25-Hydroxy- and 1α,25-Dihydroxycholecalciferol Have Greater Potencies than 25-Hydroxy- and 1α,25-Dihydroxyergocalciferol in Modulating Cultured Human and Mouse Osteoblast Activities

<div><p>Despite differences in the phamacokinetics of 25-hydroxycholecalciferol (25(OH)D₃) and 25-hydroxyergocalciferol (25(OH)D₂) in man, the effects of these and their 1α-hydroxylated forms (1,25(OH)₂D₃ and 1,25(OH)₂D₂) on cellular activity of vitamin D-responsive cells have hardly been compared. We studied differences in the effects of these metabolites on cell number, gene transcription, protein expression and mineralisation of cultured human bone marrow-derived stromal cells (hBMSC) and rapidly mineralising mouse 2T3 osteoblasts. 50–1000 nM 25(OH) and 0.05–10 nM 1,25(OH)₂ metabolites were used. At high concentrations, 25(OH)D₂/D₃ and 1,25(OH)₂D₂/D₃ suppressed cell number in both human and mouse cells. The suppression was greater with cholecalciferol (D₃) metabolites than with those of ergocalciferol (D₂). In both cell types, 25(OH)D₂ and 25(OH)D₃ increased the expression of osteopontin, osteocalcin, collagen-1, receptor activator of nuclear factor kappa-B ligand, vitamin D receptor, CYP24A1 and CYP27B1 genes. Whereas there was little or no difference between the effects of 25(OH)D₂ and 25(OH)D₃ in hBMSCs, differences were observed in the magnitude of the effects of these metabolites on the expression of most studied genes in 2T3 cells. Alkaline phosphatase (ALP) activity was increased by 25(OH)D₂/D₃ and 1,25(OH)₂D₂/D₃ in hBMSC and 2T3 cells, and the increase was greater with the D₃ metabolites at high concentrations. In hBMSCs, mineralisation was also increased by 25(OH)D₂/D₃ and 1,25(OH)₂D₂/D₃ at high concentrations, with D₃ metabolites exerting a greater influence. In 2T3 cells, the effects of these compounds on mineralisation were stimulatory at low concentrations and inhibitory when high concentrations were used. The suppression at high concentrations was greater with the D₃ metabolites. These findings suggest that there are differences in the effects of 25-hydroxy and 1α,25(OH)₂ metabolites of D₃ and D₂ on human preosteoblasts and mouse osteoblasts, with the D₃ metabolites being more potent in suppressing cell number, increasing ALP activity and influencing mineralisation.</p></div

Effects of 25(OH)D₂ and 25(OH)D₃ on 2T3 cell gene expression.

<p>Histograms depict the comparative effects of 25(OH)D₂ and 25(OH)D₃ on the expression of mouse 2T3 osteoblast signature genes, <i>opg</i> (A), <i>rankl</i> (B), <i>col1a</i> (C), <i>opn</i> (D), <i>ocn</i> (E), <i>vdr</i> (F), <i>cyp24a1</i> (G) <i>and cyp27b1</i> (H). 2T3 cells were cultured for 24 h in the presence or absence of 200 nM of either 25(OH)D₂ or 25(OH)D₃ in differentiation media. Quantified values (RQ) have been normalised to <i>gapdh</i> expression and are mean ± SEM from triplicate experiments. # <i>P</i><0.05, ## <i>P</i><0.01 and ### <i>P</i><0.001 for comparisons between 25(OH)D₂ or 25(OH)D₃ treatments and the control (vehicle); * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 for comparisons between 25(OH)D₂ and 25(OH)D₃. Western blots showing relative amounts of VDR, CYP27B1, OPN and OCN proteins in 2T3 cell lysates (20 μg protein) after 8 days of treatment with (I) 100–500 nM of either 25(OH)D₂ (D₂) or 25(OH)D₃ (D₃), and (J) 0.1–0.5 nM of either 1,25(OH)₂D₂ (1D₂) or 1,25(OH)₂D₃ (1D₃).</p

Comparative effects of 25(OH)D₂, 25(OH)D₃, 1,25(OH)₂D₂ and 1,25(OH)₂D₃ on cell numbers.

<p>Human BMSCs (panels A&B) and 2T3 cells (panels C&D) received separate treatments over 24 h with either 50–1000 nM 25(OH)D₂/D₃ or 0.05–1 nM 1,25(OH)₂D₂/D₃. Cell numbers were measured by MTS. Mean ± SEM percentage values from triplicate experiments have been presented as fluorescence units (RFU) from treated cells relative to untreated control (vehicle). # <i>P</i><0.05, ## <i>P</i><0.01, ### <i>P</i><0.001 for comparisons between the treatments and the control; * <i>P</i><0.01 for comparisons between the D₂ and D₃ metabolites.</p

Effects of 25(OH)D₂, 25(OH)D₃, 1,25(OH)₂D₂ and 1,25(OH)₂D₃ on mineralisation.

