Marijke Schreuders-Koedam

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.

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.

Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway.

Significance Osteoporosis, a disease characterized by increased bone fragility and fracture risk, affects over 20% of the ever-growing elderly population. It is important to further our knowledge of bone cell biology so we can develop new bone anabolic treatments. By combining genomic and bioinformatic tools against the backdrop of osteogenic differentiating human mesenchymal stromal cells, we have identified a previously unidentified bone anabolic compound that induces osteoblast differentiation in a subset of the hMSC population through cytoskeletal changes and increased bone morphogenetic protein 2 activity. Through this novel approach we identified an important mechanism of lineage allocation and demonstrated the significance of cytoskeletal organization in osteogenic differentiation, providing us with a novel mechanism for bone formation to target for new osteoporosis treatments. Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity.

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.

A human vitamin D receptor mutation causes rickets and impaired Th1/Th17 responses

We present a brother and sister with severe rickets, alopecia and highly elevated serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Genomic sequencing showed a homozygous point mutation (A133G) in the vitamin D receptor gene, leading to an amino acid change in the DNA binding domain (K45E), which was described previously. Hereditary vitamin D resistant rickets (HVDRR) was diagnosed. Functional studies in skin biopsy fibroblasts confirmed this. 1,25-(OH)2D3 reduced T helper (Th) cell population-specific cytokine expression of interferon γ (Th1), interleukins IL-17A (Th17) and IL-22 (Th17/Th22) in peripheral blood mononuclear cells (PBMCs) from the patients parents, whereas IL-4 (Th2) levels were higher, reflecting an immunosuppressive condition. None of these factors were regulated by 1,25-(OH)2D3 in PBMCs from the boy. At present, both patients (boy is 23 years of age, girl is 7) have not experienced any major immune-related disorders. Although both children developed alopecia, the girl did so earlier than the boy. The boy showed complete recovery from the rickets at the age of 17 and does not require any vitamin D supplementations to date. In conclusion, we characterized two siblings with HVDRR, due to a mutation in the DNA binding domain of VDR. Despite a defective T cell response to vitamin D, no signs of any inflammatory-related abnormalities were seen, thus questioning an essential role of vitamin D in the immune system. Despite the fact that currently medicine is not required, close monitoring in the future of these patients is warranted for potential recurrence of vitamin D dependence and diagnosis of (chronic) inflammatory-related diseases.

Adverse Effects of Diabetes Mellitus on the Skeleton of Aging Mice

In the present study, the possibility that a diabetic (DM) status might worsen age-related bone deterioration was explored in mice. Male CD-1 mice aged 2 (young control group) or 16 months, nondiabetic or made diabetic by streptozotocin injections, were used. DM induced a decrease in bone volume, trabecular number, and eroded surface, and in mineral apposition and bone formation rates, but an increased trabecular separation, in L1-L3 vertebrae of aged mice. Three-point bending and reference point indentation tests showed slight changes pointing to increased frailty and brittleness in the mouse tibia of diabetic old mice. DM was related to a decreased expression of both vascular endothelial growth factor and its receptor 2, which paralleled that of femoral vasculature, and increased expression of the pro-adipogenic gene peroxisome proliferator-activated receptor γ and adipocyte number, without affecting β-catenin pathway in old mouse bone. Concomitant DM in old mice failed to affect total glutathione levels or activity of main anti-oxidative stress enzymes, although xanthine oxidase was slightly increased, in the bone marrow, but increased the senescence marker caveolin-1 gene. In conclusion, DM worsens bone alterations of aged mice, related to decreased bone turnover and bone vasculature and increased senescence, independently of the anti-oxidative stress machinery.

Hydroxychloroquine affects bone resorption both in vitro and in vivo

We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC‐sorted monocytes for 14 days and treated with different HCQ doses (controls 1 and 5 μg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker β‐CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum β‐CTx was significantly decreased after 6 months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker β‐CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss.

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.

Identification of microRNAs in human plasma

In recent years, microRNAs (miRNA) have been demonstrated to be present in body fluids and may therefore serve as diagnostic markers for diseases. By characterizing miRNA profiles in plasma, a miRNA signature may potentially be developed as a diagnostic and risk assessment tool for particular (patho)physiological states. This chapter describes the isolation, purification, identification, and sequencing of human plasma miRNAs.

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.