Characterization of the effects of bile acids on 25-hydroxyvitamin D-1alpha hydroxylase (CYP27B1)

Abstract

Osteoporosis is a skeletal disorder characterized by low bone mass, deterioration of bone tissues, and hence increased risk of bone fracture. Annually, one in two postmenopausal women and one-third of older men suffer from this unbearable disorder. Recently, our team has demonstrated that bile acid membrane receptor Takeda G protein coupled receptor 5 (TGR5) can be activated by non-bile acid agonist, leading to the activation of the enzyme 25-hydroxyvitamin D-1alpha hydroxylase (CYP27B1) to induce the production of vitamin D, 1,25(OH)2D3. Bile acids not only play an essential role in lipid digestion and fat-soluble vitamins absorption, but also bind to vitamin D receptor when vitamin D is deficient to initiate calcium absorption. However, the ability of bile acids as natural agonist of bile acid receptors to affect bone health by regulating CYP27B1 is far from clear. Thus, the hypothesis of this study is that bile acids regulate vitamin D metabolism via their actions on renal and bone 25-hydroxyvitamin D-1alpha hydroxylase (CYP27B1). In the first part of this study, the most effective bile acids and their effective time-point and dosages were first identified. Human osteoblastic (MG-63) cells and human renal proximal convoluted tubule HKC-8 cells were treated with 10-8M of lithocholic acid (LCA) for 0, 1, 2, 4, 6 and 8 hours, the potent agonist of TGR5, to identify the effective time-point for activation of CYP27B1 protein expression. The results showed that the protein expressions of CYP27B1 in both HKC-8 and MG-63 cells were best induced by LCA at 4 hours (vs. 0-hour group, P<0.05). The effects of other bile acids at lower concentration on mRNA and protein expression of TGR5 and CYP27B1 in both HKC-8 and MG-63 cells were determined. LCA, deoxycholic acid (DCA), cholic acid (CA), and chenodeoxycholic acid (CDCA) ranging form 10-12M to 10-5 M were used to treat HKC-8 and MG-63 cells for 4 hours. The potency of bile acids in inducing TGR5 expression was found to match with those reported in the literature, in a descending order of LCA>DCA>CA. However, the effective dosages to induce CYP27B1 expression in the two major sites of 1,25(OH)2D3 production, HKC-8 and MG-63 cells, were different. The enzymatic activity of CYP27B1 induced by LCA was measured in both MG-63 and HKC-8 cells. LCA significantly induced 1,25(OH)2D3 production in MG-63 cells at 10-10M (vs. control group, P<0.05) and in HKC-8 cells at 10-6M (vs. control group, P<0.01). Moreover, the role of TGR5 in mediating the action of LCA on TGR5 and CYP27B1 inductions was studied by performing TGR5 knockdown in MG-63 cells. The results showed that knocking down TGR5 by siRNA would lead to the decrease in CYP27B1 protein expression in MG-63 cells. Pre-treatment of MG-63 cells with TGR5 siRNA abolished the effects of LCA (10-10 to 10-9M) on CYP27B1 protein expression. The results suggested that LCA exerted its effect on CYP27B1 through TGR5 receptor in MG-63 cells. In the last part of my study, the in vivo actions of LCA on vitamin D metabolism were investigated. The results showed that administration of LCA (25mg/kg oral gavage) for three consecutive days significantly increased serum 1,25(OH)2D3 level (vs. control group, P<0.01) and induced the protein expression of CYP27B1 in both the kidney and iliac crest (vs. control group, P<0.05) in 6-month-old C57BL/6J male mice. Moreover, a time-dependent change (2, 5, 8 hours) in serum 1,25(OH)2D3 levels was determined upon feeding the mice with a single dose of 25mg/kg LCA. The results showed that serum 1,25(OH)2D3 increased significantly at 2 (vs. control group, P<0.01) and 5 hours (vs. control group, P<0.05) and returned to the basal level at 8 hours in mature male mice. CYP27B1 protein expressions in the kidney and iliac crest were significantly induced in male mice by LCA at 8 hours (vs. control group, P<0.05) but not at 2 or 5 hours. In summary, the present study supports our hypothesis that bile acids, specifically LCA, upregulated TGR5 and CYP27B1 expression to induce 1,25(OH)2D3 production in osteoblasts and renal proximal tubules cells in vitro and in vivo. The study extends our understanding of the effects of bile acids on vitamin D metabolism and provides new insight into the mechanism by which bile acids exert bone protective effects.