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. 2010 Jul;24(7):2507–2515. doi: 10.1096/fj.09-153635

Role of alkaline ceramidases in the generation of sphingosine and its phosphate in erythrocytes

Ruijuan Xu *, Wei Sun *, Junfei Jin *, Lina M Obeid *,†,‡, Cungui Mao *,†,1
PMCID: PMC2887272  PMID: 20207939

Abstract

Plasma sphingosine-1-phosphate (S1P) has been suggested to mainly originate from erythrocytes; however, within the erythrocyte, how sphingosine (SPH) generation—the precursor to S1P—is controlled is unknown. SPH is only generated from the hydrolysis of ceramides via ceramidases. Five human ceramidases have been identified: 1 acid, 1 neutral, and 3 alkaline ceramidases (ACER1, ACER2, and ACER3). Here, we demonstrate that only alkaline ceramidase activity is expressed in erythrocytes and that it is instrumental for SPH generation. Erythrocytes have alkaline but not acid or neutral ceramidase activity on D-e-C18:1-ceramide, a common substrate of ceramidases. Not only alkaline ceramidase activity but also the generation of SPH and S1P are increased during erythroid differentiation in K562 erythroleukemic cells. Such SPH and S1P increases were inhibited by the alkaline ceramidase inhibitor D-e-MAPP, suggesting that alkaline ceramidases have a role in the generation of SPH and S1P in erythroid cells. Alkaline ceramidase activity is highly expressed in mouse erythrocytes, and intravenous administration of D-e-MAPP decreased both SPH and S1P in erythrocytes and plasma. Collectively, these results suggest that alkaline ceramidase activity is important for the generation of SPH, the S1P precursor in erythrocytes.—Xu, R., Sun, W., Jin, J., Obeid, L. M., Mao, C. Role of alkaline ceramidases in the generation of sphingosine and its phosphate in erythrocytes.

Keywords: ceramide, erythroid cells, S1P, and S1P lyase

Sphingosine-1-phosphate(S1P), a sphingolipid metabolite, is a ligand of the 5 G-protein-coupled receptors, S1P1-5, which mediate cell proliferation, survival, adhesion, and migration, in addition to various biological processes, including cardiovascular development (1), angiogenesis (2), and immunity (3).

The metabolic pathway of S1P in mammalian cells has been delineated. Sphingomyelin is hydrolyzed by sphingomyelinases to generate ceramide (4,5,6), which is further hydrolyzed via ceramidases (7) (8,9,10,11) to generate sphingosine (SPH), which, in turn is phosphorylated to form S1P by sphingosine kinases (12, 13), SPHK1 and SPHK2. Subsequently, S1P is irreversibly cleaved by S1P lyase (SPL) (14, 15) to produce ethanolamine phosphate and hexadecenal, which are incorporated into phosphatidylethanolamine and glycerolipids, respectively. S1P can also be dephosphorylated to yield SPH by S1P-specific phosphatases (16, 17), SPP1 and SPP2, or lipid phosphatases with broad specificity, LPP1–3 (18).

S1P is abundant in plasma (19,20,21), and plasma S1P has been suggested to mainly originate from erythrocytes (21, 22). However, how the generation of the S1P precursor, SPH, is regulated in erythrocytes remains unclear.

As stated earlier, SPH is only generated from ceramide by ceramidases. To date, 5 human ceramidase genes have been identified: 1 acid ceramidase gene (AC/ASAH1) (7), 1 neutral ceramidase gene (NC/ASAH2) (9), and 3 alkaline ceramidase genes, ACER1/ASAH3 (23), ACER2/ASAH3L (11), and ACER3/APHC/PHCA (24). These ceramidases have different cellular localizations and substrate specificities. AC/ASAH1 is a lysosomal ceramidase that has an optimal pH value of 4.5 for its in vitro activity. It catalyzes the hydrolysis of ceramides with saturated medium acyl chains (C10-14) or unsaturated long acyl chains (C18:1 or C18:2) (25). ASAH2 is localized to the plasma membrane or secreted from cells (26). It has an optimal pH value of ∼7.0 and uses various ceramides as substrates (27). ACER1 is localized to the endoplasmic reticulum (ER) and has an optimal pH value of ∼9.0. It only catalyzes the hydrolysis of ceramides with unsaturated long acyl chains (C18:1 and C20:1) (unpublished data) or a very long saturated (C24:0) or unsaturated (C24:1) acyl chain (23). ACER2 is a Golgi ceramidase with an optimal pH value of ∼9.0. It uses various ceramides as substrates (11, 28). ACER3 is localized to both the ER and Golgi complex, and it has an optimal pH of ∼9.0. We initially demonstrated that ACER3 preferentially catalyzes the hydrolysis of phytoceramide, an analog of ceramide (24). Our recent studies showed that ACER3 preferentially catalyzes the hydrolysis of ceramides carrying unsaturated long acyl chains (C18:1 and C20:1) (29).

