Tumor Necrosis Factor-α Inhibits Expression of CTP:Phosphocholine Cytidylyltransferase

Abstract

We investigated the effects of tumor necrosis factor ␣ (TNF␣), a key cytokine involved in inflammatory lung disease, on phosphatidylcholine (PtdCho) biosynthesis in a murine alveolar type II epithelial cell line (MLE-12). TNF␣ significantly inhibited [ 3 H]choline incorporation into PtdCho after 24 h of exposure. TNF␣ 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 TNF␣ inhibition of CCT activity was associated with a uniform decrease in the mass of CCT␣ 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 CCT␣ mRNA, and CCT␤ mRNA was not detected. Incorporation of [ 35 S]methionine into immunoprecipitable CCT␣ protein in pulse and pulsechase studies revealed that TNF␣ did not alter de novo synthesis of enzyme, but it substantially accelerated turnover of CCT␣. 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 TNF␣. TNF␣-induced degradation of CCT␣ protein was partially blocked by ALLN or lactacystin. CCT was ubiquitinated, and ubiquitination increased after TNF␣ exposure. m-Calpain degraded both purified CCT and CCT in cellular extracts. Thus, TNF␣ inhibits PtdCho synthesis by modulating CCT protein stability via the ubiquitin-proteasome and calpain-mediated proteolytic pathways.

Calmodulin Binds and Stabilizes the Regulatory Enzyme, CTP:Phosphocholine Cytidylyltransferase

“…A fulllength CCT␣ template (pCMV-CCT FL ) plasmid DNA was used as a template. The primers used to mutate Thr 25 were 5Ј-GCC-CTAATGGAGCAGCAGAGGAAGATGG-3Ј (forward) and 5Ј-CCATCTTCCTCTGCTGCTCCATTAGGGC-3Ј (reverse). The primers used to mutate Ser 32 were 5Ј-GAAGATGGAAT-TCCTGCCAAAGTGCAGCGC-3Ј (forward) and 5Ј-GCGCT-GCACTTTGGCAGGAATTCCATCTTC-3Ј (reverse).…”

“…Construction of CCT␣ PEST Mutants-A CCT␣ variant harboring point mutations in the PEST domain (CCTPEST SDM ) was constructed where Thr 25 and Ser 32 were mutated to Ala using the QuikChange site-directed mutagenesis kit. A fulllength CCT␣ template (pCMV-CCT FL ) plasmid DNA was used as a template.…”

“…Mutagenesis was performed to disrupt this signature motif and potentially reduce the ability of calpain to degrade CCT␣. Mutagenesis of highly conserved Thr 25 and Ser 32 to Ala resulted in loss of the PEST sequence as identified by this algorithm. First, three constructs, CCT␣ full-length (CCT␣ FL ) and CCT SDM and CCT SOE (mutants with disrupted PEST motifs), were synthesized in vitro and incubated with calpain, and reaction products were resolved and visualized by autoradiography.…”

“…35 S]methionine (60 Ci/ml) for 4 h at 37°C as described (25). Cells were rinsed, chased in medium replete with methionine and cysteine for 0 -8 h, and processed for CCT␣ immunoprecipitation, SDS-PAGE, and autoradiography as described (7).…”

“…Cell lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (Buffer A) at 4°C prior to analysis. Cytosolic and microsomal preparations were isolated as described (25).…”