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. 2009 Feb;23(2):656–666. doi: 10.1096/fj.08-115634

Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion

Wei Sun *,1, Wei Hu †,1, Ruijuan Xu *,1, Junfei Jin †, Zdzislaw M Szulc †, Guofeng Zhang ‡, Sehamuddin H Galadari §, Lina M Obeid *,†,‖, Cungui Mao *,†,2
PMCID: PMC2630785  PMID: 18945876

Abstract

The polypeptide core of the integrin β1 subunit (β1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form β1 precursor and mature β1, respectively. The β1 precursor to mature β1 conversion, termed β1 maturation, regulates the cell surface levels and function of β1-containing integrins, β1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates β1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited β1 maturation, thus leading to a decrease in the levels of mature β1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced β1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of β1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited β1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both β1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing β1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both β1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating β1 maturation and cell adhesion mediated by β1 integrins.—Sun, W., Hu, W., Xu, R., Jin, J., Szulc, Z. M., Zhang, G., Galadari, S. H., Obeid, L. M, Mao, C. Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion.

Keywords: retinoic acid, protein kinase C, Golgi, sphingolipid

Integrins are heterodimeric cell adhesion receptors consisting of an α- and a β-subunit (1). Integrins not only play an important role in cell-extracellular matrix (ECM) adhesion or cell-cell adhesion but also mediate signal transduction in a bidirectional mode: relaying extracellular signals into cells (outside-in signaling) or receiving intracellular signals to modulate cell-ECM or cell-cell adhesion and cell motility (inside-out signaling; refs. 1,2,3,4).

A total of 18 α and 8 β integrin subunits in mammals has been identified, and they pair to form 24 distinct integrins, 12 of which contain the β1 subunit (β1) (5). β1 is synthesized as an 87-kDa polypeptide core, which is glycosylated in the ER to generate a partially glycosylated form, immature β1 or β1 precursor (6, 7). The β1 precursor is then transported to the Golgi complex where it is further glycosylated to generate mature forms of β1, the mature β1 (6). The conversion of β1 precursor into mature β1 is termed the maturation of β1. It has been shown that mature β1, but not β1 precursor, is transported with integrin α subunits to the cell surface (6, 8,9,10), suggesting that regulation of the maturation of β1 may control cell-ECM or cell-cell adhesion and other cellular processes mediated by β1 integrins.

Although the β1 maturation process represents a mechanism for modulation of the function of β1 integrins, much remains unknown about how this process is regulated. It has been shown that TGFβ accelerates β1 maturation, thus increasing the cell surface levels of β1 integrins and enhancing cell adhesion (11, 12). It was found that knockout of low-density lipoprotein receptor-related protein-1 (LRP-1) significantly delays β1 maturation in mouse embryonic fibroblast cells and decreases cell surface levels of mature β1, leading to a decrease in cell adhesion ability (13). It has been demonstrated that antisense cDNA-mediated knockdown of talin markedly inhibits β1 maturation, leading to defective cell adhesion (14). These observations suggest that LRP-1 and talin are required for the proper maturation process of β1. In contrast to LRP-1 and talin, β1,4-galactosyltransferase V (GalT V) has been shown to inhibit β1 maturation because its knockdown by an antisense cDNA promotes β1 maturation and increases the cell surface levels of β1, thus leading to an increased cell adhesion (15). Similar to GalT V, presenilins have been shown to inhibit β1 maturation because knockout of these proteins markedly enhances β1 maturation in fibroblast cells (10). Apart from these proteinaceous modulators for β1 maturation, our recent studies (8) demonstrated that sphingosine, a bioactive lipid, potently inhibits β1 maturation, leading to defects in cell-ECM adhesion.

Sphingosine has been shown to mediate growth arrest, differentiation, and apoptosis of mammalian cells in response to various stressful stimuli (see reviews in refs. 16,17,18,19,20). Sphingosine is generated from the hydrolysis of ceramides through the action of ceramidases (21, 22). Five human ceramidases have been cloned, and they are termed acid ceramidase (AC/ASHA1; ref. 23), neutral ceramidase (NC/ASAH2; ref. 24), alkaline ceramidase 1 (ACER1/ASAH3; ref. 25), alkaline ceramidase 2 (ACER2/ASAH3L; ref. 26), and alkaline ceramidase 3 (ACER3/PHCA; ref. 27). These ceramidases are localized to various cellular compartments, including the plasma membrane (NC), lysosomes (AC), the Golgi complex (ACER2 and ACER3), and the ER (ACER1 and ACER3). Except for ACER3, all of the ceramidases have been shown to possess the ability to control the generation of sphingosine both in vitro and in cells. However, it remains unclear about the role of these ceramidases in regulating β1 maturation.

In this study, we demonstrate that sphingosine generated through the action of the human alkaline ceramidase 2, but not the other ceramidases, inhibits β1 maturation and cell adhesion mediated by β1 integrins. In contrast, RNA interference-mediated inhibition of ACER2 promotes β1 maturation and cell adhesion. Prolonged treatment with PMA inhibits both the expression of ACER2 and the generation of sphingosine, thus promoting β1 maturation. In contrast, all-trans retinoic acid (ATRA) up-regulates both the expression of ACER2 and the generation of sphingosine, inhibiting β1 maturation and its function in cells adhesion. Overexpression of ACER2 inhibits the stimulating effects of PMA on β1 maturation and cell adhesion, whereas ACER2 knockdown attenuates the inhibitory effects of ATRA on β1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway regulates β1 maturation and its function in cell adhesion.