In this study, we investigate the role of ceramidases in controlling the generation of SPH and S1P in erythrocytes. We demonstrate that erythrocytes express alkaline ceramidase activity but not acid or neutral ceramidase. Alkaline ceramidase activity, SPH, and S1P are upregulated during erythroid differentiation of K562 cells, an erythroblastic leukemia cell line. Inhibiting the activity of ACERs with the alkaline ceramidase inhibitor D-e-MAPP blocks the SPH and S1P increases in response to erythroid differentiation, and intravenous administration of D-e-MAPP also decreases SPH and S1P in erythrocytes and plasma in mice. This suggests that ACERs have an important role in controlling the generation of SPH and S1P in erythrocytes and plasma.

MATERIALS AND METHODS

Reagents

Anti-S1P lyase antibody was a kind gift from Drs. Akio Kihara and Yasuyuki Igarashi (Hokkaido University, Sapporo, Japan). MEM and RPMI 1640 medium, fetal bovine serum (FBS), trypsin-EDTA, PBS, penicillin/streptomycin solution, blasticidin, and zeocin were purchased from Invitrogen (Carlsbad, CA, USA). d-erythro (e)-C18:1-ceramide, N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-phytosphingosine (D-ribo-C12-NBD-phytoceramide), and (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-e-MAPP) were synthesized in the Lipidomics Core Facility at Medical University of South Carolina. D-e-C16-ceramide, D-e-C24:1-ceramide, SPH, S1P, and other sphingolipids were from Avanti Polar Lipids (Alabaster, AL, USA). Other unstated reagents were from Sigma (St. Louis, MO, USA).

Animals

FVB/N mice (4 to 6 wk old) were purchased from Charles River (Wilmington, MA, USA). All animal procedures were carried out in accordance with the Medical University of South Carolina animal care committee guidelines.

Cell lines and culture conditions

Human leukemia K562 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Wild-type T-REx HeLa cells (Invitrogen) were cultured in MEM supplemented with 10% FBS, 1% penicillin/streptomycin solution, and 10 μg/ml blasticidin. T-REx HeLa derivatives AC-TET-ON, ACER1-TET-ON, ACER2-TET-ON, and ACER3-TET-ON cells were cultured in the same medium supplemented with 50 μM/ml zeocin. AC-TET-ON, ACER1-TET-ON, and ACER2-TET-ON cell lines that stably overexpress AC, ACER1, and ACER2, respectively, under the control of the tetracycline-inducible promoter system, CMV-TET-ON, were constructed in our previous studies (11, 30). The same procedures were used to create the ACER3-TET-ON cell line in this study. The ACER3 coding sequence was cloned inframe with the FLAG coding sequence in pcDNA4-FLAG, as described previously (11), and the resulting expression construct pcDAN4-ACER3 was transfected into T-REx HeLa cells. Transfected cells were selected in medium containing 300 μg/ml zeocin, and zeocin-resistant clones were confirmed to express the FLAG-tagged ACER3 strictly under the control of the CMV-TET-ON promoter system.

Erythroid differentiation of K562 cells

To induce erythroid differentiation, K562 cells were treated for 72 h with 1-β-d-arabinofuranosylcytosine (Ara-C, 5×10−7 M), as described previously (31). Erythroid differentiation was determined by staining with benzidine as described previously (32).

Blood cell preparation

Mouse blood was drawn into heparinized syringes from anesthetized mice via cardiac puncture and immediately put into 15-ml tubes with heparin solution (50 U/ml blood) on ice. Blood cells were separated from plasma by centrifugation at 1200 g for 5 min at 4°C, and washed twice with ice-cold PBS. Erythrocytes were separated from other cell types by Ficoll gradient (GE Healthcare, Piscataway, NJ, USA), according to the manufacturer’s instructions and the method of Hanel et al. (22). Human erythrocytes were purchased from Innovative Research (Novi, MI, USA), and further purified by the Ficoll gradient procedure as described above.

siRNA transfection

Control siRNA (siCON) [r(UUCUCCGAACGUGUCACGU)d(TT) (sense) and r(ACGUGACACGUUCGGAGAA)d(TT) (antisense)] and a SPL-specific siRNA (siSPL) [r(GUGCCCAUGCUGCAUUUAA)d(TT) (sense) and r(UUAAAUGCAGCAUGGGCAC)d(TT) (antisense)] were chemically synthesized by Dharmacon Inc. (Lafayette, CO, USA); siRNAs at 5 nM were transfected into T-REx HeLa cells and derivatives using Oligofectamine (Invitrogen), according to the manufacturer’s instructions.

