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. © 2010 American Society for Bone and Mineral Research
Highlights
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.[26,27,28,29] We have demonstrated previously that specific oxysterols stimulate the osteogenic differentiation of pluripotent marrow stromal cells (MSCs) and inhibit their adipogenic differentiation through the activation of Hedgehog signaling in vitro[30,31,32,33] and enhance bone healing in rat criticalsized 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
Since osteogenic oxysterols are novel activators of Hedgehog,(32) as well as liver X receptor (LXR) signaling,(44) and sonic hedgehog (Shh) induces Notch receptors and HES-1 expression,(20–22) we examined whether the induction of HES-1, HEY-1, and HEY-2 mRNA expression in MSCs occurs through the Hedgehog or liver X-receptor (LXR) signaling pathway. 20S and Shh induced the expression of all three Notch target genes in M2 cells, whereas the synthetic LXR agonist TO-901317 (TO) did not induce the expression of these genes, suggesting that the induction of Notch target genes by 20S is mainly through Hedgehog signaling and not through LXR signaling (Fig. 2A–C)
Since nuclear hormone receptor conformation may vary depending on the ligand used, and since the effect of 20S on LXR conformation and activity may differ from what is caused by TO,(43) we further examined the potential role of LXR in mediating oxysterol-induced Notch target gene expression in M2 cells using siRNA to knock down LXRa and LXRb expression in these cells, as we have previously reported.[42]. Results showed that 20S-induced HES-1 expression was not affected by LXR siRNAs, whereas HEY-1 expression was significantly inhibited by LXR siRNA, suggesting the role of LXR as well as Hedgehog signaling in 20S-induced HEY-1 but not HES-1 expression
Summary
The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism that plays a prominent role in cell proliferation, differentiation, and survival.[1,2] The canonical Notch signaling pathway is activated when Notch receptors (Notch-1, -2, -3, and -4) interact with ligands [Jagged-1 and -2 and Delta-like (Dll-1, -3, and -4)] on adjacent cells, triggering proteolytic cleavage of the receptor by the presenilin– g-secretase complex.[1,2] This releases the Notch intracellular domain (NICD), which translocates to the nucleus and binds the CBF-1 DNA-binding protein, thereby inducing the expression of. In addition to canonical Notch signaling, the expression of Notch target genes is regulated by growth factors, including transforming drowth factor b (TGF-b), bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), and sonic hedgehog (Shh).(17,19–21) TGF-b induces HEY-1 and Jagged in epithelial cells from mammary gland, kidney tubules, and epidermis,(19) and BMP-9 induces HEY-1 expression in C3H10T1/2 cells.[17] Shh and VEGF induce Notch-5 and HES-1 mRNA expression in various cells, including C3H10T1/2 cells, MNS70 neural cells, and granule neuron precursors.[20,21,22] it has been suggested that regulation of HES-1 expression by c-Jun kinase signaling and Hedgehog signaling may be mediated through the activation of noncanonical Notch signaling pathways.[22,23,24] the molecular mechanisms by which growth and differentiation factors activate the Notch signaling pathway and induce the expression of Notch target genes require further elucidation. The induction of Notch target gene expression by 20S is not mediated by the canonical Notch signaling pathway but mainly by Hedgehog signaling and in part by LXR signaling, and HES-1 and HEY-1 induction appears necessary for maximal induction of osteogenesis by 20S
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
