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

Abstract

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.

Introduction

Being one of the major factors in calcium homeostasis, vitamin D is essential for the development and maintenance of healthy bones. 1α,25-Dihydroxyvitamin D3 (1,25D3), the biologically most active vitamin D receptor (VDR) agonist, is synthesized by subsequent vitamin D 25-hydroxylase (CYP2R1) and 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) activity in the liver and kidney, respectively (Christakos et al., 2003). Bone formation is indirectly influenced by 1,25D3 since it promotes calcium uptake in the intestine and promotes calcium reabsorption in the kidneys. Also direct effects of 1,25D3 on osteoblasts have been established. 1,25D3 stimulates mineralization of human osteoblast cultures (Miyahara et al., 2002, van Driel et al., 2006b) and modulates gene expression of various osteoblast differentiation and mineralization-related genes such as alkaline phosphatase (ALPL), osteocalcin (BGLAP) and osteopontin (SPP1) (Barthel et al., 2007, Shen and Christakos, 2005, van Driel et al., 2006b). 1,25D3 also enhances osteoblast-produced matrix vesicle maturation (Woeckel et al., 2010). To prevent uncontrolled 1,25D3 action and potentially hypercalcemia, 1,25D3 stimulates its own degradation by up-regulating the expression of 24,25D3-hydroxylase (CYP24A1) (Atkins et al., 2007, van Driel et al., 2006a).

The synthetic peroxisome proliferator-activated receptor γ (PPAR-γ) agonist rosiglitazone belongs to the thiazolidinediones (TZD) and is an insulin sensitizer widely prescribed to type 2 diabetic patients. Recent clinical studies have revealed detrimental effects of rosiglitazone on the aging skeleton (Grey et al., 2007). Underlying mechanisms include rosiglitazone-mediated suppression of bone formation and stimulation of bone resorption caused by a) stimulation of osteoclast differentiation from hematopoietic precursor cells (Wan et al., 2007), b) increased osteoblast and osteocyte apoptosis (Bruedigam et al., 2010, Elbaz et al., 2010, Jung et al., 2007, Kim et al., 2006, Soroceanu et al., 2004), c) preferential differentiation of mesenchymal stem cells into adipocytes at the expense of osteoblasts in the bone marrow (Jeon et al., 2003, Khan and Abu-Amer, 2003, Lecka-Czernik et al., 1999, Lecka-Czernik et al., 2002), or d) modulation of hormones important for bone metabolism, e.g. leptin (Watanabe et al., 2003), insulin (Cornish et al., 1996, Cornish et al., 1998), insulin-like growth factor I (Lecka-Czernik et al., 2007), or estrogen (Rubin et al., 2000). Recently, we published a pro-apoptotic effect of rosiglitazone causing a premature onset of mineralization in human osteoblasts and vascular smooth muscle cells (Bruedigam et al., 2011).

Both VDR and PPAR-γ are nuclear receptors that form heterodimers with the retinoid X receptor (RXR). Moreover, recent studies have revealed evidence for a cross-talk between 1,25D3- and PPAR-α/-δ-ligand-mediated signaling involving a stimulatory effect of 1,25D3 on PPAR expression that is dependent on VDR (Dunlop et al., 2005, Sertznig et al., 2009a). However, there is a current lack of knowledge regarding cross-talk between 1,25D3- and PPAR-γ-ligand-mediated signaling. In this study, we therefore investigated the interaction between 1,25D3- and PPAR-γ-ligand-mediated signaling in the context of osteoblast differentiation and mineralization.