PI3K/Akt mediates expression of TNF-alpha mRNA and activation of NF-kappaB in calyculin A-treated primary osteoblasts.
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.


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.


CONCLUSIONS
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.
Qiu
L
L
Department of Endodontics, China Medical University, Shenyang, China. drqlh@yahoo.com
Zhang
L
L
Zhu
L
L
Yang
D
D
Li
Z
Z
Qin
K
K
Mi
X
X
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects; D008387:Marine Toxins; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000187:drug effects*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000187:drug effects*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000187:drug effects*
D002867:Chromones; D004791:Enzyme Inhibitors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008387:Marine Toxins; D009025:Morpholines; D016328:NF-kappa B; D010080:Oxazoles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C059041:calyculin A