Tumor necrosis factor-alpha inhibits expression of CTP:phosphocholine cytidylyltransferase.
We investigated the effects of tumor necrosis factor alpha (TNFalpha), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNFalpha significantly inhibited [(3)H]choline incorporation into PtdCho after 24 h of exposure. TNFalpha reduced the activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-regulatory enzyme within the CDP-choline pathway, by 40% compared with control, but it did not alter activities of choline kinase or cholinephosphotransferase. Immunoblotting revealed that TNFalpha inhibition of CCT activity was associated with a uniform decrease in the mass of CCTalpha in total cell lysates, cytosolic, microsomal, and nuclear subfractions of MLE cells. Northern blotting revealed no effects of the cytokine on steady-state levels of CCTalpha mRNA, and CCTbeta mRNA was not detected. Incorporation of [(35)S]methionine into immunoprecipitable CCTalpha protein in pulse and pulse-chase studies revealed that TNFalpha did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCTalpha. Addition of N-acetyl-Leu-Leu-Nle-CHO (ALLN), the calpain I inhibitor, or lactacystin, the 20 S proteasome inhibitor, blocked the inhibition of PtdCho biosynthesis mediated by TNFalpha. TNFalpha-induced degradation of CCTalpha protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNFalpha exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNFalpha inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.
Mallampalli
R K
RK
Department of Internal Medicine and the Department of Veterans Affairs Medical Center, the University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. rama-mallampalli@uiowa.edu
Ryan
A J
AJ
Salome
R G
RG
Jackowski
S
S
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D019916:Choline-Phosphate Cytidylyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014452:Ubiquitins / Q000276:immunology
D011480:Protease Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D019916:Choline-Phosphate Cytidylyltransferase