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its sequelae. Here, we assessed intestinal Ca(2+) absorption, bone densitometry, renal Ca(2+) excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho(-/-) mice. We observed elevated serum Ca(2+) and fractional excretion of Ca(2+) (FE(Ca)) in klotho(-/-) mice. Klotho(-/-) mice also showed intestinal Ca(2+) hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D(9K) increased. In the kidney, klotho(-/-) mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D(28K), implying a failure to absorb Ca(2+) through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-alpha-hydroxylase (1alphaOHase), and calbindin-D(9K) excluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca(2+) wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca(2+) handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca(2+) loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis.

1α,25‐(OH)2D3 acts in the early phase of osteoblast differentiation to enhance mineralization via accelerated production of mature matrix vesicles

1α,25‐dihydroxyitamin D3 (1,25D3) deficiency leads to impaired bone mineralization. We used the human pre‐osteoblastic cell line SV‐HFO, which forms within 19 days of culture an extracellular matrix that starts to mineralize around day 12, to examine the mechanism by which 1,25D3 regulates osteoblasts and directly stimulates mineralization. Time phase studies showed that 1,25D3 treatment prior to the onset of mineralization, rather than during mineralization led to accelerated and enhanced mineralization. This is supported by the observation of unaltered stimulation by 1,25D3 even when osteoblasts were devitalized just prior to onset of mineralization and after 1,25D3 treatment. Gene Chip expression profiling identified the pre‐mineralization and mineralization phase as two strongly distinctive transcriptional periods with only 0.6% overlap of genes regulated by 1,25D3. In neither phase 1,25D3 significantly altered expression of extracellular matrix genes. 1,25D3 significantly accelerated the production of mature matrix vesicles (MVs) in the pre‐mineralization. Duration rather than timing determined the extent of the 1,25D3 effect. We propose the concept that besides indirect effects via intestinal calcium uptake 1,25D3 directly accelerates osteoblast‐mediated mineralization via increased production of mature MVs in the period prior to mineralization. The accelerated deposition of mature MVs leads to an earlier onset and higher rate of mineralization. These effects are independent of changes in extracellular matrix protein composition. These data on 1,25D3, mineralization, and MV biology add new insights into the role of 1,25D3 in bone metabolism and emphasize the importance of MVs in bone and maintaining bone health and strength by optimal mineralization status. J. Cell. Physiol. 225: 593–600, 2010.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/−) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a ‘survival response’, which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg−/− (also known as Ercc5−/−) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/− mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/− mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/− mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

1α,25‐dihydroxyvitamin D3 stimulates activin A production to fine‐tune osteoblast‐induced mineralization

In healthy bones, mineralization has to be tightly controlled to avoid pathological phenotypes. In this study, we investigated interactions between 1α,25(OH)2D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures, we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down‐regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3‐treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A‐mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3‐induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3‐treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength. J. Cell. Physiol. 228: 2167–2174, 2013.

IFNβ impairs extracellular matrix formation leading to inhibition of mineralization by effects in the early stage of human osteoblast differentiation.

Osteoimmunology is an emerging field of research focused on the interaction of the immune system and bone. In this study we demonstrate that human osteoblasts are sensitive to the immune cytokine interferon (IFN)β. Osteoblasts respond to IFNβ as shown by the induction of several known IFN target genes such as interferon‐induced (IFI) proteins (IFIT1, IFI44L), interferon‐stimulated gene factor 3 (ISGF3) complex and the induction of IFNβ itself. We demonstrated that IFNβ has severe inhibitory effects on mineralization of osteoblast‐derived extracellular matrix (ECM). Analysis of the timing of the IFNβ effects revealed that committed osteoblasts in early stage of differentiation are most sensitive to IFNβ inhibition of mineralization. A single IFNβ treatment was as effective as multiple treatments. During the progress of differentiation osteoblasts become desensitized for IFNβ. This pinpoints to a complex pattern of IFNβ sensitivity in osteoblasts. Focusing on early osteoblasts, we showed that IFNβ decreased gene expression of ECM‐related genes, such as type I Collagen (COL1A1), fibronectin (FN1), fibullin (FBLN1), fibrillin (FBN2), and laminin (LAMA1). Additionally, ECM produced by IFNβ‐treated osteoblasts contained less collagen protein. IFNβ stimulated gene expression of osteopontin (OPN), annexin2 (ANXA2), and hyaluronan synthase 1 (HAS1), which are important factors in the adhesion of hematopoietic stem cells (HSC) in the HSC niche. In conclusion, IFNβ directly modifies human osteoblast function by inhibiting ECM synthesis eventually resulting in delayed bone formation and mineralization and induces a HSC niche supporting phenotype. These effects are highly dependent on timing of treatment in the early phase of osteoblast differentiation. J. Cell. Physiol. 227: 2668–2676, 2012.

Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization.

It is well established that 1α‐25‐dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre‐osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon‐related and ‐regulated genes (ISG) to be overrepresented in the set of 1,25D3‐regulated genes. 1,25D3 down‐regulated ISGs predominantly during the pre‐mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ‐ and 1,25D3‐mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti‐correlated with interferon‐induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis. J. Cell. Physiol. 227: 3258–3266, 2012.

TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk

We explored the role of transient receptor potential vanilloid 4 (TRPV4) in murine bone metabolism and association of TRPV4 gene variants with fractures in humans. Urinary and histomorphometrical analyses demonstrated reduced osteoclast activity and numbers in male Trpv4(-/-) mice, which was confirmed in bone marrow-derived osteoclast cultures. Osteoblasts and bone formation as shown by serum procollagen type 1 amino-terminal propeptide and histomorphometry, including osteoid surface, osteoblast and osteocyte numbers were not affected in vivo. Nevertheless, osteoblast differentiation was enhanced in Trpv4(-/-) bone marrow cultures. Cortical and trabecular bone mass was 20% increased in male Trpv4(-/-) mice, compared to sex-matched wild type (Trpv4(+/+)) mice. However, at the same time intracortical porosity was increased and bone matrix mineralization was reduced. Together, these lead to a maximum load, stiffness and work to failure of the femoral bone, which were not different compared to Trpv4(+/+) mice, while the bone material was less resistant to stress and less elastic. The differential impacts on these determinants of bone strength were likely responsible for the lack of any changes in whole bone strength in the Trpv4(-/-) mice. None of these skeletal parameters were affected in female Trpv4(-/-) mice. The T-allele of rs1861809 SNP in the TRPV4 locus was associated with a 30% increased risk (95% CI: 1.1-1.6; p=0.013) for non-vertebral fracture risk in men, but not in women, in the Rotterdam Study. Meta-analyses with the population-based LASA study confirmed the association with non-vertebral fractures in men. This was lost when the non-population-based studies Mr. OS and UFO were included. In conclusion, TRPV4 is a male-specific regulator of bone metabolism, a determinant of bone strength, and a potential risk predictor for fractures through regulation of bone matrix mineralization and intra-cortical porosity. This identifies TRPV4 as a unique sexually dimorphic therapeutic and/or diagnostic candidate for osteoporosis.

1α,25-dihydroxyvitamin D3 and rosiglitazone synergistically enhance osteoblast-mediated mineralization.

Both vitamin D receptor (VDR) and peroxisome proliferator-activated receptor γ (PPAR-γ) are ligand-activated nuclear transcription factors that are instrumental for bone health. While 1α,25-dihydroxyvitamin D3 (1,25D3), the ligand for VDR, is essential for the development and maintenance of healthy bone, PPAR-γ agonists cause detrimental skeletal effects. Recent studies have revealed evidence for a cross-talk between 1,25D3- and PPAR-α/-δ ligand-mediated signaling but there is a current lack of knowledge regarding cross-talk between signaling of 1,25D3 and the PPAR-γ ligand mediated signaling. In this study, we investigated the cross-talk between 1,25D3- and PPAR-γ agonist rosiglitazone-mediated signaling in human osteoblasts. 1,25D3 slightly but significantly induced expression of the primary PPAR-γ target gene ANGPTL4 but did not influence FABP4. 1,25D3 did not change rosiglitazone regulation of ANGPTL4 and FABP4. The other way around, rosiglitazone reduced CYP24A1 gene expression but this did not change CYP24A1 induction by 1,25D3. The findings regarding CYP24A1 gene expression are in line with the observation that 1,25D3 levels in medium were not affected by rosiglitazone. Furthermore, rosiglitazone significantly inhibited 1,25D3-induction of BGLAP while rosiglitazone alone did not change BGLAP. Additionally, 1,25D3 and rosiglitazone increase osteoblast alkaline phosphatase activity and synergistically stimulated extracellular matrix mineralization. In conclusion, these data provide evidence for a cross-talk between rosiglitazone- and 1,25D3-mediated signaling leading to an acceleration of extracellular matrix mineralization. The data suggest that the reduction of the mineralization inhibitor BGLAP and the increased differentiation status underlie the increased mineralization.