MATERIALS AND METHODS

Reagents

Sphingosine and D-e-C24:1-ceramide were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Antibodies against GM130, integrin β1 subunit, and integrin α5 subunit were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse and anti-rabbit immunoglobulin G (IgG) antibodies conjugated with horseradish hydrogen peroxidase (HRP) were from Bio-Rad Laboratories (Hercules, CA, USA). Anti-mouse and anti-rabbit IgG antibodies conjugated with either fluorescein isothiocyanate (FITC) or rhodamine were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The ACER2 antibody was raised previously in this laboratory (26). Fibronectin, blasticidin, Zeocin, Opti-MEM, modified essential medium (MEM), and FBS were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Sulfo-NHS-LC-biotinylation kits and Micro BCA protein assay reagent kit were from Pierce Biotechnology (Rockford, IL, USA. Agarose beads conjugated with Phaseolus vulgaris lectin (PHA-L), PHA-L-agarose beads, were from Vector (Burlingame, CA, USA). Protease inhibitor cocktail tablets were from Roche Applied Science (Indianapolis, IN, USA). Endo-β-N-acetylglucominidase H (endo H) and peptide:N-glycosidase F (PNGase F) were from New England Biolabs (Ipswich, MA, USA). Orthophthalaldehyde (OPA) and other unlisted chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

Cell lines

MCF-7 cells and HeLa cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). T-REx HeLa cells that stably express the tetracycline repressor protein were purchased from Invitrogen. The ACER2-TET-ON cell line, previously called haCER2-TET-ON, was derived from T-REx HeLa cells in our previous studies (26). This cell line expresses ACER2 under the control of a tetracycline-responsive promoter system (CMV-TET-ON) so that the ectopic expression of ACER2 is induced by tetracycline (TET) but not ethanol (ET), the vehicle control. T-REx HeLa cells were also used to construct stable cell lines that express AC, NC, and ACER1, respectively, under the control of CMV-TET-ON. The coding sequence of each ceramidase was cloned into pcDNA4 (Invitrogen), and the resulting expression for each enzyme was transfected into T-REx HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The stable cell lines AC-TET-ON, NC-TET-ON, and ACER1-TET-ON, which express AC, NC, and ACER1, respectively, in the presence of 10 ng/ml TET but not the vehicle control ET, were selected in MEM containing 5 μg/ml blasticidin and 150 μg/ml Zeocin according to the method we previously developed (26).

RNA interference

A control small-interfering (si)RNA [siCON-2; 5′-UAAGGCUAUGAAGAGAUACUU-3′ (sense)/5′-GUAUCUCUUCAUAGCCUUAUU-3′(antisense)] and an ACER2-specific siRNA [siACER2; 5′-UGACCGAGCUUUCUGCGAGUU-3′ (sense)/5′-CUCGCAGAAAGCUCGGUCAUU-3′ (antisense)] were purchased from Dharmacon (Lafayette, CO, USA).siRNAs were transfected into HeLa or MCF-7 cells using oligofectamine according to the manufacturer’s instructions.

Immunostaining and confocal microscopy

Cells were fixed with 3.7% PBS-buffered paraformaldehyde, washed once with PBS, and permeabilized with 0.1% Triton X-100. After being blocked in 1% BSA in PBS, the cells were incubated with a primary antibody for 1 h at room temperature or overnight at 4°C. After being washed with PBS, the cells were incubated with a secondary antibody conjugated with FITC or rhodamine. After a final wash, the cells were analyzed under a laser-scanning confocal microscope (LSM Meta 540; Carl Zeiss, Thornwood, NY, USA) as described previously (26).

Electron microscopy

ACER2-TET-ON cells were grown to a 95% confluence in the presence of ET or TET (1 ng/ml) in T25 tissue culture flasks before being fixed for 1 h with 3.75% paraformaldehyde in PBS. Fixed cells were washed with 0.1 M sodium cacodylate buffer (pH 7.4) and postfixed for 1 h in 1% osmium tetroxide in the cacodylate buffer. After being rinsed with the cacodylate buffer, the cells were dehydrated in a series of ET and infiltrated with Epon-Aradite (Ted Pella, Redding, CA, USA). The cell samples were polymerized at 60°C for 2 days before ultrathin sections (∼80 nm) were cut on a Reichert Ultracut E microtome (Leica Microsystems, Wetzlar, Germany) and collected on 150 mesh grids. The sections were counterstained with uranyl acetate and lead citrate and examined under an FEI CM120 transmission electron microscope (FEI Co., Hillsboro, OR, USA) equipped with a Gatan GIF100 image filter (Gatan, Inc., Warrendale, PA, USA) operating at a beam energy of 120 keV. Images were acquired using a HitachiAMT camera system (Hitachi, Tokyo, Japan).

HPLC analysis of sphingosine

Cells were washed 3 times with PBS before they were subjected to Bligh-Dyer extraction (28). An aliquot of the extracted lipids was saved for the determination of the total phosphate (Pi). An internal standard (D-e-C17-sphingosine) was added to the remaining extracted lipids, which were treated with 0.125 M methanolic KOH for 30 min at 37°C to remove phospholipids. The base-hydrolyzed lipids were derivatized with OPA before HPLC analysis was performed for the content of sphingosine according to the method by Merrill et al. (29). The content of sphingosine in each sample was normalized to the total Pi, which was determined as described previously (30).