<p>Human BMSCs cells were cultured in differentiation media for 21 days in the presence of either 500–1000 nM 25(OH)D₂/D₃ (A) or 1–10 nM 1,25(OH)₂D₂/D₃ (B), stained with alizarin red and photographed. Quantification of mineralisation was by alizarin red stain extraction and colourimetry, and the effects of treatment of hBMSCs with either 500–1000 nM 25(OH)D₂/D₃ (C) or 0.1–10 nM 1,25(OH)₂D₂/D₃ (D) are presented. Values are mean ± SEM from triplicate experiments as percentage of untreated controls (vehicle). 2T3 cells were cultured in differentiation media for 8 days in the presence of either 100–500 nM 25(OH)D₂/D₃ (E) or 0.1–0.5 nM 1,25(OH)₂D₂/D₃ (F). Alizarin red staining of 2T3 was quantified and the responses to 100–500 nM 25(OH)D₂/D₃ (G) or 0.1–0.5 nM 1,25(OH)₂D₂/D₃ (H) are shown as mean ± SEM from triplicate experiments and as percentage of untreated controls. # <i>P</i><0.05, ## <i>P</i><0.01 and ### <i>P</i><0.001 for comparisons between the treatments and the control; * <i>P</i><0.05 and ** P<0.001 for comparisons between the D₂ and D₃ metabolites.</p

Representative immunofluorescence staining images of cytospin slides of healthy peripheral blood M1 and M2 MDM.

<p>Immunofluorescence staining of cytospin slides of healthy peripheral blood MDM with the nuclear stain POPO-1 (to confirm presence of cells), TSPO, the macrophage marker CD68 and merging of the three stains. <b>(A)</b> staining of MDM stimulated with LPS plus IFN<i>-</i>γ for 24 hours; <b>(B)</b> staining of MDM stimulated with IL-4 for 24 hours. x400 magnification. Scale bar = 20μm.</p

Western blotting indicates TSPO protein expression decreases on treatment of MDM with M1 stimuli LPS plus IFN-γ.

<p>(i) representative western blot of TSPO protein with β-actin acting as loading control and (ii) densitometry normalized to β-actin in (<b>A)</b> THP-1 MDM, (<b>B)</b> healthy peripheral blood MDM and (<b>C)</b> synovial fluid MDM either unstimulated (<i>unstim</i>) or treated with 10ng/mL LPS plus 20ng/mL IFN-γ (<i>LPS</i>) for 2,4,6 and 24 hours. Densitometry data is expressed as the mean of five independent experiments ± SEM. (*<i>p</i><0.05, **<i>p</i> <0.01, ***<i>p</i>≤0.001).</p

M1:M2 ratio of mean fold change of macrophage mRNA markers.

<p>M1:M2 ratio of mean fold change of macrophage mRNA markers.</p

TSPO protein expression is higher in MDM than in monocytes.

<p>TSPO protein was assessed by Western blot with β-actin as loading control <b>(Ai)</b>, with accompanying densitometry normalised to β-actin <b>(Aii)</b>. Results shown are for THP-1 monocytes, healthy peripheral blood monocytes, synovial fluid monocytes, and corresponding MDM. <b>(B)</b> [³H]PBR28 tracer binding per 1x10⁶ cells in healthy human peripheral blood monocytes and MDM. Each experiment was run in triplicate, and data expressed as mean± SEM for five independent experiments. *** p<0.001 using Student’s <i>t</i>-test, comparing data from each MDM with that of their counterpart monocytes.</p

Binding of the TSPO specific radio-ligand [³H]PBR28 in unstimulated, M1 and M2 healthy human peripheral blood MDM.

<p>Binding of [³H]PBR28 in unstimulated healthy peripheral blood MDM, in MDM stimulated with LPS plus IFN-γ, and MDM stimulated with IL-4 for 24 hours. Each experiment was performed in triplicate, with data expressed as mean ± SEM of five independent experiments. ** p<0.01 compared to unstimulated.</p

The macrophage marker translocator protein (TSPO) is down-regulated on pro-inflammatory ‘M1’ human macrophages

<div><p>The translocator protein (TSPO) is a mitochondrial membrane protein, of as yet uncertain function. Its purported high expression on activated macrophages, has lent utility to TSPO targeted molecular imaging in the form of positron emission tomography (PET), as a means to detect and quantify inflammation <i>in vivo</i>. However, existing literature regarding TSPO expression on human activated macrophages is lacking, mostly deriving from brain tissue studies, including studies of brain malignancy, and inflammatory diseases such as multiple sclerosis. Here, we utilized three human sources of monocyte derived macrophages (MDM), from THP-1 monocytes, healthy peripheral blood monocytes and synovial fluid monocytes from patients with rheumatoid arthritis, to undertake a detailed investigation of TSPO expression in activated macrophages. In this work, we demonstrate a consistent down-regulation of TSPO mRNA and protein in macrophages activated to a pro-inflammatory, or ‘M1’ phenotype. Conversely, stimulation of macrophages to an M2 phenotype with IL-4, dexamethasone or TGF-β1 did not alter TSPO expression, regardless of MDM source. The reasons for this are uncertain, but our study findings add some supporting evidence for recent investigations concluding that TSPO may be involved in negative regulation of inflammatory responses in macrophages.</p></div