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  • Title: PI3K/Akt mediates expression of TNF-alpha mRNA and activation of NF-kappaB in calyculin A-treated primary osteoblasts.
    Author: Qiu L, Zhang L, Zhu L, Yang D, Li Z, Qin K, Mi X.
    Journal: Oral Dis; 2008 Nov; 14(8):727-33. PubMed ID: 19193202.
    Abstract:
    OBJECTIVE: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor-alpha (TNF-alpha) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. MATERIALS AND METHODS: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF-alpha and GAPDH mRNA were determined by RT-PCR. Cell extracts were subjected to SDS-PAGE and the activation of Akt and NF-kappaB were analyzed by western blotting. RESULTS: Calyculin A-treatment markedly increased the expression of TNF-alpha mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF-alpha mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF-kappaB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. CONCLUSION: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF-alpha mRNA and activation of NF-kappaB. This NF-kappaB activation involves both phosphorylation and nuclear translocation of NF-kappaB.
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