Received for publication February 13, 2008.
Revised July 22, 2008.
Accepted for publication July 22, 2008.

Regulation of Neutral Sphingomyelinase-2 (nSMase2) by Tumor Necrosis Factor- involves Protein Kinase C- in Lung Epithelial Cells

Abstract

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production, but there is still limited knowledge of the regulatory mechanisms of the cloned N-SMase enzyme - nSMase2. Previously, we reported that p38 MAPK was upstream of nSMase2 in tumor necrosis- (TNF)-stimulated A549 cells (Clarke et al. (2007) J. Biol. Chem. 282, 1384-1396). Here, we report a role for protein kinase C (PKC) in mediating TNF-induced translocation of nSMase2 from the Golgi to the plasma membrane (PM). Pharmacological inhibition of PKCs prevented TNF-stimulated nSMase2 translocation to the PM in A549 cells. Utilizing phorbol esters (PMA) as a tool to dissect PKC responses, it was found that PMA induced nSMase2 translocation to the PM in a time- and dose-dependent manner. Pharmacological inhibitors and specific siRNA implicated the novel PKCs, specifically PKC-, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC- did not regulate TNF-induced N-SMase activity. Furthermore, PKC- and nSMase2 did not co-immunoprecipitate suggesting other signaling proteins may be involved. PMA-stimulated nSMase2 translocation was independent of p38 MAPK and neither PKC inhibitors nor siRNA had significant effects on TNF-stimulated p38 MAPK activation indicating that PKC- does not act through p38 MAPK in regulating nSMase2. Finally, downregulation of PKC- inhibited induction of VCAM and ICAM, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC- as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of crosstalk between the PKC and sphingolipid pathways.

Key words: Tumor necrosis factor, Protein Kinase C, Sphingolipids, P38 MAP Kinase