Lentiviral shRNA transduction

K562 cells were transduced with Mission lentiviral transduction particles (Sigma) containing a control shRNA (shCON) targeting none of the human genes or expressing a set of shRNAs that specifically target ACER1 (shACER1), ACER2 (shACER2), ACER3 (shACER3), or SPL (shSPL) (Sigma) according to the manufacturer’s instructions.

Quantitative PCR analysis

Total RNA was isolated from K562 cells using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA was reversely transcribed into cDNA and subjected to real-time PCR analysis, which was performed on an iCycler system (Bio-Rad, Hercules, CA, USA). Standard reaction volume was 25 μl, including 10 μl of iQ SYBR Green Supermix (Bio-Rad), 10 μl of cDNA template, and 2.5 μl of a primer mixture. The initial PCR step was 3 min at 95°C, followed by 40 cycles of a 10-s melting at 95°C and a 45-s annealing/extension at 60°C. The final step was 1-min incubation at 60°C. All reactions were performed in triplicate. Real-time RT-PCR results were analyzed using Q-Gene software (http://www.qgene.org), which expresses data as mean normalized expression (MNE) (33). MNE is directly proportional to the amount of mRNA of a target gene relative to the amount of mRNA of the reference gene (β-actin). Primers used in this study were 5′-TGATGCTTGACAAGGCACCA-3′ and 5′-GGCAATTTTTCATCCACCACC-3′ for ACER1; 5′-AGTGTCCTGTCTGCGGTTACG-3′ and 5′-TGTTGTTGATGGCAGGCTTGAC-3′ for ACER2; 5′-CAATGTTCGGTGCAATTCAGAG-3′ and 5′-GGATCCCATTCCTACCACTGTG-3′ for ACER3; and 5′-CAATGTTCGGTGCAATTCAGAG-3′ and 5′-GGATCCCATTCCTACCACTGTG-3′ for β-actin.

Protein concentration determination

Protein concentrations were determined with BSA as a standard using a BCA protein determination kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Western blot analysis

Proteins were resolved by SDS-PAGE, and then subjected to Western blot analysis using various antibodies, as described previously (11).

Ceramidase assays

Ceramidase activity was determined by the release of SPH from the hydrolysis of various ceramides, according to the method we previously developed (11). Acid ceramidase activity and neutral ceramidase activity in total cell lysates were determined using D-e-C18:1-ceramide at pH 5.0 and 7.0, respectively. Alkaline ceramidase activity was determined in total membranes at pH 9.4 using various ceramides as substrates. The substrate concentration was 150 μM.

SPL activity assays

SPL activity assays were performed essentially as described by Bandhuvula et al. (34) using the fluorescent analog NBD-S1P as a substrate.

Electrospray ionization/dual mass spectroscopy (ESI/MS/MS) lipid analysis

Total lipids were extracted from cell pellets or conditioned medium and sphingolipids were subjected to ESI/MS/MS analysis that was performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer (Thermo Finnegan, San Jose, CA, USA), operating in a multiple reaction monitoring (MRM) positive ionization mode, as described previously (35). Sphingolipid contents were normalized to total phosphate (P) in lipid extracts.

Statistic analysis

Student’s t test was applied for statistical analysis using the software GraphPad Prism (GraphPad, San Diego, CA, USA). Values of P < 0.05 were considered significant. Data represent mean ± sd values of ≥3 independent experiments.

RESULTS

Alkaline but not acid or neutral ceramidase activity is expressed in erythrocytes

To determine how SPH generation is controlled in erythrocytes, we first determined which ceramidase activity is expressed in these anucleated cells. We measured ceramidase activity using D-e-C18:1-ceramide, a common substrate for all known ceramidases in the presence of Ca2+, which is required for alkaline ceramidase activity. We demonstrated that erythrocytes had high alkaline ceramidase activity on D-e-C18:1-ceramide in the presence of 1 mM Ca2+, with undetectable acid or neutral ceramidase activity (Fig. 1A), suggesting that erythrocytes express alkaline ceramidase activity but not acid or neutral ceramidase activity.