Alkaline ceramidase activity assays

Alkaline ceramidase activity was determined in microsomes according to the release of sphingosine from ceramide as described previously (26). Briefly, D-e-C24:1-ceramide was dispersed into a substrate buffer (SB), 25 mM glycine-NaOH, pH 9.0, 5 mM CaCl2, and 0.3% Triton X-100, by water bath sonication. The lipid-detergent mixture was boiled for 30 s and chilled on ice immediately to form homogeneous lipid-detergent micelles, which were mixed with an equal volume of microsomes suspended in an enzyme buffer, SB with no Triton X-100. The final concentrations of D-e-C24:1-ceramide and Triton X-100 were 150 μM and 0.15%, respectively. Enzymatic reactions were initiated by incubating the substrate-enzyme mixtures at 37°C for 20 min. The reactions were stopped by boiling the substrate-enzyme mixture, and the reaction mixtures were completely dried in a 180°C oven. Sphingosine was extracted and assayed by HPLC analysis as described above.

Western blot analysis

After being washed with ice-cold PBS, cells were lysed in a lysis buffer (LB), 10 mM Tris-HCl, pH 7.4, containing 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton-X 100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, and protease inhibitor cocktail. The cell lysates were then centrifuged at 14,000 g for 15 min at 4°C, and the resulting supernatant was assayed for protein concentrations using Micro BCA protein assay reagent kit. Proteins (40–60 μg) were separated on SDS-polyacrylamide gels and were transferred onto nitrocellulose membrane blots (Bio-Rad Laboratories). After being blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature, the membrane blots were incubated with a primary antibody for 1 h. After a wash with PBST, the blots were incubated with a goat anti-rabbit IgG or anti-mouse IgG antibody conjugated with a HRP. Protein bands were detected by an enhanced chemiluminescence (ECL) system (ECL plus; Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer’s instructions.

Protein precipitation by lectin

Cell lysates (∼500 μg proteins in 500 μl) in 50 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, and protease inhibitor cocktail were incubated at 4°C with 50 μl PHA-L bead slurry for 4 h. The PHA-L beads were washed five times (15 min each) with the above buffer without protease inhibitor cocktail, and bound proteins were released from PHA-L beads by boiling them in SDS-PAGE sample loading buffer. Proteins were then resolved on an 8% SDS-polyacrylamide gel and were transferred to a nitrocellulose membrane. The membrane was incubated with the anti-β1 antibody, followed by incubation with an HPR-conjugated anti-rabbit or mouse IgG antibody, and protein bands bound with the HRP-conjugated antibody were detected by ECL as described above.

Cell surface integrin assays

Cells grown in 10 cm culture plates were washed three times with ice-cold PBS before they were treated with the membrane-impermeable biotinylation reagent sulfo-NHS-LC- biotin (0.1 mg/ml) for 30 min at 4°C according to the manufacturer’s instructions. Biotinylation reactions were terminated with glycine (100 mM) in 50 mM Tris-HCl, and 150 mM NaCl, pH 7.4, for 15 min at 25°C. After being washed 3 times with ice-cold PBS, biotinylated cells were lysed in LB and protein concentrations were determined by the BCA method. Biotinylated cell surface proteins were precipitated by streptavidin-Sepharose from the clear cell lysates with the same amount of proteins. After the precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, integrin receptors were detected by Western blot analysis using anti-integrin antibodies.

Cell adhesion assays

Cell adhesion assays were performed according to the method developed in our previous study (8). Briefly, cells were dislodged by trypsin-EDTA, which was neutralized by 10% FBS and was removed by several washes with PBS. The dislodged cells were resuspended in serum-free MEM (for HeLa or T-REx HeLa cells) or RPMI1640 medium (for MCF-7 cells) before they were plated onto 24-well plates coated with type I collagen solution (20 μg/ml) or fibronectin solution (10 μg/ml). Cells were then allowed to adhere to the fibronectin or collagen-coated surface for 20–30 min before floating cells and loosely attached cells were removed by washing with PBS. The remaining adherent cells were fixed with 3.75% paraformaldehyde, and the fixed cells were stained for 5 min with 0.25% crystal violet. After being washed with PBS, the stained cells were lysed in 70% ET. The absorbance of the clear cell lysate was measured at a wavelength of 540 nm under a spectrophotometer.

Protein deglycosylation

ACER2-TET-ON cells were lysed in 50 mM sodium citrate buffer, pH 6.0, containing 150 mM NaCl2, 1% Triton X-100, 0.5% SDS, 40 mM DTT, and protein inhibitor cocktail. Cell lysates were centrifuged for 10 min at 15,000 g at 4°C, and the resulting supernatants were boiled for 10 min before being treated with endo H according to the manufacturer’s instructions.

Statistical analysis

Student’s t test was applied for statistical analysis. Data are presented as means ± sd. Statistically significant differences are reported as P ≤ 0.001 and P ≤ 0.01.

