Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo
Department of Medicine, University of California, Los Angeles, Los Ángeles, California, United StatesJournal of Orthopaedic Research (Impact Factor: 2.99). 11/2007; 25(11):1488-97. DOI: 10.1002/jor.20437
Oxysterols, naturally occurring cholesterol oxidation products, can induce osteoblast differentiation. Here, we investigated short-term 22(S)-hydroxycholesterol + 20(S)-hydroxycholesterol (SS) exposure on osteoblastic differentiation of marrow stromal cells. We further explored oxysterol ability to promote bone healing in vivo. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, mineralization, and Runx2 DNA binding activity. To explore the effects of osteogenic oxysterols in vivo, we utilized the critical-sized rat calvarial defect model. Poly(lactic-co-glycolic acid) (PLGA) scaffolds alone or coated with 140 ng (low dose) or 1400 ng (high dose) oxysterol cocktail were implanted into the defects. Rats were sacrificed at 6 weeks and examined by three-dimensional (3D) microcomputed tomography (microCT). Bone volume (BV), total volume (TV), and BV/TV ratio were measured. Culture exposure to SS for 10 min significantly increased ALP activity after 4 days, while 2 h exposure significantly increased mineralization after 14 days. Four-hour SS treatment increased OCN mRNA measured after 8 days and nuclear protein binding to an OSE2 site measured after 4 days. The calvarial defects showed slight bone healing in the control group. However, scaffolds adsorbed with low or high-dose oxysterol cocktail significantly enhanced bone formation. Histologic examination confirmed bone formation in the defect sites grafted with oxysterol-adsorbed scaffolds, compared to mostly fibrous tissue in control sites. Our results suggest that brief exposure to osteogenic oxysterols triggered events leading to osteoblastic cell differentiation and function in vitro and bone formation in vivo. These results identify oxysterols as potential agents in local and systemic enhancement of bone formation.
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- "This implies that the mechanisms by which mammalian cells respond to oxysterols can alter during their differentiation. In addition to their cytotoxic actions, oxysterols can trigger a variety of other effects including promotion of foam cell formation by 3-hydroxycholest-5-en-7-one (7-keto) (Hayden et al., 2002); differentiation of Leydig cells by cholest-5-en-3,25-diol (25-OH) (Chen et al., 2002 ) and induction of osteoblastic differentiation of marrow stromal cells by cholest-5-en-3,20-diol (20-OH) or cholest-5-en-3,22-diol (22-OH) (Aghaloo et al., 2007 ). Oxysterols also promote differentiation of NT2 cells toward a 'neuronal' phenotype (Yao et al., 2007), stimulation of mitogen activated protein kinase in human aortic smooth muscle cells (Ares et al., 2000), induction of interleukin-8 production by THP-1 monocytes (Lemaire-Ewing et al., 2009 ) and modulation of glucocorticoid production by adipocytes (Wamil et al., 2008). "
ABSTRACT: Ionised calcium (Ca(2+)) is a key second messenger, regulating almost every cellular process from cell death to muscle contraction. Cytosolic levels of this ion can be increased via gating of channel proteins located in the plasma membrane, endoplasmic reticulum and other membrane-delimited organelles. Ca(2+) can be removed from cells by extrusion across the plasma membrane, uptake into organelles and buffering by anionic components. Ca(2+) channels and extrusion mechanisms work in concert to generate diverse spatiotemporal patterns of this second messenger, the distinct profiles of which determine different cellular outcomes. Increases in cytoplasmic Ca(2+) concentration are one of the most rapid cellular responses upon exposure to certain oxysterol congeners or to oxidised low-density lipoprotein, occurring within seconds of addition and preceding increases in levels of reactive oxygen species, or changes in gene expression. Furthermore, exposure of cells to oxysterols for periods of hours to days modulates Ca(2+) signal transduction, with these longer-term alterations in cellular Ca(2+) homeostasis potentially underlying pathological events within atherosclerotic lesions, such as hyporeactivity to vasoconstrictors observed in vascular smooth muscle, or ER stress-induced cell death in macrophages. Despite their candidate roles in physiology and disease, little is known about the molecular mechanisms that couple changes in oxysterol concentrations to alterations in Ca(2+) signalling. This review examines the ways in which oxysterols could influence Ca(2+) signal transduction and the potential roles of this in health and disease.
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- "The third line of evidence is provided by publications which demonstrated that treatment of murine MSC with specific oxysterols in vitro results in enhanced osteoblast differentiation through the activation of several signaling pathways and that LXRs are partially involved in these processes [25–27,29]. In addition, oxysterols have been reported to promote bone healing in vivo . Of note, the same group has shown that pharmacological LXR activation in murine MSC by T0901317   and GW3965  in vitro decreases the expression of markers of osteogenic differentiation. "
ABSTRACT: Liver X receptors (LXRs) are nuclear receptors that play a crucial role in the transcriptional control of lipid metabolism. Pharmacological LXR activation is an attractive concept for the treatment of atherosclerosis. Genetic LXR deficiency in mice has been shown to have an effect on bone turnover and structure and LXR activation is known to influence the osteogenic differentiation of bone marrow stromal cells. Therefore, therapeutic pharmacological LXR activation may have relevant effects on bone. Here, using two synthetic LXR ligands, T0901317 and GW3965, we investigated the effect of LXR activation on murine osteoblasts and the influence of long-term LXR activation on bone in vivo in mice. Short term (48 h) in vitro treatment of primary murine osteoblasts with T0901317 resulted in a dose-dependent decrease of osteocalcin and alkaline phosphatase mRNA and protein. In vivo, a 6-day treatment of C57BL/6J mice with T0901317 led to a 40% reduction of serum osteocalcin concentrations. Long-term (12-week) oral administration of T0901317 or GW3965 influenced the expression of established LXR target genes in liver and intestine, but did not alter trabecular and cortical bone structure or bone turnover as determined by total skeleton radiography, histomorphometric analysis of lumbar vertebral trabecular bone, micro CT analysis of femur cortical bone and biochemical determination of bone formation and resorption markers. We conclude that short-term pharmacological LXR activation has the potential to profoundly influence osteoblast function, but that long-term LXR activation in vivo has no adverse effects on the murine skeleton.
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- "Oxysterols, a large family of 27-carbon oxygenated products of cholesterol present in the circulation and in human and animal tissues,(25) are involved in various biologic and pathologic processes, including cholesterol efflux, lipoprotein metabolism, cell differentiation, atherosclerosis, and apoptosis.(26–29) We have demonstrated previously that specific oxysterols stimulate the osteogenic differentiation of pluripotent MSCs and inhibit their adipogenic differentiation through the activation of Hedgehog signaling in vitro(30–33) and enhance bone healing in rat critical-sized calvarial defects in vivo.(34) Here, we report that osteogenic oxysterols are novel activators of expression of the Notch target genes HES-1, HEY-1, and HEY-2 in MSCs. "
ABSTRACT: We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similar observations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamine blocked 20S-induced Notch target gene expression. 20S did not induce Notch target genes in Smo(-/-) mouse embryonic fibroblasts, further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR) synthetic ligand TO901317 to induce Notch target genes in M2 cells, LXR knockdown studies using siRNA showed inhibition of 20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-induced Notch target gene expression was independent of canonical Notch signaling because neither 20S nor Shh induced CBF1 luciferase reporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1 and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S are regulated in part by HES-1 and HEY-1.
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