Metabolism of vitamin D3 in human osteoblasts: Evidence for autocrine and paracrine activities of 1α,25-dihydroxyvitamin D3
Introduction
The vitamin D endocrine system plays a principal role in the maintenance of calcium and phosphate homeostasis. There are considerable data to indicate that 1α,25-dihydroxyvitamin D3 (1,25D) is the biologically active metabolite of vitamin D [1], with a relatively high affinity for the nuclear vitamin D receptor (VDR). 25-Hydroxyvitamin D3 (25D) is the major circulating metabolite of vitamin D and blood levels of this metabolite serve as the best indicator for vitamin D status. 25D is the immediate metabolic precursor of 1,25D, and is considered to exert biological effects only upon its conversion to 1,25D [1]. Hydroxylation of vitamin D metabolites at the carbon 24 position by the 25-hydroxyvitamin D-24-hydroxylase (CYP24) is the first step in their inactivation and excretion. Basal expression of CYP24 is extremely low but the gene is highly induced by 1,25D [2].
1,25D is essential for the maintenance of a healthy skeleton. Vitamin D deficiency in animals and humans produces defects in bone mineralization, such as rickets and osteomalacia, diseases characterized by an increase in osteoid and impaired calcium phosphate deposition [3]. Similar defects were produced by ablation of the genes CYP27B1, which encodes the 25-hydroxyvitamin D-1α-hydroxylase [4] or VDR [5], [6]. Short-term studies with the CYP27B1 and VDR gene ablation models suggest that a high calcium and phosphate diet can normalize bone mineralization [5], [6], [7]. However, long-term studies with adult CYP27B1-null or VDR-null/CYP27B1-null mice revealed persistent bone growth deficiencies and decreased bone mineral even when normocalcemia was restored [8], [9]. These results indicate that 1,25D plays an essential role to optimize skeletal health.
1,25D affects many aspects of bone cell biology. Numerous in vitro studies have implicated 1,25D in the regulation of both osteoblastic and osteoclastic activity [10], [11], and 1,25D thus exerts effects during both the resorptive and synthetic phases of bone remodeling. A current view is that 1,25D stimulation of osteoclast-mediated bone resorption is a mechanism by which normocalcemia is maintained, however alternative mechanisms, such as osteoblast mediated release of bone matrix calcium [12], have also been suggested. 1,25D has been demonstrated to regulate osteoblast gene transcription, proliferation, differentiation and mineralization [13], [14], [15]. We have shown in vitro that human osteoblasts respond to exogenous 1,25D by decreasing their rate of proliferation, as well as increasing their expression of mRNA species encoding osteocalcin, bone sialoprotein-1, and RANKL [15].
It is now clear that while circulating levels of 1,25D derive from the kidney [16], [17], [18], there exist numerous sites of extra-renal 1,25D synthesis, including the skin [19], [20], [21], liver [22], lymph nodes [16], activated monocytes/macrophages [23], [24] and dendritic cells [25], among other tissues [26]. Local production of 1,25D in extra-renal tissues has been postulated to regulate parameters of cell growth and differentiation in an autocrine or paracrine fashion [19], [27]. However, under normal conditions, such expression does not appear to contribute to circulating levels of 1,25D [28]. Early reports suggested that osteoblast-like cells could synthesize 1,25D [29], [30], however, a detailed molecular analysis of this has not until now been performed, and the implications for osteoblast activity have not been described. As a result, this potentially important activity of 1,25D has not been widely recognized. Recently, we demonstrated that the age-related regulation of CYP27B1 and CYP24 mRNA expression in rat bone is distinct from that in the kidney and is positively associated with bone mineralization [31]. In addition, rat growth plate chondrocytes have been shown to display 1α-hydroxylase activity [32] and express CYP27B1 mRNA [33].
Of major clinical significance is the increased risk of hip fracture in the elderly with a low vitamin D status. An association between low vitamin D status and increased risk of hip and other fractures has been demonstrated at high and moderate latitudes [34], [35], [36]. However the ability of vitamin D and calcium supplementation to reduce the risk of hip fracture by 30% to 40% in ambulant, elderly French women demonstrated a causal role of low vitamin D status in this syndrome [37]. The mechanism of this effect is unclear. As circulating 25D levels decrease, particularly below 40 nmol/L, circulating 1,25D and parathyroid hormone levels initially increase so that blood calcium levels remain unchanged [38], [39]. The average 25D level for hip fracture patients has been reported within this range at 40 nmol/L [35] and therefore it is possible that the low levels of circulating 25D are more important in this syndrome than circulating 1,25D levels.
We demonstrate here that both normal human osteoblast-like cells (NHBC) and a number of well-characterized human osteosarcoma (OS) cell lines, MG-63, SAOS-2, HOS and G-292, express mRNA encoding CYP27B1 and respond to exogenous 25D and 1,25D by upregulation of CYP24 mRNA. We demonstrate that these cells metabolize 25D into 1,25D and secrete detectable levels of de novo synthesized 1,25D. Consistent with its conversion to 1,25D, exposure to physiological levels of 25D (20–100 nM) induced CYP24, osteocalcin, osteopontin and RANKL mRNA expression. Exposure to 25D also promoted the recognized 1,25D effects of inhibition of osteoblast proliferation and promotion of in vitro mineralization, suggesting that endogenous 1,25D synthesis likely has autocrine functional consequences for osteoblasts. Blocking CYP27B1 prevented cellular responses to 25D. Our data strongly imply that vitamin D metabolism in human osteoblasts represents a physiologically important pathway.
Section snippets
Cells and culture media
Human primary osteoblasts (normal human bone-derived cells; NHBC) were isolated from femoral neck trabecular bone and passaged, as we have previously described [15]. While these cells are heterogeneous in nature with respect to their differentiation stage [40], they display many of the properties associated with osteoblasts, such as formation of a mineralized matrix [40] and support of osteoclast differentiation [41]. Different NHBC donors' cells were used for each part of this study, unless
Expression of CYP27B1, VDR and CYP24 mRNA in primary human osteoblasts
CYP27B1 and VDR mRNA were expressed by all NHBC tested, and levels were not consistently changed by 72 h exposure to exogenous 1,25D (Figs. 1A–B). Significant decreases in CYP27B1 mRNA expression in response to 1,25D occurred in two donors' cells (NHBC1 and NHBC4, p < 0.03), but levels increased in the case of NHBC3 (p < 0.01), while no effect was seen for the other two donors' cells. Similarly, VDR mRNA levels significantly decreased in response to 1,25D in the case of NHBC4 and NHBC5 (p < 0.01),
Discussion
In this study, we confirm that human primary osteoblasts and human osteoblastic cell lines possess the molecular machinery to both respond to and metabolize vitamin D3. These cells were found to constitutively express CYP27B1 mRNA, which was demonstrated to be translated to functional enzyme, as provision of cells with 25D resulted in the secretion of 1,25D, detectable in the picomolar range. The regulated expression of CYP27B1 and CYP24 are important for the maintenance of appropriate
Acknowledgments
The authors wish to thank the surgeons and nursing staff of the Dept. of Orthopaedics and Trauma, Royal Adelaide Hospital, for the provision of surgical bone samples. The authors thank Dr. P. Diamond, IMVS, for providing the PCR primers used for detection of GAPDH and K.Y. Kun for technical help. This work was supported by grants from the National Health and Medical Research Council of Australia, Eli Lilly, Eisai Japan, Osteoporosis Australia Foundation and the Dept. of Orthopaedics and Trauma,
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