Figure 1.

Figure 1.

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Alkaline but not acid or neutral ceramidase is expressed in human erythrocytes. A) Total membranes were prepared from human erythrocytes and subjected to ceramidase activity assays using D-e-C18:1-ceramide as a substrate at pH 5.0 or 7.0 in the absence of Ca2+ or at pH 9.4 in the presence of 1 mM CaCl2. B) Total membranes of human erythrocytes were assayed for alkaline ceramidase activity on D-e-C12-NBD-ceramide, D-e-C16-ceramide, or D-e-C24:1-ceramide at pH 9.4 and in the presence of 1 mM Ca2+. Data represent means ± sd of 3 experiments performed in duplicate.

To differentiate the activity of individual alkaline ceramidases, we measured erythrocyte alkaline ceramidase activity on D-e-C16-ceramide (a substrate specific for ACER2), D-e-C24:1-ceramide (a substrate for both ACER1 and ACER2 but not for ACER3), and D-ribo-C12-NBD-phytoceramide (a substrate specific for ACER3), in the presence of Ca2+. As shown in Fig. 1B, erythrocytes have high alkaline ceramidase activity on D-ribo-C12-NBD-phytoceramide and moderate alkaline ceramidase activity on either D-e-C16-ceramide or D-e-C24:1-ceramide, suggesting that erythrocyte alkaline ceramidase activity is mainly encoded by ACER3.

Alkaline ceramidases are upregulated in erythroid cells

Because erythrocytes are anucleated cells, it is impossible to genetically manipulate protein expression in vitro. Therefore, it is difficult to define the role of the alkaline ceramidases in regulating SPH and S1P directly in these anucleated cells. To circumvent this problem, we investigated the role of alkaline ceramidases in controlling the generation of SPH and S1P in an in vitro erythroid differentiation model.

It has been shown that on treatment with a chemotherapeutic agent Ara-C, K562 cells, a pluripotent erythroleukemic cell line, differentiate into erythroid cells (31). As the first step to define the role of alkaline ceramidase activity in regulating the generation of SPH and S1P in erythroid cells, we determined whether alkaline ceramidase activity and SPH and S1P levels are coregulated during erythroid differentiation.

We showed that treatment of K562 cells with Ara-C markedly increased the number of cells stained positive with benzidine, erythroid cells (Fig. 2A), confirming the erythroid differentiation of K562 cells. Similar to erythrocytes, K562 cells have higher alkaline ceramidase activity on D-ribo-C12-NBD-phytoceramide or C18:1-ceramide than alkaline ceramidase activity on C16-ceramide or D-e-C24:1-ceramide, and these activities were increased by treatment with Ara-C (Fig. 2B), suggesting that erythroid cells differentiated from K562 cells have a similar expression pattern of alkaline ceramidases as erythrocytes and that alkaline ceramidase activity is up-regulated during erythroid differentiation. qPCR analyses revealed that treatment with Ara-C increased mRNA of ACER2 and ACER3, but not ACER1 (Fig. 2C), suggesting that both ACER2 and ACER3 are up-regulated in erythroid cells. ESI/MS/MS analysis demonstrated that treatment with Ara-C caused a 3-fold and 5-fold increase in SPH and S1P, respectively, in K562 cells (Fig. 2D), suggesting that the generation of SPH and S1P is also increased during erythroid differentiation. Collectively, these results suggest that alkaline ceramidase activity and the generation of SPH and S1P are up-regulated simultaneously during erythroid differentiation of K562 cells.

Figure 2.

Figure 2.

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Both alkaline ceramidase activity and SPH and S1P levels are increased during erythroid differentiation. A) K562 cells were treated with Ara-C or H2O (vehicle control) for 72 h before erythroid differentiation was determined by benzidine staining as described in Materials and Methods. Benzidine-positive cells were counted using a microscope. B) Microsomes were isolated from K562 cells treated with Ara-C as in A, and alkaline ceramidase activity on different substrates was measured as in Fig. 1B. C) Total RNA was isolated from K562 cells treated with Ara-C or H2O and subjected to qPCR analysis to measure ACER1, ACER2, or ACER3 mRNA. D) K562 cells treated with Ara-C or H2O were harvested by centrifugation, washed with 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, and subjected to ESI/MS/MS for SPH and S1P measurement. Data represent means ± sd of 3 experiments performed in duplicate. *P < 0.05.

