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Originally published In Press as doi:10.1074/jbc.M406397200 on September 1, 2004

J. Biol. Chem., Vol. 279, Issue 48, 50382-50390, November 26, 2004
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An Inhibitor of O-Glycosylation Induces Apoptosis in NIH3T3 Cells and Developing Mouse Embryonic Mandibular Tissues*

E Tian{ddagger}§, Kelly G. Ten Hagen{ddagger}, Lillian Shum¶, Howard C. Hang||, Yoannis Imbert**, William W. Young, Jr**, Carolyn R. Bertozzi||{ddagger}{ddagger}§§, and Lawrence A. Tabak{ddagger}¶¶

From the {ddagger}Biological Chemistry Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, Cartilage Biology and Orthopaedics Branch, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, ||Department of Chemistry, University of California, Berkeley, California 94720-1460, **Department of Molecular, Cellular and Craniofacial Biology, School of Dentistry, and the Departments of Biochemistry and Molecular Biology and Pharmacology and Toxicology School of Medicine, University of Louisville, Louisville, Kentucky 40292, and the {ddagger}{ddagger}Departments of Chemistry and Molecular and Cell Biology and §§Howard Hughes Medical Institute, University of California, Berkeley, California 94720-1460

Received for publication, June 8, 2004 , and in revised form, August 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1–68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1–68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein glycosylation is a key regulator of eukaryotic processes and cell-cell interactions (1). Mucin-type O-linked glycans (GalNAc{alpha}1-O-Ser/Thr) have recently been implicated in a variety of biological roles in a number of different systems. For example, one gene responsible for initiating mucin-type O-linked glycosylation in Drosophila has been shown to be required for development and viability in flies (2, 3). Additionally, a recent report has implicated mucin-type O-linked glycosylation as being responsible for familial tumor calcinosis in humans (4). Other studies have demonstrated that mice deficient in {beta}-1,3-galactosyltransferase, which forms the core 1 O-glycan structure Gal {beta}1,3GalNAc{alpha}1-O-Ser/Thr, exhibit disrupted association of endothelial cells with the extracellular matrix, resulting in defective angiogenesis and fatal brain hemorrhages in developing mice (5). Mucin-type glycans are also known to serve as cognate receptors for selectins involved in leukocyte rolling (6) and are thought to regulate avidity of T-cell receptor/CD8 binding to ligands and activation of T-cell function (7).

The first committed step in mucin-type O-glycosylation is catalyzed by a family of enzymes termed the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGaNTases,1 EC 2.4.1.41 [EC] ) (see Fig. 1), which have been shown to be conserved through much of evolution (8, 9). To date, 14 of the potential 24 mammalian isoforms have been functionally characterized (9, 10). Additionally, nine of the potential 14 isoforms from Drosophila melanogaster and three of the potential nine isoforms from Caenorhabditis elegans have been shown to be functional biochemically. In vitro assays of enzyme activity demonstrate that some isoforms display unique substrate specificities, whereas others are more promiscuous in terms of their preferred substrates. Comparison of both preferred substrates as well as sites of addition between mammalian and Drosophila orthologues shows a great deal of similarity, indicating that some enzyme activities have been conserved during evolution (3, 11).



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  		FIG. 1.
The reaction catalyzed by ppGaNTases and the inhibitor 1–68A. ppGaNTases initiate mucin-type O-linked glycosylation by transfer of GalNAc from the nucleotide donor UDP-GalNAc to Ser/Thr residues of a polypeptide substrate. The GalNAc residue is further elaborated by glycosyltransferases to generate mature O-linked glycans. 1–68A was identified from a uridine library screen as a broad-spectrum inhibitor of ppGaNTases.