Osteoclastogenic capacity of peripheral blood mononuclear cells is not different between women with and without osteoporosis.

INTRODUCTION Peripheral Blood Mononuclear Cells (PBMCs) have been extensively used as a culture model to generate osteoclasts in vitro. The aim of this study was to assess the osteoclastogenic potential of PBMCs derived from post-menopausal women with longstanding osteoporosis and compare this with PBMCs from healthy controls. MATERIAL AND METHODS We selected from the population-based Rotterdam Study 82 participants of which 43 were diagnosed with osteoporosis (T-score below -2.5 at the lumbar spine) and the presence of at least 1 fracture and 29 healthy controls (T-score above 1; no fracture). PBMCs were differentiated into osteoclasts, and both differentiation capacity and activity were measured. Total RNA was obtained to assess gene expression of osteoclast markers. Deoxypyridinoline (DPD) was measured in plasma as a marker for bone resorption, in vivo. RESULTS Neither the number of osteoclasts nor cathepsin K (CTSK) and dendritic cell-specific transmembrane protein (TM7SF4) gene expression was significantly different between both groups. There was also no significant difference in resorption pit area and plasma DPD levels. Stratification by fracture type into a group with vertebral, non-vertebral and both vertebral and non-vertebral fractures showed no difference in osteoclast formation or osteoclastic bone resorption. However, plasma DPD, but not the RNA expression markers, was significantly lower in the group of subjects with vertebral fracture group and those with vertebral and non-vertebral fractures compared to the healthy controls. No differences in osteoclastogenesis, osteoclastic resorption and plasma DPD levels were detected also after exclusion of past or present users of bisphosphonates and glucocorticoids. Stratification into high and low DPD levels showed higher osteoclastogenesis and more osteoclastic bone resorption in the high DPD group compared to the low DPD levels within the group of osteoporotic subjects. CONCLUSION This study showed no difference in PBMC osteoclastogenic capacity and activity between women with and without osteoporosis and at least one previous fracture, who were on average 29.5years after menopause, suggesting that there is no difference in circulating osteoclast precursors. Although we cannot exclude that circulating precursors may behave differently at the bone site, it is possible that long after menopause a more stable phase of bone turnover is reached compared to earlier after the start of menopause in which differences in circulating osteoclast precursors and osteoclastogenic potential are more prominent.

Using the Connectivity Map to discover compounds influencing human osteoblast differentiation

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. Identification of factors influencing osteoblast differentiation and bone formation is very important. Previously, we identified parbendazole to be a novel compound that stimulates osteogenic differentiation of human mesenchymal stromal cells (hMSCs), using gene expression profiling and bioinformatic analyzes, including the Connectivity Map (CMap), as an in‐silico approach. The aim for this paper is to identify additional compounds affecting osteoblast differentiation using the CMap. Gene expression profiling was performed on hMSCs differentiated to osteoblasts using Illumina microarrays. Our osteoblast gene signature, the top regulated genes 6 hr after induction by dexamethasone, was uploaded into CMap (www.broadinstitute.org/cmap/). Through this approach we identified compounds with gene signatures positively correlating (withaferin‐A, calcium folinate, amylocaine) or negatively correlating (salbutamol, metaraminol, diprophylline) to our osteoblast gene signature. All positively correlating compounds stimulated osteogenic differentiation, as indicated by increased mineralization compared to control treated cells. One of three negatively correlating compounds, salbutamol, inhibited dexamethasone‐induced osteoblastic differentiation, while the other two had no effect. Based on gene expression data of withaferin‐A and salbutamol, we identified HMOX1 and STC1 as being strongly differentially expressed . shRNA knockdown of HMOX1 or STC1 in hMSCs inhibited osteoblast differentiation. These results confirm that the CMap is a powerful approach to identify positively compounds that stimulate osteogenesis of hMSCs, and through this approach we can identify genes that play an important role in osteoblast differentiation and could be targets for novel bone anabolic therapies.