RESULTS

ACER2 overexpression inhibits β1 maturation

Our previous studies (8) showed that treatment of HeLa cells with D-e-C6-ceramide inhibits β1 maturation due to an increased generation of sphingosine. Because overexpression of ACER2 increased the cellular levels of sphingosine (26), we investigated whether ACER2 has a role in controlling β1 maturation by determining the effect of an increased expression of ACER2 on the relative levels of mature β1 to β1 precursor in T-REx HeLa cells. We previously generated a stable cell line derived from T-REx HeLa cells, ACER2-TET-ON cells, in which the ectopic expression of ACER2 is induced by TET at concentrations from 1–100 ng/ml in a dose-dependent manner (26). Because a high ectopic expression of ACER2 might generate artifacts, the minimum concentration (1 ng/ml) of TET was used to induce low ectopic expression of ACER2 in ACER2-TET-ON cells. Western blot analysis showed that treatment with 1 ng/ml TET, but not the vehicle control ET, induced a moderate increase in the expression of ACER2 (Fig. 1A). HPLC analysis showed that the low ectopic expression of ACER2 caused a 3-fold increase in the levels of sphingosine in ACER2-TET-ON cells (Fig. 1B). Western blot analysis revealed that TET-induced low ectopic expression of ACER2 decreased the levels of the β1 band (high β1 band) with a high apparent molecular weight, presumably mature β1, while increasing the levels of the β1 band with a low apparent molecular weight (low β1 band), presumably β1 precursor (Fig. 1C). It has been shown that mature β1 carries complex-type glycans, whereas the β1 precursor contains high-mannose-type glycans. To determine whether the two β1 protein bands, the high and low β1 bands revealed by Western blot analysis indeed represent β1 precursor and mature β1, respectively, the lysates of ACER2-TET-ON cells grown in the presence of ET or TET were subjected to deglycosylation analysis with endo H, which removes high-mannose or hybrid-type glycans from glycoproteins, while it leaves complex-type glycans intact. Western blot analysis demonstrated that treatment with endo H converted the low β1 band, but not high β1 band, to a new β1 band with the lowest apparent molecular weight, presumably the unglycosylated β1 core, in cell lysates of ACER2-TET-ON cells grown in the presence of ET or TET (Fig. 1D). It was also found that the levels of the endo H resistant β1 band were higher in ACER2-TET-ON cells grown in the presence of ET than in ACER2-TET-ON cells grown in the presence of ET (Fig. 1D). These results suggest that ACER2 expression inhibits β1 maturation.

Figure 1.

Figure 1.

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ACER2 overexpression inhibits β1 maturation and trafficking to the cell surface and cell-ECM adhesion. A) ACER2-TET-ON cells grown to an 80% confluence were treated with 1 ng/ml TET or ET for 48 h before microsomes were isolated from these cells. Microsomes were subjected to Western blot analysis using the anti-ACER2 antibody. The same membrane blot was reprobed with anti-β-actin, which serves as a control for protein loading. B) ACER2-TET-ON cells grown as described in A were harvested and subjected to HPLC analysis for the levels of sphingosine. C) ACER2-TET-ON cells were grown and treated with TET or ET as described in A before they were lysed in LB. Total cell lysates were subjected to Western blot analysis using the anti-β1 antibody. D) Cell lysates were prepared from ACER2-TET-ON cells grown in the presence of ET or TET before being boiled for 10 min. Denatured cell lysates were then treated overnight with endo H or buffer only before being subjected to Western blot analysis using the anti-β1 antibody. E) Glycoprteins were precipitated with PHA-L agarose beads from the cell lysates (500 μg proteins) of ACER2-TET-ON cells grown in the presence of ET or TET and subjected to Western blot analysis with the anti-β1 antibody. mat. β1, integrin β1 subunit mature form; pre. β1, integrin β1 subunit precursor form. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent 3 independent experiments with similar results. *P ≤ 0.001.

It has been shown that mature β1 is a major carrier of tri- and tetra-antennary β1,6-N-acetylglucosamine (β1,6-GlcNAc)-bearing N-glycans. We determined whether ACER2 expression decreases the levels of β1 carrying this type of glycans by lectin precipitation assays. β1 with β1,6-GlcNAc-bearing N-glycans was precipitated from ACER2-TET-ON cell lysates with agarose beads conjugated with PHA-L, which specifically binds to β1,6- GlcNAc-bearing N-glycans. Western blot analysis showed that ACER2 expression markedly decreased the levels of β1 bound to PHA-L (Fig. 1E), suggesting that ACER2 expression decreases the levels of mature β1 carrying β1,6-GlcNAc-bearing N-glycans.

ACER2 expression inhibits cell-ECM adhesion by decreasing the cell surface levels of β1 integrin

Because mature β1 but not β1 precursor is transported to the cell surface, we determined whether ACER2 overexpression decreases the cell surface levels of β1. ACER2-TET-ON cells were grown to an 80% confluence in the presence of 1 ng/ml TET or ET before the surface proteins were biotinylated. After being precipitated by biotin-binding agarose beads, the biotinylated mature β1 was detected by an anti-β1 antibody. Western blot analysis demonstrated that ACER2 overexpression indeed decreased the surface levels of mature β1 (Fig. 2A).

Figure 2.

Figure 2.

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ACER2 overexpression inhibits cell-ECM adhesion by reducing cell surface levels of β1 integrin. A) ACER2-TET-ON cells were grown to an 80% confluence in the presence of 1 ng/ml TET or ET before they were treated with the sulfo-NHS-LC-biotinylation reagent. Biotinylated proteins were immunoprecipitated with avedin-argarose beads, and biotinylated β1 integrin was detected by Western blot analysis using the anti-β1 integrin antibody. B) ACER2-TET-ON cells grown in the presence of ET or TET were subjected to Western blot analysis with the anti-α5 antibody for the total and cell surface levels of α5. C, D) ACER2-TET-ON cells were grown in the presence of 1 ng/ml TET or ET for 48 h before they were dislodged and replated onto 24-well plates coated with fibronectin (C) or collagen I (D). At 30 min postreplating, unattached cells were removed by washing with PBS, and adherent cells were immediately fixed with 3.75% paraformaldehyde and analyzed with crystal violet staining. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent at least 3 independent experiments with similar results. *P ≤ 0.001.

It has been shown that integrin α subunits are cotransported to the cell surface after they are associated with mature β1 (31), we determined whether ACER2 expression affected levels of integrin α subunits. As we previously demonstrated that the integrin α5 subunit (α5), which pairs with β1 to form the α5β1 integrin, is expressed in T-Rex HeLa cells (8), the cell surface levels of the biotinylated α5 were assayed by Western blot analysis with the anti-α5 antibody. It was found that ACER2 expression decreased the cell surface levels of α5 without affecting its total levels (Fig. 2B), suggesting that ACER2 expression decreases cell surface levels of α subunits associated with β1.

