Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes.

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3(+)CD8(+) cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P(5) is the predominant S1P receptor in leukemic LGLs, whereas S1P(1) is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo.

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

Acid ceramidase and human disease.

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago, and is deficient in the inherited lipid storage disorder, Farber Lipogranulomatosis (Farber disease). Importantly, AC not only hydrolyzes ceramide into sphingosine, but also can synthesize ceramide from sphingosine and free fatty acids in vitro and in situ. This "reverse" enzymatic activity occurs at a distinct pH from the hydrolysis ("forward") reaction (6.0 vs. 4.5, respectively), suggesting that the enzyme may have diverse functions within cells dependent on its subcellular location and the local pH. Most information concerning the role of AC in human disease stems from work on Farber disease. This lipid storage disease is caused by mutations in the gene encoding AC, leading to a profound reduction in enzymatic activity. Recent studies have also shown that AC activity is aberrantly expressed in several human cancers, and that the enzyme may be a useful cancer drug target. For example, AC inhibitors have been used to slow the growth of cancer cells, alone or in combination with other established, anti-oncogenic treatments. Aberrant AC activity also has been described in Alzheimer's disease, and overexpression of AC may prevent insulin resistant (Type II) diabetes induced by free fatty acids. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.

The N-acylethanolamine-hydrolyzing acid amidase (NAAA).

Bioactive N-acylethanolamines, including the endocannabinoid anandamide and anti-inflammatory N-palmitoylethanolamine, are hydrolyzed to fatty acids and ethanolamine in animal tissues by the catalysis of fatty acid amide hydrolase (FAAH). We recently cloned cDNA of N-acylethanolamine-hydrolyzing acid amidase (NAAA), another enzyme catalyzing the same reaction, from human, rat, and mouse. NAAA reveals no sequence homology with FAAH and belongs to the choloylglycine hydrolase family. The most striking catalytic property of NAAA is pH optimum at 4.5-5, which is consistent with its immunocytochemical localization in lysosomes. In rat, NAAA is highly expressed in lung, spleen, thymus, and intestine. Notably, the expression level of NAAA is exceptionally high in rat alveolar macrophages. The primary structure of NAAA exhibits 33-35% amino acid identity to that of acid ceramidase, a lysosomal enzyme hydrolyzing ceramide to fatty acid and sphingosine. NAAA actually showed a low, but detectable ceramide-hydrolyzing activity, while acid ceramidase hydrolyzed N-lauroylethanolamine. Thus, NAAA is a novel lysosomal hydrolase, which is structurally and functionally similar to acid ceramidase. These results suggest a unique role of NAAA in the degradation of N-acylethanolamines.

KLF6 is one transcription factor involved in regulating acid ceramidase gene expression.

Acid ceramidase (AC; E.C.3.5.1.23) activity is required to hydrolyze ceramide into sphingosine. An inherited deficiency of this enzymatic activity leads to the lipid storage disorder, Farber Lipogranulomatosis. Aberrant AC activity also has been demonstrated in several human cancers. We have characterized a 1931-bp putative promoter region of the murine AC gene by Luciferase reporter assays, electrophoretic mobility shift assays, and mutational analysis. A 143-bp sequence essential for AC promoter activity was found, and mobility shift and super-shift experiments using nuclear extracts of NIH3T3 cells demonstrated that a 34-bp, GC-rich sub-region could bind the transcription factors KLF6, Sp1, and AP2. Transient over-expression of KLF6 in NIH3T3 cells significantly increased the activity of a co-transfected Luciferase reporter construct containing the wild-type AC promoter, and a positive correlation was observed between AC and KFL6 RNA and protein expression in two different human cancer cell lines in which KLF6 expression was either "knocked-down" by RNAi or increased by retroviral-mediated gene transfer. Northern blot analysis also revealed a correlation of KLF6 and AC gene expression in various human tissues. These results provide the first characterization of the AC promoter from any species and demonstrate that KLF6 is one transcription factor involved in the regulation of AC gene expression.

Fenretinide cytotoxicity for Ewing's sarcoma and primitive neuroectodermal tumor cell lines is decreased by hypoxia and synergistically enhanced by ceramide modulators.

Patients with disseminated Ewing's family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O(2)) affected cytotoxicity, mitochondrial membrane potential (DeltaPsi(m)) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by l-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O(2)), that the mean 4-HPR LC(99) (the drug concentration lethal to 99% of cells) = 6.1 +/- 5.4 microm (range, 1.7-21.8 microm); safingol (1-3 microm) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC(99) to 3.2 +/- 1.7 microm (range, 2.0-8.0 microm; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O(2)) reduced ceramide species increase, DeltaPsi(m) loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC(99), 19.7 +/- 23.9 microm; range, 2.3-91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC(99), 4.9 +/- 2.3 microm; range, 2.0-8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-l-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.

Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier functions induced by tumor necrosis factor-alpha and interferon-gamma in human epidermis.

