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Regulation of TNF--induced IL-6 Production in MG-63 Human Osteoblast-like Cells

¹ Formerly Dept. of Oral Biology, UNMC, and presently Dept. of Orthodontics, The Ohio State University, Columbus, OH;
² formerly Dept. of Oral Biology, UNMC, and presently in private practice, Roseburg, OR;
³ Dept. of Oral Biology, UNMC, 40th & Holdrege Sts., Lincoln, NE 68583;

View larger version (13K): [in a new window]   Figure 1. Topographic representation of the human IL-6 gene promoter (phIL-6 WT) along with 5'deletions phIL-6A (-490 to +10) and phIL-6B (-260 to +10).

View larger version (13K): [in a new window]   Figure 2. TNF- stimulates IL-6 secretion by MG-63 cells. (Panel A) Dose-response for 24-hour and 48-hour TNF- stimulation. (Panel B) Secretion of IL-6 after pre-incubation with p38 MAPK inhibitor SB203580HCl for 1 hr prior to stimulation with TNF- (20 ng/mL) for 24 hrs. *Statistically different from control (unstimulated) cultures (p < 0.05). Bars represent mean ± standard deviation (n = 4).

View larger version (13K): [in a new window]   Figure 3. Activation of p38 MAPK by TNF-. (Panel A) Time course (min) of TNF- stimulation (20 ng/mL). (Panel B) Inhibition of p38 MAPK phosphorylation by SB203580 HCl. Cells serum-starved overnight, pre-incubated with SB203580HCl (10 µM) for 1 hr, followed by TNF- stimulation (20 ng/mL) for 5 min.

View larger version (14K): [in a new window]   Figure 4. TNF- stimulates the IL-6 gene promoter via p38 MAPK, NFB, and C/EBPß. (Panel A) MG-63 cells were transiently transfected with respective plasmid (phIL-6 wild-type [phIL-6WT] or 5'-deletion constructs [phIL-6A and phIL-6B]), serum-starved for 24 hrs, and then stimulated with TNF- (10 ng/mL) for 24 hrs. p38 MAPK inhibition was achieved by pre-incubation with SB203580HCl (10 µM) for 1 hr immediately prior to TNF- stimulation (10 ng/mL). Luciferase activity within cell lysates was measured and corrected for transfection efficiency. *Statistically significant difference from TNF--stimulated cultures (p < 0.05). Bars represent mean ± standard deviation (n = 3), relative to control (unstimulated cultures). (Panel B) TNF- modulates NFB- and C/EBPß-sensitive promoters via p38MAPK. Cells were transiently transfected with respective plasmid (or vector only), serum-starved for 24 hrs, and then stimulated with TNF- (10 ng/mL) for 24 hrs. p38 MAPK inhibition was achieved by pre-incubation with SB203580HCl (10 µM) for 1 hr immediately prior to TNF- stimulation. Luciferase activity within cell lysates was measured and corrected for transfection efficiency. *Statistically significant difference from control (unstimulated) cultures (p < 0.05). Bars represent mean ± standard deviation (n = 4).