Because cell surface levels of β1 integrins are linked to the ability of cells to adhere to the ECM, we determined the effects of ACER2 expression on cell adhesion to the surface coated with collagen-I or fibronectin, ECM ligands of β1 integrins. ACER2-TET-ON cells were grown in the presence of 1 ng/ml TET or ET for 72 h before they were dislodged and replated onto 24-well plates coated with fibronectin or collagen I. Thirty minutes after cell replating, the number of adherent cells was determined by crystal violet straining. The results showed that TET-induced ectopic expression of ACER2 inhibited the adhesion of T-REx HeLa cells to the surface coated with either fibronectin (Fig. 2C) or collagen (Fig. 2D), suggesting that ACER2 overexpression inhibits cell-ECM adhesion mediated by β1 integrins by decreasing their cell surface levels.

Overexpression of AC, NC, or ACER1 has no effect on β1 maturation

As mentioned earlier, in addition to ACER2, AC, NC, or ACER1 has been shown to catalyze the generation of sphingosine in cells. This prompted us to test whether overexpression of each of these ceramidases has the same effect as overexpression of ACER2 on β1maturation. Using the same expression system for ACER2, we established stable cell lines, AC-Tet-On, NC-TET-ON, and ACER1-TET-ON, which ectopically express AC, NC, and ACER1, respectively, under the control of the inducible promoter CMV-TET-ON. To facilitate the detection of their expression, NC and ACER1 were tagged with an epitope tag, the FLAG tag. Western blot analysis with an anti-FLAG M2 antibody demonstrated that the ectopic expression of NC and ACER1 was induced with 1 ng/ml TET, but not ET, in NC-TET-ON and ACER1-TET-ON cells, respectively (Fig. 3A). Western blot analysis with an anti-AC antibody showed that TET at 1 ng/ml also increased the expression of AC in AC-TET-ON cells (Fig. 3A). HPLC analysis showed that overexpression of AC or ACER1 caused a severalfold increase in the cellular levels of sphingosine, whereas overexpression of NC had no effect on the cellular levels of sphingosine (Fig. 3B). Western blot analysis revealed that overexpression of ACER1 or AC slightly increased the ratio of mature β1 to β1 precursor in T-REx HeLa cells, whereas overexpression of NC had no effect (Fig. 3C), suggesting that the ceramidases other than ACER2 have a minimal role or no role in regulating β1 maturation.

Figure 3.

Figure 3.

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Overexpression of AC, NC, or ACER1 does not inhibit β1 maturation. A) AC-TET-ON, NC-TET-ON, and ACER1-TET-ON cell lines that ectopically express AC, NC, and ACER1, respectively, under the control of a tetracycline (Tet) responsive promoter, CMV-TET-ON, were established as described in Materials and Methods. These cell lines were grown for 48 h in the presence of 1 ng/ml TET or ET. Ectopic expression of ACER1 and NC was determined by Western blot analyses using the anti-FLAG antibody, and the expression of AC was detected by the anti-AC antibody. B) AC-TET-ON, NC-TET-ON, and ACER1-TET-ON cells were grown in the presence of 1 ng TET or ET as described in A were subjected to HPLC analysis of the levels of sphingosine. C) AC-TET-ON, NC-TET-ON, and ACER1-TET-ON cells grown in the presence of TET or ET were analyzed for β1 maturation by Western blot analysis with the anti-β1 antibody. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent at least 3 independent experiments with similar results. *P ≤ 0.001.

ACER2 knockdown promotes β1 maturation

Because overexpression of ACER2 inhibited β1 maturation, we investigated whether inhibition of ACER2 has the opposite effect. With quantitative reverse-transcriptase-polymerase chain reaction (qRT-PCR) analysis, we showed that ACER2 is expressed at a much higher level in MCF-7 cells than in T-REx HeLa cells (data not shown). Therefore, we determined the effect of knockdown of ACER2 on β1 maturation in MCF-7 cells. MCF-7 cells were transfected with the control siRNA (siCON) or the ACER2-specific siRNA (siACER2). Western blot analysis demonstrated that transfection with siACER2 markedly inhibited the expression of ACER2 in MCF-7 cells compared with transfection with siCON (Fig. 4A). HPLC analysis showed that ACER2 knockdown decreased the cellular levels of sphingosine (Fig. 4B). Western blot analysis revealed that ACER2 knockdown markedly increased the levels of mature β1 but decreased those of β1 precursor (Fig. 4C), suggesting that ACER2 down-regulation significantly enhances β1 maturation.

Figure 4.

Figure 4.

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Knockdown of ACER2 enhances β1 maturation and trafficking to the cell surface and cell-ECM adhesion. A) MCF-7 cells were transfected with siCON or siCER2 for 48 h before Western blot analyses were performed to determine the expression of ACER2. B) MCF-7 cells transfected with siCON or siACER2 were subjected to HPLC analysis to measure sphingosine. C) MCF-7 cells transfected with siCON or siACER2 were subjected to Western blot analysis using the anti-β1 antibody. D) MCF-7 cells were transfected for 48 h with siCON or siACER2before the cell surface β1 integrin was detected by biotinylation as described in Fig. 1D. E, F) MCF-7 cells were transfected with siCON or siACER2 for 48 h before they were subjected to assays for cell adhesion to fibronectin (E) or collagen I (F) as described in Fig. 2C, D. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent at least 3 independent experiments with similar results. *P ≤ 0.001; **P ≤ 0.01.