Alkaline ceramidases have a compensatory role in controlling SPH and S1P generation in erythroid cells

To determine whether alkaline ceramidase activity is responsible for the Ara-C-induced generation of both SPH and S1P in erythroid cells, we knocked down the expression of ACER1, ACER2, or ACER3 in K562 cells using their specific short hairpin RNAs (shRNAs) delivered by a lentiviral vector system. Interestingly, we found that compared to transduction with lentiviral particles expressing a control shRNA (shCON), transduction with lentiviral particles expressing an ACER3-specific shRNA (shACER3) decreased ACER3 mRNA but increased ACER2 mRNA in K562 cells; and transduction with lentiviral particles expressing ACER2-specific shRNA (shACER2) decreased ACER2 mRNA but increased ACER1 mRNA and vice versa (Fig. 3A), suggesting that knocking down the expression of one alkaline ceramidase up-regulates another. Because of this complementary effect, we attempted to knock down the expression of ACER1, ACER2, and ACER3 simultaneously in K562 cells. However, cotransduction of K562 cells with shACER1, shACER2, and shACER3 lentiviral particles knocked down none of the alkaline ceramidases (data not shown).

Figure 3.

Figure 3.

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Inhibition of alkaline ceramidase activity suppresses SPH and S1P increases in response to erythroid differentiation. A) K562 cells were transduced with Mission lentiviral particles expressing a control nontargeting shRNA (shCON) or a shRNA specific for ACER1 (shACER1), ACER2 (shACER2), or ACER3 (shACER3) at a MOI of 5. At 72 h after lentiviral transduction, ACER1 or ACER2 mRNA levels were determined by qPCR analysis as described in Materials and Methods. B, C) ACER1-TET-ON cells (B) or ACER2-TET-ON cells (C) were grown in the presence of TET (10 ng/ml) or ET (vehicle control) for 24 h before being treated with D-e-MAPP (10 μM) or DMSO (vehicle control) for 4 h. SPH in these cells was measured by HPLC. D) ACER3-TET-ON cells, created as described in Materials and Methods, were grown in the presence of ET and TET for 48 h before expression of the FLAG-tagged ACER3 (FLAG-ACER3) was analyzed by Western blot with anti-FLAG antibody. E) ACER3-TET-ON cells were grown in the presence of ET or TET for 24 h before being treated with D-e-MAPP or DMSO for 4 h. SPH was measured by HPL. F) AC-TET-ON cells were grown in the presence of ET or TET for 24 h before being treated with D-e-MAPP or DMSO for 4 h. SPH was measured by HPLC. G) K562 cells were treated with Ara-C or H2O in the presence of D-e-MAPP or DMSO for 72 h before SPH and S1P were measured by ESI/MS/MS. Data represent means ± sd of 3 experiments performed in duplicate. *P < 0.05.

To block all alkaline ceramidase activity, we used the alkaline ceramidase inhibitor, D-e-MAPP, that potently and specifically inhibits alkaline ceramidase activity (32). To confirm that D-e-MAPP indeed inhibits ACER1, ACER2, and ACER3 activity in cells, we tested whether treatment with D-e-MAPP inhibited the SPH generation catalyzed by ACER1, ACER2, or ACER3 in cells. We previously established the T-REx-HeLa-based stable cell lines, ACER1-TET-ON and ACER2-TET-ON cells that overexpress ACER1 and ACER2, respectively, under the control of a tetracycline-inducible promoter system, CMV-TET-ON (30). In these stable cell lines, the overexpression of ACER1 or ACER2 was induced by the addition of tetracycline (TET) to medium but not by the addition of ethanol (ET), the vehicle control, resulting in the increased generation of SPH in cells (30). ACER1-TET-ON or ACER2-TET-ON cells grown in the presence of ET or TET were treated with D-e-MAPP or DMSO, the vehicle control. HPLC analyses showed that treatment with D-e-MAPP at 10 μM completely inhibited the TET-induced increase in SPH generation in either ACER1-TET-ON or ACER2-TET-ON cells (Fig. 3B, C), suggesting that D-e-MAPP indeed inhibits cellular ACER1 and ACER2 activity. To determine whether D-e-MAPP also inhibited cellular ACER3 activity, we generated the cell line ACER3-TET-ON, a T-REx HeLa derivative that stably overexpresses ACER3 under the control of the TET-ON promoter system. Western blot confirmed that the overexpression of ACER3 was induced by TET but not ET in ACER3-TET-ON cells (Fig. 3D). ACER3 overexpression increased SPH, and this was inhibited by D-e-MAPP (Fig. 3E), suggesting that D-e-MAPP also inhibits ACER3 cellular activity. We previously demonstrated that overexpression of AC also increases the generation of SPH in T-REx HeLa cells (30). To confirm that D-e-MAPP specifically inhibits alkaline ceramidase activity but not AC, we investigated whether D-e-MAPP inhibits the AC-catalyzed generation of SPH in cells. We previously generated the AC-TET-ON cell line that overexpresses AC under the control of the CMV-TET-ON system, and TET-induced overexpression of AC increased the generation of cellular SPH (30). AC-TET-ON cells grown in the presence of ET or TET were treated with D-e-MAPP or DMSO. HPLC analysis revealed that AC overexpression increased SPH to a similar extent in AC-TET-ON cells treated with DMSO and those treated with D-e-MAPP (Fig. 3F), confirming that D-e-MAPP does not inhibit AC activity in cells.