 
Efforts to define the biological role of this enzyme family in mammalian systems have been hampered by the potential redundancy among the many isoforms present. To date, ablation of ppGaNTase-T1, -T4, -T5 or -T13 in mice have yielded no obvious phenotypes or only subtle phenotypes (9, 12, 13), likely because of overlapping or compensatory activity by other members of this family. Whereas recent studies in Drosophila and humans indicate that certain members of this gene family are important for various biological processes, dissection of the role of each family member may prove difficult to challenge in mammalian systems using standard genetic approaches as it may be necessary to ablate multiple ppGaNTases before obvious phenotypes are observable. Small molecules have proven very useful in situations where a genetic approach is not feasible (14). Recently, we identified a small molecule inhibitor (1–68A) of ppGaNTases from a uridine-based library (Fig. 1) (15). In an effort to begin to define the biological roles of the ppGaNTases, we exposed NIH3T3 cells to 1–68A and monitored changes in glycosylation and cell viability. We observed a decrease in the O-glycosylation of cells treated with inhibitor followed by induction of apoptosis. Overexpression of certain ppGaNTase isoforms restored cell surface glycosylation and rescued the inhibitor-induced apoptosis. Additionally, exposure of mandibular organ cultures to 1–68A produced similar decreases in lectin staining followed by induction of apoptosis. These studies suggest that the activity of the ppGaNTases may be required for cell viability and normal development and may be linked with the regulation of apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—The NIH3T3 mouse embryonic fibroblast cell line (ATCC) was grown in Dulbecco's modified Eagle's medium with 10% bovine calf serum (Invitrogen) and 0.1 mM non-essential amino acids at 37 °C in 6% CO2.

Mouse Mandibular Organ Culture—Mouse mandibular culture was performed as described previously (16, 17). Briefly, mouse embryos were dissected from time-pregnant Swiss Webster mice and staged according to Theiler stages (18). Mandibles of the first branchial arch were isolated by dissection and then cultured on a supporting filter (6-mm diameter, 0.8 µm, Millipore, Bedford, MA) and stainless steel grid at the air/medium interface. Mandibular explants were cultured in BGJb serum-less medium (Invitrogen) and supplemented with 0.1 µg/ml ascorbic acid, 100 units/ml penicillin, and 100 units/ml streptomycin. Three mandibles were cultured on each filter at 37 °C in 5% CO2 with the media changed every 2 days. All experiments were repeated at least three times.

1–68A Inhibitor Treatment and Specimen Preparation—A 200 mM stock of inhibitor (1–68A) was prepared using Me2SO (Sigma). NIH3T3 cells were seeded at a density of 2–4 x 104 cells/well into a 24-well format without (for flow cytometry) or with (for immunohistochemistry) cover slips (Fisher) 18–24 h prior to 1–68A treatment. Following transfection, serial concentrations of 1–68A were added to NIH3T3 cells. Serial concentrations of 1–68A were added to culture medium at the beginning of mandibular culture. Control cells and mandibles were cultured with an equal volume of the vehicle Me2SO. After incubation with 1–68A, cells and mandibular explants were washed twice with PBS (pH 7.4) and fixed with 4% paraformaldehyde-PBS for 1 h (cells) or at least 2 h (mandibles) at 4 °C and then washed with PBS again and stored at 4 °C.

Immunohistochemistry—Fixed cells on cover slips or paraffin sections of developing mandibles were blocked with 1% bovine serum albumin in PBS for 20 min at room temperature and then stained with fluorescein isothiocyanate-jacalin, biotinylated-ConA (EY Laboratories, San Mateo, CA), or Alexa Fluor 488 conjugated peanut agglutinin (PNA) (Molecular Probes, Eugene, OR) at a concentration of 5–10 µg/ml using conditions recommended by the manufacturer. Counterstaining was performed with Hochest 33342 (Molecular Probes) (for jacalin and PNA) or Methyl Green (for ConA). Carbohydrate inhibition controls were performed by preincubation of the lectin with 0.2 M galactose (for jacalin and PNA) or 0.2 M mannose (for ConA) for 30–60 min at room temperature before applying. For PNA staining, incubation with 0.2 units/ml neuraminidase (Sigma) was performed at 37 °C for 1 h (cells on cover slips) or 6–8 h (tissue sections) before PNA incubation. For O-GlcNAc detection, fixed cells were incubated overnight at 4 °C with mouse monoclonal anti-O-GlcNAc antibody (CTD110.6, Covance, Berkeley, CA) (19) at a dilution of 1:200 in 2% bovine serum albumin-PBS, then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:200. 50 mM N-acetylglucosamine (Sigma) was used for the carbohydrate competition during the primary antibody incubation. Slides were mounted with Fluoromount-G aqueous medium (Electron Microscopy Sciences). Immunofluorescence was examined with a Zeiss Axiovert 135 microscope, and the image was captured with a CoolSNAP digital camera. Images were analyzed and presented using Photoshop. The fluorescent pixels from three selected presumptive tooth and cartilage regions of each mandible were measured and expressed as a ratio of the total area of the mandible. Five different microscopic fields of cells or three microscopic fields of developing mandibles were scored to quantitate the cellular response.