Since ACER2 knockdown promotes β1 maturation, we investigated whether ACER2 knockdown promotes the trafficking of β1 to the cell surface. MCF-7 cells were transfected with siCON or siACER2 for 72 h before they were subjected to biotinylation assays for the cell surface β1. Western blot analysis demonstrated that ACER2 knockdown significantly increased the cell surface levels of β1 in MCF7 cells (Fig. 4D), suggesting that ACER2 down-regulation promotes the trafficking of β1 to the cell surface.

Next, we determined whether ACER2 knockdown promotes cell adhesion. MCF-7 cells were transfected with siCON or siACER2 for 2 days before they were assayed for cell adhesion. MCF-7 cells transfected with siACER2 adhered to the surface coated with either fibronectin (Fig. 4D) or collagen (Fig. 4E) better than MCF-7 cells transfected with siCON, suggesting that ACER2 knockdown promotes cell-ECM adhesion mediated by β1 integrins.

Retinoic acid (ATRA) inhibits β1 integrin maturation and cell adhesion by up-regulating the ACER2/sphingosine pathway in HeLa cells

Thus far we have demonstrated that genetic manipulations of the expression of ACER2 significantly altered β1 maturation and the function of β1 integrins in cell adhesion. Following these findings, we investigated whether stimuli that regulate the ACER2/sphingosine pathway have roles in controlling β1 maturation and its function in cell adhesion. Our studies on ACER2 promoter activity revealed that ATRA increased ACER2 transcription in HeLa cells (unpublished data). In vitro activity assays confirmed that ATRA increased ACER2 activity in HeLa cells in a time-dependent manner, and this activity increase reached a plateau at 10 h (Fig. 5A). Western blot analysis demonstrated that treatment with ATRA increased the expression of ACER2 in HeLa cells (Fig. 5B). Consistent with ACER2 up-regulation, ATRA caused a >3-fold increase in the levels of sphingosine in HeLa cells (Fig. 5C). These results suggest that ATRA up-regulates the ACER2/sphingosine pathway.

Figure 5.

Figure 5.

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ATRA inhibits β1 and cell adhesion by up-regulating the ACER2/sphingosine pathway. A) HeLa cells were treated with 1 μM ATRA or DMSO for different time periods before microsomes were isolated and subjected to alkaline ceramidase activity assays. B) HeLa cells were treated with 1 mM ATRA or DMSO for 12 h before they were subjected to HPLC analysis for the levels of sphingosine. C) HeLa cells were treated with 1 μM ATRA or DMSO for 12 h before the cells were harvested and lysed in LB buffer. Cell lysates from HeLa cells treated with 1 μM ATRA or DMSO were adjusted to have the same protein concentration. ACER2 was then immunoprecipitated with the anti-ACER2 antibody from an equal volume of the cell lysates from cells treated with ATRA or DMSO before being detected by Western blot analysis with the anti-ACER2 antibody. D) HeLa cells treated with 1 μM ATRA or DMSO as described in A before β1 maturation was determined by Western blot analysis. E, F) HeLa cells were transfected with 5 nM siCON or siACER2 for 48 h before they were treated with 1 μM ATRA or DMSO for 12 h. Cells were subjected to Western blot analysis for the expression of ACER2 (E) or β1 integrin (F). G) HeLa cells were transfected with 5 nM siCON or siACER2 for 48 h before they were treated with 1 μM ATRA or DMSO for 24 h. These cells were dislodged and subjected to assays for cell adhesion to fibronectin as described in Fig. 1E. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent at least 3 independent experiments with similar results. *P ≤ 0.001.

Consistent with an increase in the levels of sphingosine, Western blot analysis showed that ATRA markedly decreased mature β1 with a concomitant increase in β1 precursor (Fig. 5D), suggesting that ATRA inhibits β1 maturation. To investigate whether ACER2 up-regulation plays a role in the ATRA-induced inhibition of the maturation of β1, we determined the effect of ACER2 knockdown on the inhibitory effect of ATRA on β1 maturation. HeLa cells were transfected with siCON or siACER2 for 48 h before they were treated for 12 h with 1 μM ATRA or dimethyl sulfoxide (DMSO), the vehicle control. Western blot analysis showed that transfection with siACER2 markedly inhibited the ATRA-induced up-regulation of ACER2 in HeLa cells compared with transfection with siCON (Fig. 5E). Treatment with ATRA inhibited β1 maturation in HeLa cells transfected with siCON but not in HeLa cells transfected with siACER2 (Fig. 5F), suggesting that ACER2 up-regulation is responsible for the ATRA-induced inhibition of the maturation of β1 in HeLa cells.

It was observed that prolonged treatment with ATRA caused cell rounding and detachment from culture vessels, indicating that ATRA inhibits cell adhesion. To determine whether up-regulation of the ACER2/sphingosine pathway plays a role in ATRA-induced defective cell adhesion, we investigated the effect of ACER2 knockdown on the inhibitory effect of ATRA on cell adhesion. HeLa cells were transfected with siCON or siACER2 for 48 h before they were treated with ATRA or DMSO for 12 h. The cells were then subjected to assays for cell adhesion to fibronectin. The results showed that ATRA inhibited the adhesion of HeLa cells transfected with siCON and that this effect was attenuated by transfection with siACER2 (Fig. 5G), suggesting that ATRA inhibits cell adhesion at least in part through up-regulating the ACER2/sphingosine pathway.