Subsequently, we tested whether treatment with D-e-MAPP inhibited the generation of SPH and S1P in erythroid cells. K562 cells were treated with Ara-C or H2O (the vehicle control) in the presence of D-e-MAPP and its vehicle, DMSO. Treatment with Ara-C increased both SPH and S1P in K562 cells treated with DMSO but failed to do so in K562 cells treated with D-e-MAPP (Fig. 3G), suggesting that alkaline ceramidase activity is responsible for the Ara-C-induced generation of SPH and S1P in erythroid cells.

Inhibition of alkaline ceramidase activity blocks SPL knockdown-induced accumulation of S1P in erythroid cells

Although erythroid cells differentiated from K562 cells have more S1P than undifferentiated K562 cells, they produce less S1P than erythrocytes. It has been suggested that anucleated erythrocytes accumulate S1P due to a lack of SPL activity (20). This prompted us to investigate whether inhibiting SPL would also lead to S1P accumulation in erythroid cells, and if so, whether blocking alkaline ceramidase activity inhibits S1P accumulation. We thus knocked down SPL expression in K562 cells by RNA interference (RNAi). K562 cells were transduced with lentiviral particles expressing a SPL-specific shRNA (shSPL) or shCON lentiviral particles. Compared to transduction with shCON, transduction with shSPL markedly decreased SPL protein in K562 cells (Fig. 4A). The decrease in SPL protein led to a marked decrease in SPL activity (Fig. 4B). ESI/MS/MS analyses demonstrated that SPL knockdown or Ara-C treatment alone caused a 7- to 8-fold increase in S1P in K562 cells, whereas the combination of SPL knockdown and Ara-C treatment caused a 40-fold increase in S1P (Fig. 4C) and that the SPL knockdown-induced accumulation of S1P was inhibited by treatment with D-e-MAPP (Fig. 4D), suggesting that alkaline ceramidase activity is required for S1P accumulation in erythroid cells, in which SPL activity is inhibited.

Figure 4.

Figure 4.

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Inhibition of alkaline ceramidase activity blocks S1P accumulation in response to knockdown of SPL in erythroid cells. A, B) K562 cells were transduced with Mission lentiviral particles expressing a SPL-specific shRNA (shSPL) or the control siRNA (shCON) for 72 h before being analyzed by Western blot with anti-SPL antibody (A) or by in vitro activity assays (B). C) K562 cells were transduced with shCON or shSPL Mission lentiviral particles in the presence of Ara-C or H2O for 72 h before S1P was measured by ESI/MS/MS. D) K562 cells transduced with shSPL lentiviruses were treated with Ara-C or H2O in the presence of D-e-MAPP or DMSO for 72 h before S1P was measured. Data represent means ± sd of 3 experiments performed in duplicate. *P < 0.05.

Alkaline ceramidase overexpression along with SPL knockdown results in S1P accumulation in cells

To further confirm that high alkaline ceramidase activity is sufficient for S1P accumulation in cells in which SPL is inhibited, we determined whether SPL knockdown along with ACER1, ACER2, or ACER3 overexpression also results in S1P accumulation in T-REx HeLa cells. ACER1-TET-ON, ACER2-TET-ON cells, or ACER3-TET-ON cells were transfected with siCON or siSPL for 24 h before the overexpression of ACER1, ACER2, or ACER3 was induced by treatment with TET or not induced by treatment with ET. Compared to transfection with siCON, transfection with a SPL-specific siRNA (siSPL) markedly decreased SPL activity in ACER1-TET-ON, ACER2-TET-ON, or ACER3-TET-ON cells (Fig. 5A, C, E). ESI/MS/MS analyses revealed that knocking down the expression of SPL caused a several-fold increase in S1P in ACER1-TET-ON and ACER2-TET-ON cells grown in the presence of ET, but greater than a 20-fold increase in S1P in either ACER1-TET-ON or ACER2-TET-ON cells grown in the presence of TET (Fig. 5B, D). SPL knockdown caused a 2-fold and 3-fold increase in S1P in ACER3-TET-ON cells grown in the presence of ET and TET, respectively (Fig. 5F). These results suggest that high alkaline ceramidase activity, especially encoded by ACER1 or ACER2, is sufficient for S1P accumulation in cells in which SPL is inhibited.

