Substrate Specificity and Some Other Enzymatic Properties of Dihydroceramide Desaturase (Ceramide Synthase) in Fetal Rat Skin

Mikami, Takeshi; Kashiwagi, Motoi; Tsuchihashi, Keiko; Akino, Toyoaki; Gasa, Shinsei

doi:10.1093/oxfordjournals.jbchem.a022023

Cited by 29 publications

(11 citation statements)

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“…Dihydroceramide desaturase 1 (DEGS1; with delta-4 desaturase activity) plays the predominant role in human cells, whereas DEGS2 (with an additional hydroxylase activity), which is less characterized, appears to play a minor role (31,32). DEGS1 prefers short-chain dhCer substrates (33,34). Incomplete inhibition of these enzymes, possibly by indirect mechanisms, could be mediated by changes in the membrane fluidity, owing to celecoxib (unpublished observations).…”

Section: Discussionmentioning

confidence: 98%

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Schiffmann

Sandner

Schmidt

et al. 2009

Journal of Lipid Research

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Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C 16:0 -, C 24:0 -, C 24:1 -dihydroceramide (dhCer) and led to a depletion of C 24:0 -, C 24:1 -Cer in a time-and concentrationdependent manner, whereas other coxibs had no effect. Using 13 C, 15 N-labeled L-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC 50 of 78.9 6 1.5 mM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of L-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.-Schiffmann, S., J. Sandner, R. Schmidt, K. Birod, I. Wobst, H. Schmidt, C. Angioni, G. Geisslinger, and S. Grösch. The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis. J. Lipid Res. 2009. 50: 32-40. Supplementary key words cancerSphingolipids constitute an essential component of the eucaryotic plasma membrane and are also utilized as an important second messenger in a variety of cellular events, including cell senescence, cellular differentiation, apoptosis, and proliferation (1-3). Endogenous sphingolipid levels can be controlled by activation of sphingomyelinases (SMases) and de novo synthesis as well as by specific degradation mechanisms (e.g., ceramidases, lyases). The sphingolipid biosynthesis commences in the endoplasmic reticulum with the condensation of palmitoyl-CoA and L-serine by L-serine palmitoyltransferase (L-SPT). The intermediate 3-ketosphinganine is rapidly converted into sphinganine (dhSph) by 3-ketosphinganine reductase. Acyl-CoA thioesters of variable chain lengths are then attached to dhSph by chain-length-specific (dihydro)ceramide synthases (CerSs) (4). Dihydroceramide desaturase (DEGS) introduces a 4, 5-trans double bond in dihydroceramides (dhCers), resulting in formation of the final product, Cer. Via sphingosine (Sph), Cer is metabolized by the enzymes ceramidase and sphingosine kinase to sphingosine-1-phosphate (Sph1P

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Section: Discussionmentioning

confidence: 98%

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Schiffmann

Sandner

Schmidt

et al. 2009

Journal of Lipid Research

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“…These results are consistent with previous reports (38). However, other sources of mitochondria seem to have substantial amounts of DDase activity that may not be accounted for by MAM (39). The mitochondrial proteome varies from tissue to tissue, and it is also possible that DDase is activated or introduced into mitochondria under specific physiological conditions, such as early in apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins

Stiban

Caputo

Colombini

2008

Journal of Lipid Research

139

114

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Increased mitochondrial ceramide levels are associated with the initiation of apoptosis. There is evidence that ceramide is causal. Thus, the conversion of the precursor, dihydroceramide, to ceramide by the enzyme dihydroceramide desaturase may be important in preparing the cell for apoptosis. Ceramide can initiate apoptosis by permeabilizing the mitochondrial outer membrane to apoptosisinducing proteins. However, the mitochondrion's ability to produce ceramide may be limited by its proteome. Here, we show that ceramide synthesized in isolated mammalian endoplasmic reticulum (ER) vesicles from either C 8 -dihydroceramide or sphingosine to produce long-chain ceramide can transfer to isolated mitochondria. The rate of transfer is consistent with a simple collision model. The transfer of the long-chain ceramide is faster than expected for an uncatalyzed process. Sufficient ceramide is transferred to permeabilize the outer membrane to cytochrome c and adenylate kinase. The mitochondria-associated membranes, ER-like membranes that are tightly associated with isolated mitochondria, can produce enough ceramide to permeabilize the outer membrane transiently. Thus, this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria to increase ceramide levels to the critical value required for functional changes, such as ceramide channel self-assembly followed by protein release.-Stiban, J., L. Caputo, and M. Colombini. Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins. J. Lipid Res. 2008. 49: 625-634.

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“…We modified the conditions used in a radiometric assay for desaturase activity in fetal rat skin (33) to look for optimal assay conditions including: substrate specificity for the chain length of dihydroceramides, C16:0 or a more natural C24:0; pH dependence of activity in the range of 5.5-8.5; the effects of detergents, α-and β-NADH, and FAD on activity; and isolation and preparation methods of enzyme sources. FAD is a coenzyme in the microsomal electron transport system containing the desaturases.…”

Section: Discussionmentioning

confidence: 99%

Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function

Takagi¹,

Tojo²,

Tomita³

et al. 2003

J. Clin. Invest.

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Aryl hydrocarbon receptor nuclear translocator (ARNT), a transcription factor of the Per/AHR/ ARNT/Sim family, regulates gene expression in response to environmental stimuli including xenobiotics and hypoxia. To examine its role in the epidermis, the Cre-loxP system was used to disrupt the Arnt gene in a keratinocyte-specific manner. Gene-targeted, newborn mice with almost normal appearance died neonatally of severe dehydration caused by water loss. Histology showed small changes in the architecture of cornified layers, with apparently preserved intercorneocyte lamellar structures responsible for the skin barrier function. In contrast, HPLC/ion-trap mass spectrometry revealed significant alterations in the compositions of ceramides, the major components of the lamellae. The murine epidermal ceramides normally contain 4-sphingenine and 4-hydroxysphinganine. In Arnt-null epidermis, 4-sphingenine was largely replaced by sphinganine and the amounts of ceramides with 4-hydroxysphinganine were greatly decreased, suggesting deficiency of dihydroceramide desaturases that catalyze the formation of both 4-sphingenyl and 4-hydroxysphinganyl moieties. A desaturase isoenzyme, DES-1, prefers desaturation, but DES-2 catalyzes both reactions to a similar extent. Transcript levels of Des-2, but not Des-1, were considerably decreased in cultured keratinocytes from Arntnull epidermis. These results indicate that proper ceramide compositions through 4-desaturation regulated by ARNT are crucial for maintaining the epidermal barrier function. .jp. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: aryl hydrocarbon receptor nuclear translocator (ARNT); basic helix-loop-helix (bHLH); aryl hydrocarbon receptor (AHR); Per/AHR/ARNT/Sim (PAS); hypoxia-inducible factor 1α (HIF1α); stratum corneum (SC); high-performance TLC (HPTLC); tandem mass spectrometry (MS/MS); embryonic day (E); electrospray ionization (ESI).

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Substrate Specificity and Some Other Enzymatic Properties of Dihydroceramide Desaturase (Ceramide Synthase) in Fetal Rat Skin

Cited by 29 publications

References 0 publications

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins

Alteration of the 4-sphingenine scaffolds of ceramides in keratinocyte-specific Arnt-deficient mice affects skin barrier function

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