Marco Eijken

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)

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).

Systems biology towards life in silico: mathematics of the control of living cells

Systems Biology is the science that aims to understand how biological function absent from macromolecules in isolation, arises when they are components of their system. Dedicated to the memory of Reinhart Heinrich, this paper discusses the origin and evolution of the new part of systems biology that relates to metabolic and signal-transduction pathways and extends mathematical biology so as to address postgenomic experimental reality. Various approaches to modeling the dynamics generated by metabolic and signal-transduction pathways are compared. The silicon cell approach aims to describe the intracellular network of interest precisely, by numerically integrating the precise rate equations that characterize the ways macromolecules’ interact with each other. The non-equilibrium thermodynamic or ‘lin–log’ approach approximates the enzyme rate equations in terms of linear functions of the logarithms of the concentrations. Biochemical Systems Analysis approximates in terms of power laws. Importantly all these approaches link system behavior to molecular interaction properties. The latter two do this less precisely but enable analytical solutions. By limiting the questions asked, to optimal flux patterns, or to control of fluxes and concentrations around the (patho)physiological state, Flux Balance Analysis and Metabolic/Hierarchical Control Analysis again enable analytical solutions. Both the silicon cell approach and Metabolic/Hierarchical Control Analysis are able to highlight where and how system function derives from molecular interactions. The latter approach has also discovered a set of fundamental principles underlying the control of biological systems. The new law that relates concentration control to control by time is illustrated for an important signal transduction pathway, i.e. nuclear hormone receptor signaling such as relevant to bone formation. It is envisaged that there is much more Mathematical Biology to be discovered in the area between molecules and Life.

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.

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.

Synergistic induction of local glucocorticoid generation by inflammatory cytokines and glucocorticoids: implications for inflammation associated bone loss

Objectives Synovial fibroblasts and osteoblasts generate active glucocorticoids by means of the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. This activity increases in response to proinflammatory cytokines or glucocorticoids. During inflammatory arthritis synovium and bone are exposed to both these factors. This study hypothesised that glucocorticoids magnify the effects of inflammatory cytokines on local glucocorticoid production in both synovium and bone. Methods The effects of inflammatory cytokines (IL-1β/tumour necrosis factor alpha; TNFα) and glucocorticoids, alone or combined, were assessed on the expression and activity of 11β-HSD1 in primary synovial fibroblasts, primary human osteoblasts and MG-63 osteosarcoma cells. A range of other target genes and cell types were used to examine the specificity of effects. Functional consequences were assessed using IL-6 ELISA. Results In synovial fibroblasts and osteoblasts, treatment with cytokines or glucocorticoids in isolation induced 11β-HSD1 expression and activity. However, in combination, 11β-HSD1 expression, activity and functional consequences were induced synergistically to a level not seen with isolated treatments. This effect was seen in normal skin fibroblasts but not foreskin fibroblasts or adipocytes and was only seen for the 11β-HSD1 gene. Synergistic induction had functional consequences on IL-6 production. Conclusions Combined treatment with inflammatory cytokines and glucocorticoids synergistically induces 11β-HSD1 expression and activity in synovial fibroblasts and osteoblasts, providing a mechanism by which synovium and bone can interact to enhance anti-inflammatory responses by increasing localised glucocorticoid levels. However, the synergistic induction of 11β-HSD1 might also cause detrimental glucocorticoid accumulation in bone or surrounding tissues.

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP1) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

GPM6B regulates osteoblast function and induction of mineralization by controlling cytoskeleton and matrix vesicle release.

Neuronal membrane glycoprotein gene (GPM6B) encodes a membrane glycoprotein that belongs to the proteolipid protein family. We identified GPM6B as a gene that is strongly upregulated during osteoblast differentiation. To investigate the role of GPM6B in the process of bone formation, we silenced GPM6B expression during osteogenic differentiation of human mesenchymal stem cells (hMSCs). GPM6B silencing in hMSCs resulted in reduced alkaline phosphate (ALP) activity along with reduced mineralization of extracellular matrix (ECM). Microarray expression analysis of GPM6B‐depleted osteogenic hMSCs revealed significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry results confirm changes in the distribution of actin filaments, as well as the shape and size of focal adhesions on GPM6B silencing. Moreover, we demonstrated that production and release of ALP‐positive matrix vesicles (MVs) were reduced. In conclusion, we identified GPM6B as a novel regulator of osteoblast function and bone formation. This finding demonstrates the significance of cytoskeleton organization for MV production and subsequent mineralization.

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.