Figure 5.

Figure 5.

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Increasing alkaline ceramidase activity results in S1P accumulation in cells with SPL knockdown. A, B) ACER1-TET-ON cells grown in the presence of ET or TET were transfected with a SPL-specific siRNA (siSPL) or a control siRNA (siCON) for 72 h before SPL activity (A) and S1P levels (B) were measured. C, D) ACER2-TET-ON cells grown in the presence of ET or TET were transfected with siSPL or siCON for 72 h before SPL activity (C), and the levels of S1P (D) were determined. E, F) ACER3-TET-ON cells grown in the presence of ET or TET were transfected with siSPL or siCON for 72 h before SPL activity (E), and the levels of S1P (F) were determined. Data represent means ± sd of 3 experiments performed in duplicate. *P < 0.05.

Interestingly, we showed that SPL knockdown along with ACER1 or ACER2 overexpression also markedly increased DHS1P in cells (Fig. 5B, D), suggesting that ACER1 and ACER2 also play an important role in controlling DHS1P levels by catalyzing the hydrolysis of dihydroceramides into DHS in cells.

Alkaline ceramidase activity is important for the generation of SPH and S1P in mouse erythrocytes and plasma

Our in vitro studies suggest that alkaline ceramidase activity is important for the generation of both SPH and S1P in human erythroid cells. To determine whether mouse alkaline ceramidase activity is important for the generation of S1P in mouse erythrocytes and plasma, we investigated whether inhibiting alkaline ceramidase activity in vivo inhibits the generation of S1P in mouse erythrocyte plasma. First, we determined whether mouse erythrocytes also express alkaline ceramidase activity. We detected high alkaline ceramidase activity on D-e-C18:1-ceramide in membranes isolated from mouse erythrocytes (Fig. 6A). To inhibit alkaline ceramidase activity in mouse erythrocytes in vivo, mice were administered D-e-MAPP (2.5 nmol/g iv) or vehicle control DMSO for 6 h before the measurement of SPH and S1P. ESI/MS/MS analyses revealed that compared to those animals treated with DMSO, mice given D-e-MAPP had less SPH (Fig. 6B) and S1P (Fig. 6C) in erythrocytes or plasma (Fig. 6B), suggesting that alkaline ceramidase activity is important for the generation of SPH and S1P in mouse erythrocytes.

Figure 6.

Figure 6.

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Inhibition of alkaline ceramidase activity decreases mouse plasma S1P and DHS1P. A) Blood was drawn from mice, and blood cells were separated from plasma. Erythrocytes were separated from other blood cells by Ficoll gradient and subjected to alkaline ceramidase activity assays with indicated substrates. B, C) D-e-MAPP (2.5 or 5.0 nmol/g) or vehicle control DMSO was injected into mice through tail veins. At 12 h postinjection, blood was drawn from mice, and erythrocytes and plasma were subjected to ESI/MS/MS to measure SPH (B) and S1P (C). Data represent means ± sd in 3 mice. *P < 0.05.

DISCUSSION

Erythrocytes were found to be the major source of plasma S1P (21). However, how erythrocytes generate SPH, the S1P precursor, remains unclear. In this study, we provide strong evidence that alkaline ceramidase activity plays an important role in controlling the generation of SPH in erythrocytes, and thereby S1P in plasma.