Detection of Apoptosis—Apoptosis was detected using the phycoerythrin-conjugated polyclonal active caspase-3 antibody kit (BD Bioscience) by flow cytometry or by TUNEL staining using an In Situ Cell Death Detection Kit, TMR red (Roche Applied Science). Permeabilization was achieved using freshly made 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice (cells) or for 10 min at room temperature (mandibular sections). The glass cover slips or sections were counter-stained with Hochest 33342. Immunofluorescence was examined as above.

Flow Cytometry—Inhibitor-treated cells were fixed by 4% paraformaldehyde-PBS for 20 min on ice and then subjected to neuraminidase digestion (10 milliunits/105 cells) at 37 °C for 30 min followed by incubation with 25–50 µg/ml Alexa Fluor 488 conjugated PNA at room temperature for 30 min. Cells were analyzed on the FLH-1 channel using the FACSCaliber (BD Instruments).

Cloning of ppGaNTase Genes into Eukaryotic Expression Vectors— Each ppGaNTase gene was cloned into the eukaryotic expression vector pIRES2-EGFP (BD Biosciences), yielding a bicistronic mRNA from the cytomegalovirus promoter containing both a full-length ppGaNTase and enhanced green fluorescent protein (EGFP), separated by an internal ribosome entry site (IRES). This construct allows detection of cells expressing the polypeptide-GalNAc transferases through monitoring the EGFP signal. The ppGaNTase-mT1 expression vector (pIR-mT1EGFP) was produced by cloning the 2-kb SacII/BbvCI fragment from pBS-mT1 into the SacII/SmaI sites of pIRES2-EGFP. pIR-mT2EGFP was constructed by cloning the 1.9-kb EcoRI/AflII fragment from the vector pBS-mT2 into the EcoRI/SmaI sites of pIRES2-EGFP. pIR-mT4EGFP was produced by cloning the 2-kb EcoRV fragments from pBS-mT4 into the SmaI site of pIRES2-EGFP. pIR-rT5EGFP was constructed by cloning the 3.1-kb EcoRI/BstEII fragment of pBS-rT5(TB) into the EcoRI/SmaI sites of pIRES2-EGFP. pIR-rT10EGFP was constructed by cloning the 2-kb EcoRI/ScaI fragment of pBS-rT9(T10) into the EcoRI/SmaI sites of pIRES2-EGFP. Constructs were used for rescue assays of 1–68A-treated NIH3T3 cells.

Rescue Assay of 1–68A Treated NIH3T3 Cells—NIH3T3 cells were seeded at 5 x 104 cells/well in a 24-well plate the day before transfection. LipofectAMINE 2000 (Invitrogen) was used to transfect 1.5 µg of DNA per well of cells according to the manufacturer's instructions. 24 h after transfection, cells were split in half, allowed to incubate for 12 h, and then exposed to 1–68A (50 µM 1–68A) for 10 h. Cells were dissociated using enzyme-free PBS (Invitrogen) at room temperature and then stained with lectins (Alexa Fluor 647 conjugate) and/or caspase-3 (phycoerythrin conjugate), and analyzed by FACSCaliber under multiple channels (FLH-1 for EGFP, FLH-2 for phycoerythrin, and FLH-4 for Alexa Fluor 647).

Quantitative mRNA Analysis by Real-time Reverse Transcriptase-PCR—cDNA was synthesized from total RNA and real-time reverse transcriptase-PCR performed as described previously (20). Results are expressed as the-fold difference between the expression value for the ppGaNTase isoform and the endogenous control, glyceraldehyde-3-phosphate dehydrogenase. The data are presented as 1/(-fold difference) so that stronger expression is represented by a higher value (20).