PMA promotes β1 maturation and cell adhesion by down-regulating the ACER2/sphingosine pathway

ATRA inhibited β1 maturation and cell adhesion by up-regulating the ACER2/sphingosine pathway. This prompted us to investigate whether a stimulus that down-regulates the ACER2/sphingosine pathway promotes β1 maturation. Our analysis of ACER2 promoter activity revealed that PMA markedly down-regulated ACER2 transcription in T-REx HeLa cells (unpublished data). Alkaline ceramidase activity assays confirmed that treatment with 10 nM PMA decreased alkaline ceramidase activity in T-Rex HeLa cells in a time-dependent manner and that the maximal inhibitory effect was seen at 6 h and sustained thereafter (Fig. 6A). Western blot analysis revealed that treatment with PMA significantly decreased ACER2 protein levels in T-REx HeLa cells (Fig. 6B). HPLC analysis showed that treatment with PMA also decreased sphingosine in T-REx HeLa cells (Fig. 6C). These results suggest that PMA down-regulates the ACER2/sphingosine pathway in T-REx HeLa cells.

Figure 6.

Figure 6.

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PMA/PKC enhances β1 maturation and cell adhesion by down-regulating the ACER2/sphingosine pathway. A) T-REx HeLa cells grown to a 60% confluence were treated with 10 nM PMA or DMSO for different time periods before they were subjected to assays for alkaline ceramidase activity as described in Materials and Methods. B) T-REx HeLa cells were treated with 10 nM PMA or DMSO for 8 h as described in A before they were subjected to immunoprecipitation and Western blot analysis for ACER2 as described in Fig. 4C. C) T-REx HeLa cells were treated with PMA or DMSO as described in B before they were subjected to HPLC analysis for the levels of sphingosine. D) ACER2-TET-ON cells grown in the presence of ET or TET were treated with 10 nM PMA, 4 μM bisindolylmaleimide I (BIS), or DMSO for 6 h before they were analyzed for β1 maturation by Western blot analysis. E) ACER2-TET-ON cells grown in the presence of ET or TET were treated with 10 nM PMA or DMSO for 6 h before they were dislodged for assays of cell adhesion to fibronectin. Data represent means ± sd of 3 independent experiments performed in duplicate. Images represent at least 3 independent experiments with similar results. *P ≤ 0.001.

Subsequently, we determined whether PMA-induced down-regulation of the ACER2/sphingosine pathway enhances β1 maturation in T-REx HeLa cells. Western blot analysis showed that treatment with PMA for 6 h significantly increased mature β1 with a concomitant decrease in β1 precursor in T-Rex HeLa cells grown in the presence of ET and that this stimulatory effect was completely blocked by treatment with bisindolylmaleimide I (BIS), a PKC inhibitor, suggesting that PMA enhances β1 maturation by activating PKC (Fig. 6D). Treatment with PMA had no effect on β1 maturation in ACER2-TET-ON cells grown in the presence of TET (Fig. 6D), indicating that TET-induced expression of ACER2 inhibits the PMA-induced enhancement in β1 maturation in T-REx HeLa cells.

These results suggest that PMA/PKC promotes β1 maturation at least in part by suppressing the ACER2/sphingosine pathway. This prompted us to investigate whether there is a causal relationship between down-regulation of the ACER2/sphingosine pathway and the PMA-induced cell adhesion. ACER2-TET-ON cells grown in the presence of ET or TET were treated with 10 nM PMA for 6 h before they were subjected to assays for cell adhesion to the surface coated with fibronectin. Treatment with PMA enhanced cell adhesion in ACER2-TET-ON cells grown in the presence of ET, but not in those cells grown in the presence of TET (Fig. 6E), suggesting that PMA/PKC enhances cell adhesion in part by down-regulating the ACER2/sphingosine pathway.

ACER2 expression causes the dilation of the Golgi complex

Thus far we have demonstrated that sphingosine generated by the action of ACER2 inhibits both β1 maturation, but the mechanism for this action remains unclear. We previously demonstrated that treatment with D-e-C6-ceramide caused fragmentation of the Golgi complex (8). Because Golgi fragmentation may affect the function of the Golgi complex, including protein glycosylation, we investigated the effect of ACER2 expression on the structure of the Golgi complex by both confocal and electron microcopy. ACER2-TET-ON cells grown in the presence of ET or TET (1 ng/ml) were immunostained with an antibody against GM130, a Golgi membrane protein, followed by a secondary antibody, the anti-rabbit IgG antibody conjugated with rhodamine. The morphology of the Golgi complex was examined by confocal microscopy. Consistent with our previous finding (26), treatment with 1 ng/ml TET did not alter the overall morphology of the Golgi complex compared with treatment with ET (Fig. 7A). It has been shown that a subtle alteration in the structure of the Golgi complex can block the sialylation of β1 integrin (32). Thus, we examined the effect of ACER2 expression on the ultrastructure of the Golgi complex by electron microscopy. Treatment with TET caused a dilation of vesicles and cisternae especially in the trans-Golgi region compared with treatment with ET (Fig. 7B), suggesting that low ectopic expression of ACER2 alters the ultrastructure of the Golgi complex.

Figure 7.

Figure 7.

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ACER2 expression induces dilation of the Golgi complex. A) ACER2-TET-ON cells were grown to a 80% confluence in 1.5 cm culture plate with a coverslip bottom before they were treated for 48 h with 1 ng/ml TET or ET. The above cells were fixed with 3.75% paraformaldehyde in PBS and were labeled with the anti-GM130 antibody followed by the anti-mouse IgG antibody conjugated with rhodamine. Golgi complex was analyzed by confocal microscopy. B) ACER2-TET-ON cells were grown to a 95% confluence in the presence of ET or TET (1 ng/ml) in T25 tissue culture flasks before being processed for electron microscopic analysis. Golgi complex is labeled by arrowheads. Images represent 2 independent experiments with similar results.

