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

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Abstract

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 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1).

Introduction

Osteoblast differentiation is a complex process regulated by many endocrine, paracrine and autocrine factors. In human and rat bone marrow stromal cells GCs are crucial for the induction of osteoblast differentiation and formation of a mineralized extracellular matrix (Bellows et al., 1987, Cheng et al., 1996, Herbertson and Aubin, 1995, Iba et al., 1995, Jorgensen et al., 2004, Maniatopoulos et al., 1988). Although the exact role of GCs in regulating bone formation is unclear, it might be dependent on the duration and concentration of GC treatment and on the differentiation stage of both osteoblasts and osteoclasts (Hirayama et al., 2002, Ishida and Heersche, 1998, Pockwinse et al., 1995, Smith et al., 2000). At a molecular level GC signaling is mediated via the GC receptor (GRα). GRα is expressed in almost all cell-types including osteoblasts (Liesegang et al., 1994) where it regulates gene expression by binding to GC responsive elements in the regulatory regions of several target genes, including osteocalcin, collagenIα1 and transforming growth factor-β1 (Heinrichs et al., 1993, Parrelli et al., 1998, Peterkofsky et al., 1999). Despite clear stimulation of bone formation by GCs in vitro, GCs are still interpreted as negative regulators of bone formation. This idea is mainly caused by the clinical use of GCs. GC therapy frequently results in bone loss and increased risk for fractures (Canalis, 1996, Clowes et al., 2001, Weinstein, 2001). This negative effect on bone is caused by high levels of GCs for longer periods and might not reflect the normal in vivo role of GCs in bone. Furthermore, most studies on osteoblast differentiation are performed using mouse osteoblasts that lack the need for GC treatment for the induction of differentiation (Ecarot-Charrier et al., 1983, Sudo et al., 1983), which is in contrast to the human situation. Therefore we emphasize in this paper the importance of GCs for proper human osteoblast differentiation and matrix mineralization. Moreover we want to highlight the significance of pre-receptor regulation of GCs in osteoblasts by expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). 11β-HSD1 is found in almost all GC target tissues including osteoblasts (Bland et al., 1999, Cooper et al., 2000) and primarily displays reductase activity converting relative high levels of free cortisone into the biologically active cortisol. As a consequence 11β-HSD1 activity in osteoblasts provides an efficient mechanism for the local activation of GCs in bone and is an important autocrine determinant of osteoblast proliferation, differentiation and function (Canalis and Delany, 2002a, Cooper et al., 1999, Eijken et al., 2005).

Section snippets

Cell culture

SV-HFO cells were cultured in αMEM (GIBCO, Paisley, UK) supplemented with 20 mM HEPES, pH 7.5 (Sigma, St. Louis, MO); streptomycin/penicillin; 1.8 mM CaCl2 (Sigma); and heat-inactivated FCS (GIBCO) at 37 °C and 5% CO2 in a humidified atmosphere. Thawed cells were precultured for 1 week in the presence of 10% FCS. In this preculture, cells were seeded in a density of 5 × 103 vital cells/cm2 and were subcultured every week. During this preculture, SV-HFO cells remained in an undifferentiated stage.

The essential role of glucocorticoids for proper osteoblast function

To demonstrate the importance of GCs for human osteoblast differentiation in more detail we used the pre-osteoblast model SV-HFO (Chiba et al., 1993), which is a well-characterized osteoblast cell-line showing a controlled GC-induced differentiation process in a 3-week period. During this differentiation process an extracellular matrix (ECM) is formed and the process of mineralization is initiated around day 14 (Fig. 1A). Moreover, GC-induced differentiation leads to increased sensitivity to

Conclusion

The major aim of this study was to underscore the significance of GCs for proper human osteoblast differentiation. GCs are essential for human osteoblast differentiation, which is in marked contrast to the widely studied murine osteoblast differentiation. The human GC-induced differentiation process is summarized in Fig. 6A. In the beginning of osteoblast development precursor cells need to be directed by GCs to differentiate into bone forming osteoblasts. During this process several genes are

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