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Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta 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. We have reported that p38 mitogen-activated protein kinase (MAPK) was upstream of nSMase2 in tumor necrosis-alpha (TNF-alpha)-stimulated A549 cells ( J Biol Chem 282: 1384-1396, 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-delta, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC-delta did not regulate TNF-induced N-SMase activity. Furthermore, PKC-delta 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-delta does not act through p38 MAPK in regulating nSMase2. Finally, down-regulation of PKC-delta inhibited induction of vascular cell and intercellular adhesion molecules, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC-delta as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of cross-talk between the PKC and sphingolipid pathways.

SUBMITTER: Clarke CJ

PROVIDER: S-EPMC4698891 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.

Clarke Christopher J CJ Guthrie J Michael JM Hannun Yusuf A YA

Molecular pharmacology 20080724 4

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-alpha (TNF-alpha)-stimulated A549 cells ( J Biol Chem 282: 1384-1396, 2007 ). Here, we report a role for protein kinase C (PKC) in mediating TNF-induced translocation of nSMase2 from the Gol ...[more]

PMID: 18653803

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Project description:The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] and interferon gamma (IFN-gamma) in HL-60 cells were investigated. IFN-gamma was found to increase selectively the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)(2)D(3) and IFN-gamma had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)(2)D(3) and IFN-gamma. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca(2+)-dependent PKC isoforms, inhibited the effect of 1,25(OH)(2)D(3) but had no effect on the IFN-gamma-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)(2)D(3), arachidonic acid (AA) and PMA. The depletion of PKC isoforms beta(1), beta(2), epsilon, eta, mu, zeta and lambda had no effect on the activation of sphingomyelinase induced by 1,25(OH)(2)D(3) or by AA. The depletion of PKC alpha from the cytosol completely abolished the effect of 1,25(OH)(2)D(3) on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or alpha-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC delta. Moreover, PMA inhibited the effect of 1,25(OH)(2)D(3) on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC delta from the cytosol. These studies demonstrate that 1,25(OH)(2)D(3)-induced activation of sphingomyelinase is mediated by PKC alpha. Furthermore, PKC delta had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)(2)D(3)- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC alpha and PKC delta isoforms.

Dataset Information

Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.

Publications

Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.

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