DISCUSSION

The maturation of β1 is critical for its trafficking to the cell surface and for the activity of β1 integrins in binding to ECM proteins. Our previous studies (8) identified sphingosine as a potent inhibitor of β1 maturation. In this study, we identified the alkaline ceramidase 2 as the only known ceramidase with the ability to regulate β1 maturation by controlling the generation of sphingosine in the Golgi complex.

Sphingosine is generated in different cellular compartments and its role in cellular processes appears to be spatially specific. We demonstrated that overexpression of ACER2 but not AC or ACER1 inhibited β1 maturation, although overexpression of these ceramidases causes a similar increase in the cellular levels of sphingosine. This suggests that sphingosine that inhibits β1 maturation must be generated in the Golgi complex. This also indicates that sphingosine generated by the action of AC or ACER1 cannot traffic in the free form from other cellular compartments to the Golgi complex. Consistent with this notion, sphingosine exogenously added to cells was shown to be trapped in the lysosomes (33). Sphingosine generated in the ER may be locally phosphorylated to form S1P, which would be efficiently cleaved by the S1P lyase that is localized to the ER as well. Therefore, sphingosine generated in the ER may not be transported to the Golgi complex either. NC has been shown to generate sphingosine on the plasma membrane or in the extracellular milieu (34). As mentioned earlier, exogenous D-e-sphingosine can cross the plasma membrane but will be trapped in the lysosomes, and it is expected that sphingosine generated by the action of NC would have the same fate as exogenous sphingosine. Therefore, sphingosine generated by NC may not reach the Golgi complex in its free form. We showed that overexpression of NC failed to increase the cellular levels of sphingosine in T-REx HeLa cells, suggesting that overexpression of NC fails to inhibit β1 maturation integrin in T-REx HeLa cells due to its inability to generate sphingosine in this cell system. These observations suggest that sphingosine generated in the Golgi complex but not in the ER or the lysosomes inhibits β1 maturation.

Accumulating evidence suggests that β1 maturation enhances the trafficking of β1 integrins to the cell surface. Kim et al. (35) demonstrated that keratinocytes cultured in a serum-containing medium have a higher ratio of mature β1 to the precursor, leading to an increase in the levels of mature β1 on the cell surface, where the β1 precursor is absent. Zou et al. (10) showed that knockout of both presenilin 1 and presenilin 2 markedly enhances β1 maturation and increases the cell surface levels of mature β1. We (8) have previously shown that treatment with D-e-C6-ceramide inhibits the conversion of β1 precursor to mature β1, resulting in a decrease in mature β1 on the cell surface. In agreement with these studies, we showed that ACER2 overexpression inhibits mature β1, leading to a decrease in the cell surface levels of mature β1. Since cell adhesion activity correlates with the cell surface levels of functional integrins, a reduction of β1 integrins in the cell surface is a major mechanism by which ACER2 expression inhibits cell adhesion.

The mechanism by which sphingosine inhibits maturation β1 is not entirely clear but an alteration in the structure of the Golgi complex may be involved. It has been shown that the glycosylation of β1 in the Golgi complex is especially susceptible to a subtle change in the Golgi complex (32). We show that low ectopic expression of ACER2 caused a significant dilation of the trans-Golgi without affecting the overall integrity of the Golgi complex. This indicates that a structural change in the Golgi complex may be a possible mechanism for the inhibition of the maturation of β1 in response to ACER2 expression.

ATRA has been shown to inhibit cell-ECM adhesion by down-regulating the cell surface levels of β1 in many cell types (36,37,38,39) although the mechanism for this down-regulation is unclear. In this study, we demonstrated that ATRA markedly up-regulated the ACER2/sphingosine pathway and inhibited β1 maturation. RNAi-mediated knockdown of ACER2 inhibited not only the ATRA-induced up-regulation of ACER2 but also the inhibitory effect of ATRA on β1 maturation and cell adhesion. These results suggest that ATRA inhibits cell-ECM adhesion at least in part through up-regulating the ACER2/sphingosine pathway that negatively regulates β1 maturation.

Prolonged treatment with PMA has been shown to promote cell adhesion by increasing the biosynthesis of β1 integrins and the cell surface levels of β1 integrins (40). However, it remains unclear whether β1 maturation enhancement is a mechanism by which PMA increases the cell surface levels of β1 integrins and cell-ECM adhesion. In this study, we demonstrated that, by inhibiting the ACER2/sphingosine pathway, prolonged treatment with PMA enhanced β1 maturation in T-REx HeLa cells. Suppression of the PMA-induced down-regulation of the ACER2/sphingosine by overexpressing ACER2 inhibited both the PMA-induced maturation of β1 and cell adhesion, suggesting that enhancing β1 maturation by down-regulating the ACER2/sphingosine pathway represents a novel mechanism by which PMA increases the cell surface levels of β1 integrins and promotes cell-ECM adhesion. PMA has also been shown to enhance cell adhesion acutely by inducing the phosphorylation of integrins. Our unpublished data showed that short-term treatment with PMA neither inhibits ACER2 expression nor affects the activity of preexisting ACER2, suggesting that ACER2 may not modulate the short-term effect of PMA on cell adhesion.

The fact that the ACER2/sphingosine pathway regulates β1 maturation and cell adhesion in response to different stimuli highlights the importance of this pathway in controlling cell adhesion and integrin signaling.

Acknowledgments

This work is supported by the National Institutes of Health grant R01CA104834 (to C.M.) and a Veterans Affairs Merit award (to L.M.O.).

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