The study by Ito et al. (20) suggested that erythrocytes have no ceramidase activity. In contrast, our results indicate that erythrocytes have alkaline ceramidase activity—a discrepancy brought about by differences in activity assay conditions. First, the substrates were different. Ito et al. (20) used a synthetic fluorescent ceramide, D-e-C12-NBD-ceramide, whereas we used natural ceramides (D-e-C18:1-ceramides, D-e-C16-ceramide, and D-e-C24:1-ceramide). Our unpublished data showed that D-e-C12-NBD-ceramide is a poor substrate for ACER2 or ACER3, whereas C18:1-ceramide is an excellent substrate for all alkaline ceramidases. Second, the use of a cation was different. Our assay buffer contained 1 mM Ca2+ and their assay buffer did not. We demonstrated that Ca2+ is required for ACER2 activity and that it markedly activates both ACER1 and ACER3. Therefore, erythrocyte alkaline ceramidase activity was underestimated in the study by Ito’s group. Of note, we confirm that erythrocytes have no acid and neutral ceramidase activity by using a different ceramide substrate (D-e-C18:1-ceramide), suggesting that erythrocytes only express alkaline ceramidase activity.

Our data suggest that alkaline ceramidase activity is important for the generation of S1P in erythrocytes. First, we demonstrated that overexpression of each of the alkaline ceramidases markedly increased S1P in HeLa cells in which SPL is inhibited by RNAi. Second, we showed that SPL knockdown caused S1P accumulation in differentiated erythroid cells with high alkaline ceramidase activity but not in undifferentiated K562 cells expressing lower alkaline ceramidase activity. Third, blocking alkaline ceramidase activity with the alkaline ceramidase inhibitor D-e-MAPP not only inhibited the generation of S1P in cultured human erythroid cells but also significantly decreased S1P in mouse erythrocytes and plasma in vivo.

It is noteworthy that the inhibitory effect of D-e-MAPP on the generation of both SPH and S1P is solely due to an inhibition of alkaline ceramidase activity but not acid or neutral ceramidase activity. This is supported by the following observations. First, it has been well established that D-e-MAPP does not inhibit in vitro acid ceramidase activity (36), and we confirm that D-e-MAPP does not inhibit AC cellular activity. Second, although it remains unclear whether D-e-MAPP inhibits neutral ceramidase activity, its inhibition—if any exists—would not cause the D-e-MAPP-induced decrease in SPH and S1P generation in cultured cells or mouse erythrocytes or plasma because we previously showed that overexpression of NC has no effect on SPH levels in T-REx HeLa cells (30), and knocking out the mouse neutral ceramidase has no effect on SPH and S1P levels in most tissues, including mouse plasma (37). Therefore, treatment with D-e-MAPP decreases S1P in cultured cells and mouse plasma exclusively by inhibiting alkaline ceramidase activity.

We demonstrated that two different concentrations of D-e-MAPP decreased the levels of SPH and S1P in erythrocytes or plasma to similar levels, suggesting that D-e-MAPP at these two concentrations may completely inhibit alkaline ceramidase activity in erythrocytes. However, a complete inhibition of alkaline ceramidase activity did not totally block the generation of SPH and S1P in erythrocytes and plasma. The remaining SPH and S1P in erythrocytes may be from other sources and generated by other ceramidase activity. Ito et al. (20) demonstrated that erythrocytes can readily take up exogenous SPH, suggesting that erythrocytes may acquire plasma SPH. How plasma SPH is generated remains unclear. Although the neutral ceramidase activity is detected in plasma, its involvement in the generation of plasma SPH is unlikely because knocking out the neutral ceramidase does not decrease plasma S1P. No other ceramidases have been shown to be in the plasma. Possibly, other hematopoietic cells or nonhematopoietic cells secrete SPH into the plasma, and if so, alkaline ceramidase activity may also supply erythrocytes with SPH via generation of SPH in other cell types—in an indirect way. Acid ceramidase activity has been detected in other hematopoietic cells (38, 39), and we demonstrated that AC also generates cellular SPH, so AC activity may also contribute to the generation of SPH secreted into plasma. In this sense, AC activity may also be important for S1P generation in erythrocytes and plasma by controlling the generation of SPH taken up by erythrocytes. This also explains why blocking alkaline ceramidase activity substantially decreased but did not abolish SPH and S1P in mouse erythrocytes and plasma.

In summary, we demonstrated that alkaline ceramidase activity is the only ceramidase activity found in erythrocytes and that it plays an important role in the generation of plasma S1P by controlling the generation of SPH in erythrocytes. Alkaline ceramidase activity is expected to play important roles in mediating plasma S1P-mediated biological responses, such as cardiovascular development, angiogenesis, and immunity.

Acknowledgments

This work was supported by U.S. National Institutes of Health grants R01CA104834 (C.M.), P20RR017677 (C.M.), and GM062887 (L.M.O.), and a Veterans Affairs merit award (L.M.O.). The authors thank Dr. Jennifer Schnellmann for English proofreading and editing of the manuscript.

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