Risk factors for GC-DM.

<p>The multivariate analysis revealed an older age (≥45 years) and a family history of diabetes to be independent risk factors for the development of GC-DM during hospitalization. In a logistic regression analysis, older age (≥45 years) and a family history of diabetes emerged as independent risk factors for the development of GC-DM (odds ratio [OR], 6.3 and 95% confidence interval [CI], 1.6–27.6; OR, 4.4 and 95% CI, 1.2–16.6, respectively).</p

Comparison of the baseline characteristics of the patients with and without GC-DM treatment at one year.

<p>Comparison of the baseline characteristics of the patients with and without GC-DM treatment at one year.</p

Comparison of the baseline characteristics of the patients with and without GC-DM during hospitalization.

<p>Comparison of the baseline characteristics of the patients with and without GC-DM during hospitalization.</p

Schematic illustration of the protocol.

<p>Patients received methylprednisolone (mPSL) pulse (500 mg daily) administered intravenously for 3 consecutive days followed by oral prednisolone (30 mg daily) on 4 consecutive days, with the course repeated 3 times during hospitalization. Oral prednisolone (30 mg) was then given on every alternate day and gradually tapered and discontinued at 1 year.</p

ED71 or 1,25(OH)₂D₃ activity requires the VDR.

<p>(<b>A, B</b> and <b>C</b>) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type (WT) or VDR-deficient (VDR KO) mice and cultured in the presence of M-CSF alone (50 ng/ml) or M-CSF + RANKL (25 ng/ml) with or without indicated concentrations of ED71 or 1,25(OH)₂D₃ for 5 days. Cells were then stained with TRAP (<b>A</b>), and multi-nuclear TRAP-positive cells were counted (<b>B</b>). Expression of <i>Ctsk</i>, <i>NFATc1</i> and <i>DC-STAMP</i> was assessed by realtime PCR (<b>C</b>). Data represent mean <i>Ctsk</i>, <i>NFATc1</i> or <i>DC-STAMP</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5).</p

1,25(OH)₂D₃ is a more potent inhibitor of osteoclastogenesis <i>in vitro</i> than is ED71.

<p>(<b>A, B</b> and <b>C</b>) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type mice and cultured in the presence of M-CSF (M, 50 ng/ml) + RANKL (R, 25 ng/ml) with or without indicated concentrations of ED71 or 1,25(OH)₂D₃ (1,25D) for 5 days. Cells were then stained with TRAP (<b>A</b>) and the number of multi-nuclear TRAP-positive cells was counted (<b>B</b>). Expression of <i>Ctsk</i>, <i>NFATc1</i> and <i>DC-STAMP,</i> all of which are osteoclastic genes, was analyzed by realtime PCR (<b>C</b>). Expression of <i>Blimp1</i>, <i>Bcl6</i> and <i>Irf8</i> was analyzed by realtime PCR (<b>D</b>). Data represent mean expression of each relative to <i>Actb</i> ± SD (<i>n</i> = 5). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; NS, not significant.</p

HIF1α protein is suppressed by ED71 but not by 1,25(OH)₂D₃.

<p>(<b>A</b>) Western analysis of Raw264.7 cells cultured in hypoxic conditions with or without 10⁻⁷ M of ED71 or 1,25(OH)₂D₃ (1,25D). (<b>B</b>) <i>Hif1α</i> mRNA levels in Raw264.7 cells cultured in hypoxic conditions were analyzed by realtime PCR in the presence or absence of 10⁻⁷ M ED71 or 1,25(OH)₂D₃. Data represent mean <i>Hif1α</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). (<b>C</b>) Levels of <i>VDR</i> transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean <i>VDR</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). (<b>D</b>) Western analysis of control (shControl) or VDR-suppressed (shVDR#1 or shVDR#2) Raw264.7 transformants cultured in hypoxic conditions with ED71 or 1,25(OH)₂D₃ (1,25D), both at 10⁻⁷ M. (<b>E</b>) M-CSF-dependent Ctsk Cre/<i>Hif^flox/flox</i> cells were cultured in normoxic conditions to suppress HIF1α in the presence of M-CSF (50 ng/ml) plus RANKL (25 ng/ml) with either ED71 or 1,25(OH)₂D₃ (1,25D) both at 10⁻⁷M for 4 days. Expression of <i>Ctsk</i> and <i>NFATc1</i> was then assessed by realtime PCR. Data represent mean <i>Ctsk</i> or <i>NFATc1</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

1,25(OH)₂D₃ is more active in promoting c-Fos protein inhibition and <i>Ifnβ</i>-induction in osteoclasts compared with ED71.

<p>(A and B) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type mice and cultured in the presence of M-CSF alone (50 ng/ml) or M-CSF + RANKL (25 ng/ml) with or without 10⁻⁷ M ED71 or 1,25(OH)₂D₃ (1,25D) for 5 days. c-Fos protein was then assessed by western blot (A). <i>Ifnβ</i> expression was analyzed by realtime PCR (B). Data represent mean <i>Ifnβ</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). ***<i>P</i><0.001.</p