Regulation of Neutral Sphingomyelinase-2 (nSMase2) by Tumor Necrosis Factor-α Involves Protein Kinase C-δ in Lung Epithelial Cells (original) (raw)
Research ArticleArticle
Molecular Pharmacology October 2008, 74 (4) 1022-1032; DOI: https://doi.org/10.1124/mol.108.046250
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. We have reported that p38 mitogen-activated protein kinase (MAPK) was upstream of nSMase2 in tumor necrosis-α (TNF-α)-stimulated A549 cells (
J Biol Chem**:**-1396, 2007
). 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. Using phorbol 12-myristate 13-acetate (PMA) as a tool to dissect PKC responses, we 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-δ, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC-δ did not regulate TNF-induced N-SMase activity. Furthermore, PKC-δ and nSMase2 did not coimmunoprecipitate, suggesting that other signaling proteins may be involved. PMA-stimulated nSMase2 translocation was independent of p38 MAPK, and neither PKC inhibitors nor small interfering RNA had significant effects on TNF-stimulated p38 MAPK activation, indicating that PKC-δ does not act through p38 MAPK in regulating nSMase2. Finally, down-regulation of PKC-δ inhibited induction of vascular cell and intercellular adhesion molecules, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC-δ as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of cross-talk between the PKC and sphingolipid pathways.
Footnotes
This work was supported in part by National Institutes of Health grant GM43825 (Y.A.H.) and a Mid-Atlantic Affiliate Postdoctoral Fellowship from the American Heart Association (C.J.C.)
ABBREVIATIONS: SMase, sphingomyelinase; N-SMase, neutral sphingomyelinase; PM, plasma membrane; TNF, tumor necrosis factor-α; PKC, protein kinase C; DAG, diacylglycerol; PS, phosphatidylserine; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; Scr, scrambled; PBS, phosphate-buffered saline; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5_H_-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1_H_-imidazole.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.- Received February 11, 2008.
- Accepted July 22, 2008.
The American Society for Pharmacology and Experimental Therapeutics
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