lithium-chloride and lactacystin

It is recognized that Wnt pathways regulate bone metabolism. We have previously shown that tumor necrosis factor-α (TNF-α) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase)/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TNF-α-stimulated IL-6 synthesis in these cells. Wnt3a, which alone did not affect the IL-6 levels, significantly suppressed the TNF-α-stimulated IL-6 release. Lithium Chloride (LiCl), which is an inhibitor of GSK3β, markedly reduced the TNF-α-stimulated IL-6 release, similar to the results with Wnt3a. The suppression by Wnt3a or LiCl was also observed in the intracellular protein levels of IL-6 elicited by TNF-α. Wnt3a failed to affect the TNF-α-induced phosphorylation of p44/p42 MAP kinase, Akt, IκB or NFκB. Either Wnt3a or LiCl failed to reduce, rather increased the IL-6 mRNA expression stimulated by TNF-α. Lactacystin, a proteasome inhibitor, and bafilomycin A1, a lysosomal protease inhibitor, significantly restored the suppressive effect of Wnt3a on TNF-α-stimulated IL-6 release. Taken together, our results strongly suggest that Wnt3a regulates IL-6 release stimulated by TNF-α at post-transcriptional level in osteoblasts.

Topics: Acetylcysteine; Animals; Cell Line; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; I-kappa B Proteins; Interleukin-6; Lithium Chloride; Macrolides; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Osteoblasts; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Messenger; Tumor Necrosis Factor-alpha; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

In cultured bovine adrenal chromaffin cells, 12-h treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser(9) phosphorylation of GSK-3beta by approximately 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by approximately 38 and approximately 62% in a concentration-dependent manner. Treatment with SB216763 (0.1-30 microM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by approximately 38 and approximately 48%, while increasing beta-catenin protein level by approximately 47%, due to the prevention of GSK-3-induced degradation of beta-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser(9) phosphorylation of GSK-3beta by approximately 104%, while decreasing IRS-1 and IRS-2 levels by approximately 41 and approximately 72%; the insulin-induced Ser(9) phosphorylation of GSK-3beta, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin beta-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by approximately 100 and approximately 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3beta in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.

Topics: Acetylcysteine; Adjuvants, Immunologic; Adrenal Glands; Animals; Blotting, Northern; Cattle; Cells, Cultured; Chromaffin Cells; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Gene Expression Regulation; Glycogen Synthase Kinase 3; Indoles; Lactones; Lithium Chloride; Maleimides; Phosphoproteins; Phosphorylation; Proteasome Endopeptidase Complex; Serine; Time Factors