M. Koedam

Evidence for auto/paracrine actions of vitamin D in bone: 1α-hydroxylase expression and activity in human bone cells

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1α‐Hy‐droxylation of 25‐(OH)D3 to form the bioactive vitamin D hormone, 1α,25‐(OH)2D3, is classically considered to take place in the kidney. However, 1α‐hydroxylase has been reported at extrarenal sites. Whether bone is a 1α,25‐(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/ paracrine function for 1α,25‐(OH)2D3 in bone. We show that 1α‐hydroxlase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1α‐hydoxylase substrate 25‐(OH)D3, the osteoblasts can produce sufficient 1α,25‐(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1α‐hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1α‐hydroxylase in osteoblasts. In contrast, interleukin (IL)‐1β strongly induces 1α‐hydroxylase. Besides the bone‐forming cells, we demonstrate 1α‐hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1α‐hydoxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.—van Driel, M., Koedam, M., Buurman, C. J., Hewison, M., Chiba, H., Uitterlinden, A. G., Pols, H. A. P., van Leeuwen, J. P. T. M. Evidence for auto/paracrine actions of vitamin D in bone: 1α‐hydroxylase expression and activity in human bone cells. FASEB J. 20, E1811–E1819 (2006)

The activin A-follistatin system: potent regulator of human extracellular matrix mineralization

Bone quality is an important determinant of osteoporosis, and proper osteoblast differentiation plays an important role in the control and maintenance of bone quality. We investigated the impact of activin signaling on human osteoblast differentiation, extracellular matrix formation, and mineralization. Ac‐tivins belong to the transforming growth factor‐β su‐perfamily and activin A treatment strongly inhibited mineralization in osteoblast cultures, whereas the ac‐tivin antagonist follistatin increased mineralization. Os‐teoblasts produced activin A and follistatin in a differentiation‐dependent manner, leading to autocrine regulation of extracellular matrix formation and mineralization. In addition, mineralization in a vascular smooth muscle cell‐based model for pathological calcification was inhibited. Comparative activin A and fol‐listatin gene expression profiling showed that activin signaling changes the expression of a specific range of extracellular matrix proteins prior to the onset of mineralization, leading to a matrix composition with reduced or no mineralizing capacity. These findings demonstrate the regulation of osteoblast differentiation and matrix mineralization by the activin A‐follista‐tin system, providing the possibility to control bone quality as well as pathological calcifications such as atherosclerosis by using activin A, follistatin, or analogs thereof.—Eijken M., Swagemakers, S., Koedam, M., Steenbergen, C., Derkx, P., Uitterlinden, A. G., van der Spek P. J., Visser, J. A., de Jong F. H., Pols, H. A. P., van Leeuwen J. P. T. M. The activin A‐follistatin system: potent regulator of human extracellular matrix mineralization. FASEB J. 21, 2949–2960 (2007)

Evidence that both 1α,25‐dihydroxyvitamin D3 and 24‐hydroxylated D3 enhance human osteoblast differentiation and mineralization

Vitamin D plays a major role in the regulation of mineral homeostasis and affects bone metabolism. So far, detailed knowledge on the vitamin D endocrine system in human bone cells is limited. Here we investigated the direct effects of 1α,25‐(OH)2D3 on osteoblast differentiation and mineralization. Also, we studied the impact of 24‐hydroxylation, generally considered as the first step in the degradation pathway of vitamin D, as well as the role of the nuclear and presumed membrane vitamin D receptor (VDR). For this we used a human osteoblast cell line (SV‐HFO) that has the potency to differentiate during culture forming a mineralized extracellular matrix in a 3‐week period. Transcriptional analyses demonstrated that both 1α,25‐(OH)2D3 and the 24‐hydroxylated metabolites 24R,25‐(OH)2D3 and 1α,24R,25‐(OH)3D3 induced gene transcription. All metabolites dose‐dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1α,24R,25‐(OH)3D3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1α,25‐(OH)2D3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1β,25‐(OH)2D3, an inhibitor of membrane effects of 1α,25‐(OH)2D3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1α,25‐(OH)2D3, but also the presumed 24‐hydroxylated “degradation” products stimulate differentiation of human osteoblasts. 1α,25‐(OH)2D3 as well as the 24‐hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1β,25‐(OH)2D3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR‐dependent way. J. Cell. Biochem. 99: 922–935, 2006.

The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization

Glucocorticoids (GCs) exert profound effects on bone and are essential for human osteoblast differentiation. However, GCs are still interpreted as negative regulators of bone formation, mainly caused by the detrimental effects on bone after clinical use of GCs. In this paper we emphasize the importance of GCs for proper human osteoblast differentiation and matrix mineralization. We show that human osteoblast differentiation needs to be triggered by GCs in a specific time-window during the early stages of development. Exposure to GCs in the beginning of osteoblast development induces a dose dependent increase in alkaline phosphatase activity and matrix mineralization. GC-induced differentiation stimulated expression of genes involved in bone formation and suppressed genes that negatively regulate bone formation and mineralization. Furthermore we highlight the importance of local cortisol activation in osteoblasts by expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1).