Microarray Analysis—Total RNA was extracted from untreated or 1–68A-treated NIH3T3 cells using NucleoSpin purification kits (BD Biosciences) according to the manufacturer's instructions. Radioactive probes were generated using 3 µg of DNase I-treated total RNA labeled with [33P]dATP (Amersham Biosciences). AtlasTM plastic mouse 5K oligo microarrays (BD Biosciences) were hybridized overnight at 60 °C and then washed and exposed to a phosphorimaging screen overnight. The arrays were analyzed using Atlas Image 2.7 and Atlas Navigator 2.0 software. The array was stripped and reused three times. The intensity of each gene was averaged from two individual spots. A cDNA synthesis control was used as a positive control and for grid template alignment. All array experiments were normalized by glyceraldehyde-3-phosphate dehydrogenase and {beta}-actin. Genes were excluded if they were detected in only one spot or at levels near or below background.

Whole-mount Cartilage Staining and Histology—Three-dimensional architecture of developing cartilage was examined by Alcain blue staining as described previously (16). For histology, after fixing with 4% paraformaldehyde-PBS, mandibular tissues were dehydrated in graded series of ethanol, cleared in xylene, and embedded in paraffin. The paraffin blocks were sectioned at 5 µm and stained with hematoxylin and eosin (Sigma).

Statistical Analysis—Statistical comparisons were conducted by Student's t test under the conditions of two-tailed distribution and two-sample equal variance. A significant difference was present if p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Inhibitor 1–68A Reduces O-Glycosylation in NIH3T3 Mouse Fibroblast Cells—In an effort to characterize the role played by mucin-type O-glycosylation on cellular physiology, we exposed NIH3T3 cells to a compound (1–68A) that was previously shown to inhibit ppGaNTases in vitro (Fig. 1) (15). Cells exposed to 1–68A for 1–6 h were stained with jacalin (a lectin specific for O-linked glycosylation that recognizes sialylated and non-sialylated core 1) and PNA (a lectin that recognizes unmodified core 1). Diminution of staining with both lectins was observed microscopically after1hof exposure using 100 µM 1–68A (Fig. 2A–D). However, staining using antibodies to O-GlcNAc (the product of the UDP-GlcNAc:polypeptide O-{beta}-N-acetylglucosaminyltransferase or OGT enzyme) and the lectin ConA (that specifically recognizes N-linked glycans) was unaffected by this treatment (Fig. 2, E–H).



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  		FIG. 2.
1–68A diminishes O-glycosylation in NIH3T3 cells. Cells were treated with Me2SO (A, C, E, G, and I), 100 µM 1–68A (B, D, F, and H) or 4 µg/ml camptothecin (J) for 1 h. Cells were stained with jacalin (A and B) (green), PNA (C and D) (green), O-GlcNAc (E and F) (green), or ConA (G–J) (brown). Blue represents nuclear counterstain. Scale bar for A–J = 50 µm.

 
1–68A Induces Apoptosis in NIH3T3 Mouse Fibroblast Cells—Cytological examination revealed that cells exposed to 1–68A undergo apoptosis (as determined by TUNEL staining) at concentrations as low as 50 µM and after exposure for as short as 1 h (Fig. 3B). The number of cells undergoing apoptosis was seen to increase as the concentration of 1–68A was increased and the time of exposure was increased with very high percentages of cells staining positive for TUNEL after6hatall concentrations of inhibitor tested (Fig. 3). Following treatment with camptothecin, a known inducer of apoptosis, staining with all three lectins (jacalin, PNA and ConA) was abolished (Fig. 2, I and J, and data not shown).



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  		FIG. 3.
Loss of lectin staining prior to induction of apoptosis in NIH3T3 cells in the presence of 1–68A inhibitor. Cells were treated with Me2SO (A, E, and I), 50 µM 1–68A (B, F, and J), 100 µM 1–68A (C, G, and K), or 200 µM 1–68A (D, H, and L) for 1 h (A–D), 3 h (E–H) or 6 h (I–L). TUNEL staining is shown in red. Blue represents nuclear counterstain. Scale bar, 100 µm. Also depicted is the effect of time and concentration of 1–68A exposure on PNA staining (M) and caspase-3 activation (N). Me2SO- and 1–68A-treated cells were stained for PNA and caspase-3 and analyzed by flow cytometry at various time points. MFI, mean fluorescence intensity of gated cell population. Each bar represents the average of three repeats. *, p < 0.05; **, p < 0.001.

