24R,25-dihydroxyvitamin D3 promotes the osteoblastic differentiation of human mesenchymal stem cells

Abstract

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.

Figures

Figure 1.

Schematic representation depicting vitamin D3 metabolite treatment(s) and subsequent time points for analysis during in vitro osteoblastic differentiation.

Figure 2.

24R,25(OH)2D3 regulates the osteoblastic differentiation of hMSCs. 24R,25(OH)2D3 (10 nM) and/or 1α,25(OH)2D3 (10 nM) were added at day 0 and every 3 days when medium was changed (Figure 1). A and B, hMSCs were cultured under expansion conditions (seeded at 500–1000 cells/cm2) and collected for cell counting (A) or BrdU incorporation (B). B, BrdU incorporation was assayed at days 3 and 5. Relative fold change compared with day 3 control samples is shown (set to equal 1). Abs, absorbance. C, hMSCs were cultured in osteogenic medium (OG) with no dexamethasone, unless otherwise stated (seeded at 10 000 cells/cm2) and assayed for alkaline phosphatase activity. pNP, p-nitrophenol. D, hMSCs were seeded at 10 000 cells/cm2 in osteogenic medium with or without dexamethasone (Dex; 10 nM). Alizarin Red S stain was used to determine Ca2+ mineralization. ★, P ≤ .05 compared with the respective “linked samples” (A and B) or relative to the respective day controls (B).

Figure 3.

24R,25(OH)2D3 and 25(OH)D3 but not 1α,25(OH)2D3 induces Ca2+ mineralization in the absence of dexamethasone. A and B, hMSCs were seeded at 10 000 cells/cm2 in osteogenic medium (OG) with no dexamethasone, unless otherwise stated, and 5% FBS. Treatments were started at day 0, and medium was changed every 3 days (Figure 1). Alizarin Red S stain was used to assess Ca2+ mineralization at day 14. B, Cytochrome P450 inhibitor ketoconazole (10 μM) was added 1 hour before treatment or medium change at day 0 and at every medium change. C, Quantification of Ca2+ mineralization (from A): cetylpyridinium chloride was used to solubilize Alizarin Red S staining, and the OD550 readings were normalized against total protein for each well (per milligram of protein) to account for differences in cell number. D, Quantification of Ca2+ mineralization (from B) with addition of ketoconazole. n = ≥3 independent experiments. ★, P ≤ .05 compared with osteogenic conditions without ketoconazole. 1α,25-, 1α,25(OH)2D3.

Figure 4.

24R,25(OH)2D3 decreased 1α,25(OH)2D3-stimulated genes: osteocalcin and VDR. A–C, Using RT-qPCR, the mRNA expression of osteogenic genes was assessed after vitamin D3 treatment up to the time of mineralization (day 14). hMSCs were seeded at 10 000 cells/cm2. A 10 nM concentration was used for all vitamin D3 metabolites. Treatments started at day 0, and medium was change every 3 days (Figure 1). B, Western blot analysis of the 1α,25(OH)2D3 receptor (VDR) levels. α-Tubulin was used as a loading control. n = 3 independent experiments in triplicate. ★, P ≤ .05 compared to the respective day control. 1α,25-, 1α,25(OH)2D3.

Figure 5.

24R,25(OH)2D3 decreases 25(OH)D3-1α-hydroxylase (CYP27B1), whereas 1α,25(OH)2D3 increases 25-hydroxylase (CYP2R1) expression. With use of RT-qPCR, mRNA expression of the cytochrome P450 genes (CYP27A1 and CYP2R1, 25-OHases [A and C]; CYP27B1, 1α-OHase [B]; and CYP24A1, 24-OHase [D]) involved in vitamin D3 metabolism was assessed after vitamin D3 treatment up to the time of mineralization (day 14). hMSCs were seeded at 10 000 cells/cm2. A 10 nM concentration was used for all vitamin D3 metabolites. Treatments started with day 0, and medium was changed every 3 days (Figure 1). ★, P ≤ .05 compared with the respective day control. 1α,25-, 1α,25(OH)2D3.

Figure 6.

24R,25(OH)2D3 decreases the biosynthesis of 1α,25(OH)2D3 from 25(OH)D3 in hMSCs. A, Diagram of the cytochrome P450 hydroxylases (OHases) involved in vitamin D3 metabolism. B, Biosynthesis of 1α,25(OH)2D3 from 25(OH)D3 was assessed after 14 days of 24R,25(OH)2D3 pretreatment, with treatments on day 0 and every 3 days (Figure 1). hMSCs were seeded at 10 000 cells/cm2. Medium was collected 12, 24, and 48 hours after 25(OH)D3 (1 μM) stimulation and assayed for 1α,25(OH)2D3. The rate of 1α,25(OH)2D3 biosynthesis is represented as femtomoles of 1α,25(OH)2D3, normalized against total cellular protein per hour, and is the average from the 12-, 24-, and 48-hour time points. Normalization against total cellular protein (per milligram of protein) was used to account for changes in cell number over time. N.D., none detected. C, RT-qPCR analysis was used to assess the levels of cytochrome P450s (CYP24A1, 24-OHase; CYP27A1 and CYP2R1, 25-OHases; and CYP27B1, 1α-OHase), after pretreatment with 24R,25(OH)2D3 (10 nM) for 14 days under confluent conditions with treatments on day 0 and every 3 days. D, RT-qPCR analysis was used to assess the level of 24-OHase (CYP24A1) mRNA expression after treatment with 24R,25(OH)2D3 (1 pM, 10 pM, 0.1 nM, 1 nM, and 10 nM) for 7 days. Treatments started at day 0 and continued every 3 days. ★, P ≤ .05 compared with the untreated control.

Figure 7.

24R,25(OH)2D3 decreases whereas 1α,25(OH)2D3 increases ROS production. ROS production in the culture media was measured after 14 days of osteoblastic differentiation conditions (osteogenic medium [OG]). hMSCs were seeded at 10 000 cells/cm2. Treatments were started at day 0 and continued every 3 days (A and B) (Figure 1). B, Cytochrome P450 inhibitor ketoconazole (10 μM) was added 1 hour before the treatment/medium change. The medium was changed to serum-free conditions 24 hours before the collection for ROS measurement. The medium was passed through a 10-kDa exclusion filter. The flowthrough was used for analysis of ROS. ★, P ≤ .05 compared with the no ketoconazole treatment; #, P ≤ .05 comparing 1α,25(OH)2D3 vs the combination of 1α,25(OH)2D3 + 24R,25(OH)2D3 treatment groups; $, P ≤ .05 comparing treatments vs the untreated osteogenic control.