1,25-dihydroxyvitamin D₃ dose dependently decreases MC medium-induced phosphorylation of p38 MAPK in human adipocytes.

<p>Adipocytes were incubated with increasing doses of 1,25(OH)₂D₃ (10⁻¹¹ M, 10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M) or without (control) for 72 h, followed by stimulation with macrophage conditioned (MC) medium (25%) for another 24 h. Protein expression of phosphorylated p38 MAPK in cell lysates was analysed by western blotting, with GAPDH used as loading controls. (A) Representative western blots. (B) Signals were quantified by densitometry; data are means ± SEM, normalised to total Akt levels, n = 3 per group. ^†<i>P</i><0.05 vs 10⁻¹¹ M dose group, ***<i>P</i><0.001 vs MC group.</p

1,25-dihydroxyvitamin D₃ reduces monocyte migration.

<p>Human adipocytes growing in maintenance medium were pretreated with 1,25(OH)₂D₃ (10⁻⁸ M) or without (control) for 24 h and the culture medium (150 µl) was harvested. The maintenance medium (without cells) (150 µl) was also collected. The medium was added to the lower chamber of the transwells and THP-1 monocytes (2×10⁶/ml; 100 µl) were placed to the upper chamber of the transwells. After incubation for 4 h at 37°C, the migration of monocytes was determined by the MTT assay. (A) Representative images of monocyte migration. (B) The number of monocytes migrated; data are means ± SEM, n = 3 per group. ***<i>P</i><0.001 vs maintenance medium; ^†††<i>P</i><0.001 vs controls. The results were confirmed by three independent experiments.</p

Effects of 1,25-dihydroxyvitamin D₃ on MC medium-induced expression of the chemokines/cytokines by human adipocytes.

<p>Adipocytes were pretreated with 1,25(OH)₂D₃ (10⁻⁸ M) or without for 48 h, followed by the incubation with RPMI-1640 medium (control) or macrophage conditioned (MC) medium (25%) for another 4 h. mRNA levels of IL-8, MCP-1, RANTES, IL-1β and IL-6 were measured by real-time PCR and normalised to β-actin. Data are means ± SEM, n = 6 per group. ^†††<i>P</i><0.001 vs controls; *<i>P</i><0.05, **<i>P</i><0.01 vs MC group. The results were verified by three independent experiments.</p

Effects of 1,25-dihydroxyvitamin D₃ on MC medium-induced secretion of the chemokines/cytokine by human adipocytes.

<p>Adipocytes were pretreated with 1,25(OH)₂D₃ (10⁻⁸ M) or without for 48 h, followed by the incubation with RPMI-1640 medium (control) or macrophage conditioned (MC) medium (12.5% or 25%) for another 24 h. A separate group (25% MC medium only without cells) was included to show basal levels of chemokine/cytokines in the MC medium. Protein release of IL-8 (A), MCP-1 (B), RANTES (C) and IL-6 (D) was determined using ELISAs in supernatants. Data are means ± SEM, n = 6 per group. ^†††<i>P</i><0.001 vs controls; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs MC group. The results were confirmed by three independent experiments.</p

1,25-dihydroxyvitamin D₃ attenuates MC medium-induced phosphorylation of Erk1/2 in human adipocytes.

<p>Adipocytes were pretreated with 1,25(OH)₂D₃ (10⁻⁸ M) or without for 48 h, followed by incubation with RPMI-1640 medium (control) or macrophage conditioned (MC) medium (25%) for another 24 h. Protein expression of phosphorylated Erk1/2 in cell lysates was analysed by western blotting, with GAPDH used as loading controls. (A) Representative western blots. (B) Signals were quantified by densitometry; data are means ± SEM, normalised to GAPDH levels, n = 3 per group. ^††<i>P</i><0.01 vs controls; ***<i>P</i><0.01 vs MC group. The results were confirmed by three independent experiments.</p

1,25-dihydroxyvitamin D₃ reduces MC medium-induced phosphorylation of p38 MAPK in human adipocytes.

<p>Effect of 1,25(OH)₂D₃ on basal level of p38 MAPK was studied in adipocytes incubated with vitamin D₃ (10⁻¹¹ M and 10⁻⁸ M) or without (control) for 72 h. (A) Phosphorylated p38 MAPK protein content in cell lysates was analysed by western blotting, with GAPDH used as loading controls. (B) Signals were quantified by densitometry. Effect of 1,25(OH)₂D₃ on MC medium-induced phosphorylation of p38 MAPK was studied in adipocytes pretreated with 1,25(OH)₂D₃ (10⁻⁸ M), followed by incubation with RPMI-1640 medium (control) or macrophage conditioned (MC) medium (25%) for another 6 h. Protein expression of phosphorylated p38 MAPK in cell lysates was analysed by western blotting. (C) Representative western blots. (D) Signals were quantified by densitometry. Data are means ± SEM, normalised to GAPDH levels, n = 3 per group. ^†<i>P</i><0.05, ^†††<i>P</i><0.001 vs controls; **<i>P</i><0.01 vs MC group. The results were confirmed by three independent experiments.</p

Schematic diagram of mechanisms of 1,25-dihydroxyvitamin D₃ action in human adipocytes.

<p>1,25(OH)₂D₃ has an inhibitory effect on the activation of the NFκB and MAPK signalling pathways, with increased IκBα expression while decreased phosphorylation of NFκB p65, p38 MAPK and Erk1/2. Consequently, there is a reduction in gene transcription and protein release of proinflammatory chemokines/cytokines, such as IL-8, MCP-1, RANTES and IL-6, by adipocytes, which may lead to reduced chemotaxis of monocytes/macrophages and adipose tissue inflammation.</p