 
Reduction of O-Glycosylation in NIH3T3 Cells Occurs Prior to Apoptosis—Next we determined the relative time course of O-glycosylation inhibition and the induction of apoptosis by flow cytometry. Significant reduction in PNA staining was detected by flow cytometry as early as 30 min after treatment (Fig. 3M). Cells exposed to 1–68A were also stained with an antibody to the activated form of caspase-3 (an early marker of apoptosis). As seen in Fig. 3N, activated caspase-3 was first detected after 45 min of treatment with both 100 µM and 200 µM 1–68A (Fig. 3N). Therefore, a reduction in the amount of cell surface O-linked glycosylation is detected before early events in the initiation of apoptosis.

Rescue of Apoptosis by Overexpression of ppGaNTase Isoforms—To begin investigation of a causal role for O-linked glycosylation in the induction of apoptosis, we examined whether or not overexpression of ppGaNTase isoforms could inhibit or reduce the amount of apoptosis seen upon 1–68A exposure. Real-time reverse transcriptase-PCR results showed that ppGaNTase-T1 and -T2 are expressed to the highest degree in NIH3T3 cells, with ppGaNTase-T8, -T10, and -T4 at moderate levels and ppGaNTase-T7, -T11, -T12, and -T13 at lower levels (Fig. 4A). ppGaNTase-T3, -T5, -T6, and -T14 were not detected. For the overexpression studies, we cloned full-length ppGaNTase-T1, -T2, -T4, -T5, and -T10 into the pIRES2-EGFP expression vector. Additionally, we cloned two ppGaN-Tase-T1 mutants, ppGaNTase-T1E213Q and -T1E322Q (which display <0.3% and <1% of wild type activity, respectively (21)) into this expression system. This vector contains a cytomegalovirus promoter driving expression of each transferase followed by an IRES and the EGFP gene. This results in a bicistronic message from the cytomegalovirus promoter where cells expressing the transferases will also be expressing EGFP. We then examined caspase-3 staining in GFP-positive cells (i.e. cells expressing various transferases) and plotted the ratio of caspase-3 positive to negative cells when cells were exposed to Me2SO alone or 1–68A. As seen in Fig. 4B, transfection with vector alone does not reduce the amount of apoptosis seen in the presence of 1–68A to levels obtained with Me2SO alone. However, overexpression of ppGaNTase-T1, -T2, -T5, or -T10 results in a reduction in apoptosis to levels seen with Me2SO treatment. In contrast, expression of ppGaNTase-T4 or either of the mT1 mutants (ppGaNTase-T1E213Q and -T1E322Q) did not result in statistically significant reductions in apoptosis.



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  		FIG. 4.
Effects of overexpression of ppGaNTases on inhibitor induced apoptosis and lectin staining. A, endogenous ppGaNTase expression levels examined by real-time reverse transcriptase-PCR. Values are the mean of triplicate data points. Also shown are the effects of overexpression of ppGaNTases on apoptosis (B) and PNA staining (C). Cells were transfected with transferases, and EGFP-positive cells were then stained for the activated form of caspase-3 and for PNA after exposure to Me2SO or 1–68A. Ratio, caspase-3 positive/caspase-3 negative cells; MFI, mean fluorescence intensity of gated cell population. Six individual repeats of each transfection were performed for caspase-3 analysis, and five repeats were performed for PNA analysis. *, p < 0.05; **, p < 0.001.

 
We also examined whether or not the overexpression of each isoform was sufficient to restore cell surface lectin staining. Transfected cells were stained for PNA and analyzed for both EGFP and PNA by flow cytometry. Over-expression of ppGaNTase-T1, -T2, -T4, -T5, or -T10 restored the levels of PNA staining in the presence of inhibitor to those seen with Me2SO alone (Fig. 4C). The two mutants, ppGaNTase-T1E213Q and -T1E322Q, did not restore PNA staining, which is consistent with their low in vitro activity (21).