Ceramide is an integral part of the extracellular lipid bilayer of the stratum corneum (SC) that forms the permeability barrier of the skin. The production of SC ceramides is catalyzed by sphingomyelinase (SMase) and glucocerebrosidase (GCase). Acid-ceramidase (acid-CDase) catalyzes the hydrolysis of ceramide in the SC. We examined the effects of T helper (Th)1 and Th2 cytokines on levels of transcripts of genes for acid-CDase, acid-SMase, and GCase, on levels of ceramide, and on the extent of transepidermal water loss (TEWL) in the human epidermis in an effort to determine whether these cytokines affect the permeability barrier functions. Levels of transcripts for acid-SMase and GCase and the amount of ceramide in human epidermal sheets were enhanced by tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma and these effects were inhibited in the presence of interleukin (IL)-4. In epidermal keratinocytes cultured under submerged conditions, however, no similar inhibitory effects of IL-4 were observed. Consistent with these results, TEWL was suppressed by TNF-alpha and IFN-gamma, and these effects were also inhibited by IL-4. The balance between Th1 and Th2 might affect the construction and/or the repair of the epidermal permeability barrier via regulation of the production of ceramide.

Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism.

During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.

Upregulation of the human alkaline ceramidase 1 and acid ceramidase mediates calcium-induced differentiation of epidermal keratinocytes.

Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs. haCER1 is the human ortholog of mouse alkaline ceramidase 1 that we previously identified. haCER1 catalyzed the hydrolysis of very long-chain ceramides to generate sphingosine (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER1 in HEKs decreased the levels of D-e-C(24:1)-ceramide and D-e-C(24:0)-ceramide but elevated the levels of both SPH and its phosphate (S1P), whereas RNA interference-mediated knockdown of haCER1 caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER1 overexpression, Ca2+(o) increased the levels of SPH and S1P, and this was attenuated by haCER1 knockdown. haCER1 knockdown also inhibited the Ca2+(o)-induced growth arrest of HEKs and the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Ca2+(o); and its knockdown attenuated the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. These results strongly suggest that upregulation of haCER1 and AC mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs by generating SPH and S1P.

Acid ceramidase overexpression prevents the inhibitory effects of saturated fatty acids on insulin signaling.

Recent studies indicate that insulin resistance and type 2 diabetes result from the accumulation of lipids in tissues not suited for fat storage, such as skeletal muscle and the liver. To elucidate the mechanisms linking exogenous fats to the inhibition of insulin action, we evaluated the effects of free fatty acids (FFAs) on insulin signal transduction in cultured C2C12 myotubes. As we described previously (Chavez, J. A., and Summers, S. A. (2003) Arch. Biochem. Biophys. 419, 101-109), long-chain saturated FFAs inhibited insulin stimulation of Akt/protein kinase B, a central regulator of glucose uptake and anabolic metabolism. Moreover, these FFAs stimulated the de novo synthesis of ceramide and sphingosine, two sphingolipids shown previously to inhibit insulin action. To determine the contribution of either sphingolipid in FFA-dependent inhibition of insulin action, we generated C2C12 myotubes that constitutively overexpress acid ceramidase (AC), an enzyme that catalyzes the lysosomal conversion of ceramide to sphingosine. AC overexpression negated the inhibitory effects of saturated FFAs on insulin signaling while blocking their stimulation of ceramide accumulation. By contrast, AC overexpression stimulated the accrual of sphingosine. These results support a role for aberrant accumulation of ceramide, but not sphingosine, in the inhibition of muscle insulin sensitivity by exogenous FFAs.

Mutation analysis of the acid ceramidase gene in Japanese patients with Farber disease.

Farber disease is a rare lysosomal storage disease, characterized by the accumulation of ceramide in tissues due to acid ceramidase deficiency. Here we report the identification of three novel mutations in the acid ceramidase gene from two Japanese patients. Patient 1 showed joint problems at around 10 months of age and the patient is now emaciated, with multiple nodules and mild neurological problems at 10 years of age. Patient 2 had consanguineous parents and showed joint contractures at around 8 months of age. He showed neurological symptoms around 2 years of age and died at 6 years owing to respiratory failure. The diagnosis was made clinically and was confirmed by enzymatic assay of acid ceramidase. Molecular analysis of cultured skin fibroblasts showed normal mRNA levels expressed in both patients. By direct sequencing of cDNA, missense mutations of V97E in exon 4 and G235R in exon 9 were detected in patient 1 and 96delV in exon 4 was homozygously identified in patient 2. These mutations were also confirmed in genomic DNA. Expression of mutated acid ceramidase cDNA in COS-1 cells showed acid ceramidase activity decreased to 35%, 2% and 37% of control value, respectively. We also found a new polymorphism V3691 in exon 14 in the allele from the mother of patient 1. To date, 13 mutations, including our newly identified mutations, have been reported. All these mutations were genetically private and genotype-phenotype correlations could not be made.

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.

Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity.

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.