| Author: | Yu, Wenxuan |
| Title: | Investigation of the role of extra-renal 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) in mediating the bone protective effects of oleanolic acid |
| Advisors: | Wong, Man-sau (ABCT) |
| Degree: | Ph.D. |
| Year: | 2021 |
| Subject: | Bones Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xvi, 197 pages : illustrations |
| Language: | English |
| Abstract: | 1,25(OH)2D3 plays important role in regulating systemic calcium homeostasis and bone mineralization. It is synthesized from its precursor 25OHD3 through the hydroxylation by 25-hydroxyvitamin D3 1-alpha-hydroxylase (CYP27B1). With the discovery of CYP27B1 in other extra-renal sites such as bone, regulation of extra-renal CYP27B1 and the paracrine or autocrine activities of 1,25(OH)2D3 has become the focus of current research. Our previous study showed oleanolic acid (OA), a naturally occurring pentacyclic triterpenoid with bone protective effects, was capable to modulate calcium balance and renal CYP27B1 expression in aged rats. However, the ability of OA to regulate CYP27B1 in extra-renal sites is far from clear. The present study aimed to characterize the effects of OA on modulating extra-renal CYP27B1 expression and activity in extra-renal sites such as osteoblasts and adipocytes, as well as their involvement in the bone protective effects of OA. In the present study, we first demonstrated that OA effectively elevated circulating 1,25(OH)2D3 levels by upregulating renal CYP27B1 expression, which contributed to the positive calcium balance and the improvement of bone properties in ovariectomized (OVX) mice. Moreover, the expressions of CYP27B1 in iliac crests were significantly increased in response to OA, indicating the potential action of OA to modulate paracrine or autocrine activities of 1,25(OH)2D3 in skeletal microenvironment. The modulatory activities of OA on CYP27B1 expressions and cellular production of 1,25(OH)2D3 in bone cells were confirmed by in-vitro experiments using human and rat osteoblast-like cells. In the following part, OA was shown to significantly induce CYP27B1 expression and cellular synthesis of 1,25(OH)2D3 as well as the expression of bone sialoprotein 2 (BSP2) and osteopontin (OPN), two vitamin D-dependent osteogenic markers in mature osteoblastic lineages. Moreover, the stimulatory effects of OA on OPN expression and ALP activity in human osteoblast-like MG-63 cells were attenuated, at least in part, in the presence of CYP27B1 siRNA. The results indicated the role of CYP27B1 and local 1,25(OH)2D3 production in mediating the effects of OA on the maturation and mineralization of osteoblasts. The third part of my study attempted to delineate the underlying mechanisms by which OA regulate CYP27B1 in osteoblasts. Our results showed that the modulatory effects of OA on the expression and activity of CYP27B1 in osteoblasts were mimicked by lithocholic acid (LCA), a bile acid and an agonist of G protein-coupled bile acid receptor 1 (TGR5), indicating the potential role of TGR5 in regulating CYP27B1 in osteoblasts. This speculation was supported by our findings that the effects of OA on CYP27B1 expression were potentiated by overexpression of TGR5 but were attenuated when TGR5 was silenced in MG-63 cells. Further studies demonstrated that OA modulated transcriptional levels of CYP27B1 through activation of TGR5 and subsequent phosphorylation of cAMP-responsive elements-binding protein (CREB). In the last part, we demonstrated that OA significantly suppressed the accumulation of bone marrow adipose tissues and serum lipids levels, in addition to improvement of bone properties, in OVX mice. Such actions of OA were believed to be associated with its improvement in the balance between adipogenesis and osteogenesis of bone marrow stem cells (BMSCs). In-vitro studies showed that OA, act like 1,25(OH)2D3, inhibited adipogenesis and lipid droplets accumulation in adipogenic differentiated hMSCs as well as 3T3-L1 preadipocytes. OA also consistently upregulated CYP27B1 expression and phosphorylated levels of AMP-activated protein kinase alpha (AMPK α) in 3T3- L1 cells, which was mediated by activation of TGR5. In summary, the present study supported our hypothesis that OA exerts its bone protective effects by modulating extra-renal CYP27B1 expression and activities in osteoblasts and adipocytes. The study extended our understandings of the paracrine or autocrine actions of 1,25(OH)2D3 in bone microenvironment and provides new insights into the approach for modulating CYP27B1 in extra-renal tissues. |
| Rights: | All rights reserved |
| Access: | open access |
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