Microarray Analysis of Gene Expression Changes in Cells Treated with 1–68A—NIH3T3 cells treated with 1–68A for 1 and 3 h were compared with untreated cells using an AtlasTM plastic mouse 5K microarray. The data in Table I show genes whose expression was altered greater than 2-fold at both time points. Fig. 5 summarizes the classes of genes that were either up- or down-regulated after exposure to 1–68A. After 1 h of exposure, genes involved in translation and transcription were primarily affected. Additionally, genes involved in cell growth, adhesion, transport, nucleic acid binding, and stress response as well as those encoding membrane and structural proteins were also altered. After 3 h of 1–68A exposure, genes that were up-regulated consisted primarily of those involved in translation and stress response and those encoding membrane proteins. Genes that were down-regulated after 3 h were those responsible for translation, transcription, transport, cell growth, structure, signal transduction, and nucleic acid binding and those encoding membrane proteins.


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  		TABLE I
BD AtlasTM plastic mouse 5K microarray analysis of NIH3T3 cells treated with 1–68A The intensities were the average value of two duplicated spots and were normalized by AtlasImage software. The ratio was set at a 2-fold increase (red numbers) or decrease (blue numbers).

 



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  		FIG. 5.
Gene categories affected by 1–68A exposure of NIH3T3 cells. Shown are up-regulated (A) and down-regulated (B) genes after 1 h of 1–68A treatment, and up-regulated (C) and down-regulated (D) genes after 3 h of 1–68A treatment.

 
1–68A Inhibitor Disrupts Normal Mouse Mandibular Development in Organ Culture—A number of ppGaNTase family members are expressed in the mandibular region during murine development (22). To explore the potential role of O-glycosylation during early development and to examine whether the effects of 1–68A in cell culture are also seen in organ culture systems, we treated developing mouse mandibular organ cultures with 1–68A. Mandibular processes derived from the embryonic stage embryonic day 10 show an increased anterior-posterior growth by day 3 (Fig. 6, E and F) and a significantly reduced size by day 6 of treatment (Fig. 6, G and H). There is almost complete loss of Meckel's cartilage (Fig. 6, I–L) and tooth bud growth (Fig. 6L and data not shown), but the tongue appears to develop normally in most cases (Fig. 6, K and L).



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  		FIG. 6.
1–68A-induced morphological defects in mandibular explants. An embryonic day 10 mouse embryo at Theiler stage 18 (A) was dissected. Arrow indicates the mandibular process. The isolated mandibular explant from a first branchial arch (1st ba) at day 0 (B) and after 9 days in culture (C) in serumless, chemically defined medium is shown. D, whole-mount Alcian Blue staining shows Meckel's cartilage of mandibular explants cultured for 9 days in vitro. Also shown are embryonic day 10-derived mandibular explants treated with Me2SO (E) or 200 µM 1–68A (F) for 3 days. Pictured are embryonic day 10-derived mandibular explants treated with Me2SO (G) or 200 µM 1–68A (H) for 6 days, and Alcian blue staining of embryonic day 10-derived mandibular explants treated with Me2SO (I) or 200 µM 1–68A (J) for 6 days. K and L, hematoxylin and eosin staining of E10-derived mandibular explants treated with Me2SO (K)or200 µM 1–68A (L) for 6 days. Scale bar for A, 1 mm; for B–J, 100 µm; and for K and L, 100 µm. mc, Meckel's cartilage; tg, tongue.

 
1–68A Reduces Cell Surface O-Glycosylation in Mandibular Cultures—Embryonic day 10-derived mandibular explants were stained with jacalin and PNA lectins to determine whether 1–68A exposure results in a decrease in cell surface glycosylation. Both jacalin and PNA staining were significantly reduced in both the epithelium and mesenchyme after 6 h of 1–68A treatment (Fig. 7, A–P). ConA staining was unchanged by 1–68A (Fig. 7, Q–X). Thus, the inhibitory effect of 1–68A is related to O-linked glycans and does not significantly affect N-glycosylation.



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  		FIG. 7.
Lectin staining of mandibular explants at different time points after 1–68A treatment. Embryonic day 10 explants were exposed to Me2SO (A, C, E, G, I, K, M, O, Q, S, U, and W) or 200 µM 1–68A (B, D, F, H, J, L, N, P, R, T, V, and X) for 1 h (A, B, I, J, Q, and R), 3 h (C, D, K, L, S, and T), 6 h (E, F, M, N, U, and V), or 12 h (G, H, O, P, W, and X). Jacalin staining (A–H) and PNA staining (I–P) are shown in green. ConA staining (Q–X) is shown in brown. Nuclear counterstaining is shown in blue (A–P) or in green (Q–X). Scale bar, 50 µm.