A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone

Bone‐marrow adipogenesis is an aging‐related phenomenon and is correlated with osteoporosis. The latter is a prevalent bone disease in the elderly leading to increased fracture risk and mortality. It is widely hypothesized that the underlying molecular mechanism includes a shift in the commitment of mesenchymal stem cells (MSCs) from the osteogenic lineage to the adipogenic lineage. Lineage skewing is at least partially a result of transcriptional changes. The nuclear transcription factor peroxisome proliferator‐activated receptor γ (PPAR‐γ) has been proposed as a major decision factor in MSC lineage commitment, promoting adipogenesis at the expense of osteogenesis. Here we found that PPAR‐γ acted unexpectedly to stimulate osteoblast differentiation from human bone marrow‐derived MSCs. Both rosiglitazone‐mediated activation and overexpression of PPAR‐γ caused acceleration of osteoblast differentiation. Conversely, shRNAi‐mediated PPAR‐γ knockdown diminished osteoblast differentiation. MSCs that were treated with rosiglitazone did not preferentially differentiate into adipocytes. However, the rosiglitazone‐mediated acceleration of osteoblast differentiation was followed by increased accumulation of reactive oxygen species and apoptosis. In contrast to the osteogenic lineage, cells of the adipogenic lineage were protected from this. Our data support a new concept on bone health that adds to the explanation of the clinically observed suppressive action of activated PPAR‐γ on bone and the associated phenomenon of bone marrow adipogenesis. This concept is based on a higher susceptibility of the osteogenic than the adipogenic lineage to oxidative stress and apoptosis that is preferentially triggered in the osteoblasts by activated PPAR‐γ. STEM CELLS 2010;28:916–927

Wnt signaling acts and is regulated in a human osteoblast differentiation dependent manner

The Wnt signaling pathway is an important regulator of cellular differentiation in a variety of cell types including osteoblasts. In this study, we investigated the impact of Wnt signaling on the function of human osteoblasts in relation to the stage of differentiation. Differentiating osteoblasts were created upon glucocorticoid (GC) treatment, whereas nondifferentiating osteoblasts were created by excluding GCs from the culture medium. GC‐induced differentiation suppressed endogenous β‐catenin levels and transcriptional activity. During GC‐induced osteoblast differentiation, activation of Wnt signaling slightly decreased alkaline phosphatase activity, but strongly suppressed matrix mineralization. In addition, mRNA expression of several Wnt signaling related genes was strongly regulated during GC‐induced osteoblast differentiation, including frizzled homolog 8, dickkopf homolog 1, and secreted frizzled‐related protein 1. In contrast, in the absence of GC‐induced differentiation, Wnt signaling acted positively by stimulating basal alkaline phosphatase activity. Interestingly, pre‐stimulation of Wnt signaling in early osteoblasts enhanced their differentiation capacity later on during the GC‐induced differentiation process. In conclusion, we showed a differentiation‐dependent effect of Wnt signaling on osteoblasts. Wnt signaling stimulated early osteoblasts in their capacity to differentiate, whereas mature osteoblasts were strongly inhibited in their capacity to induce mineralization. Moreover, osteoblast differentiation suppressed endogenous Wnt signaling and changed the expression of multiple Wnt signaling related genes. J. Cell. Biochem. 104: 568–579, 2008.

Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation.

Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre‐osteoblastic cell line SV‐HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60–70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down‐regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix‐metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization. J. Cell. Physiol. 227: 1309–1318, 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.

Evidence for multiple peroxisome proliferator-activated receptor γ transcripts in bone: fine-tuning by hormonal regulation and mRNA stability

The expression, regulation and functional significance of multiple peroxisome proliferator‐activated receptor γ transcript variants in bone were studied. PPARG transcripts giving rise to PPARg‐1 protein were expressed in human osteoblasts, whereas PPARG‐2 transcript and protein remained virtually absent. PPARG expression underwent homologous regulation, was upregulated during differentiation and directly induced by the osteogenic hormone dexamethasone, suggesting a role for PPARg‐1 in osteogenesis. Differences between the stabilities of PPARG‐1, ‐3 and ‐4 were observed. We hypothesize that cell‐specific expression patterns of multiple PPARG transcript variants encoding for the same protein but differing in mRNA stabilities enable a fine‐tuning of PPARG action, which eventually supports a well‐adjusted signal transduction between the cell and its environment.

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.