 
1–68A Induces Apoptosis in Mandibular Explants—To investigate whether 1–68A exposure induced apoptosis in embryonic day 10-derived mandibular cultures, TUNEL staining was performed at different time points after inhibitor treatment. Following exposure to 1–68A, significant apoptosis was seen after 12 h (Fig. 8, G and H), 24 h (Fig. 8, I and J), and 48 h (Fig. 8, K and L) within the epithelial region of the developing mandible (Fig. 8M, p < 0.001). A statistical summary of four individual explants treated with either Me2SO or 1–68A shows a significant increase in dental epithelial apoptosis after 12 h, 24 h, and 48 h as well as increased apoptosis in the mesenchyme after 12 h and 24 h (Fig. 8N, p < 0.05).



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  		FIG. 8.
1–68A induces apoptosis in mandibular explants. Embryonic day 10 explants were treated with Me2SO (A, C, E, G, I, and K) or 200 µM 1–68A (B, D, F, H, J, and L) for 1 h (A and B), 3 h (C and D), 6h(E and F), 12 h (G and H), 24 h (I and J), and 48 h (K and L). TUNEL staining is shown in red, and nuclear counterstaining is shown in blue. Also represented is a statistical summary of epithelial (M) and mesenchymal (N) apoptosis at different time points. **, p < 0.001; *, p < 0.05. Percentage, number of TUNEL staining cells/total number of cells (detected by nuclear staining). Scale bar, 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mucin-type, O-linked glycosylation is a ubiquitous posttranslational modification. The large number of genes encoding enzymes responsible for O-glycosylation coupled with overlap in both the expression patterns and in vitro specificities of these glycosyltransferases makes it very challenging to employ a standard genetic approach to discern the functional roles played by this modification. Therefore we have made use of a small molecule inhibitor of the ppGaNTases that has previously been shown to induce apoptosis in two human cell lines, Jurkat and HEK293 cells (15). Here we show that 1–68A reduces cell surface mucin-type O-linked glycosylation in NIH3T3 cells prior to induction of apoptosis (while having no effect on N-linked glycosylation or cytoplasmic O-GlcNAc addition). Additionally, the effects of 1–68A can be rescued by overexpression of ppGaNTases. These results suggest a direct causative role for suppression of mucin-type O-glycosylation in the induction of apoptosis.

Overexpression of ppGaNTase-T1, -T2, -T5, or -T10 resulted in both a restoration of cell surface glycosylation as well as a reduction of apoptosis in NIH3T3 cells. However, ppGaNTase-T4, although able to restore cell surface glycosylation, was unable to rescue apoptosis. There are a number of possible models to explain our observations. First, overexpression of most (but not all) of the ppGaNTase isoforms restores the glycosylation of a key substrate(s) that triggers apoptosis when inappropriately O-glycosylated. There are a number of studies that suggest a link between O-glycosylation and apoptosis. For example, Yin et al. (23) reported that intracelluar oxidative stress-induced apoptosis can be prevented by expression of MUC1 (a highly glycosylated protein). Another study indicated that MUC1 attenuates the intrinsic apoptotic pathway (24). Recently, Zachara et al. (25) have shown that inhibiting O-GlcNAc addition to proteins renders cells more susceptible to stress and reduces cell survival. However, our current study has shown that 1–68A has no effect on O-GlcNAc addition in NIH3T3 cells (Fig. 2) nor on O-{beta}-N-acetylglucosaminyltransferase activity in vitro (data not shown) but rather inhibits mucin-type O-linked addition. Given that overexpression of ppGaNTase-T1, -T2, -T5, or -T10 rescues cells from inhibitor induced apoptosis but ppGaNTase-T4 overexpression does not, glycosylation of specific molecules by certain GalNAc transferases may be responsible for the rescue.

The connection between apoptosis and ppGaNTase inhibition may also be explained by the ppGaNTases themselves being critical "sensing" molecules, and once they are inhibited apoptosis is triggered. For example, ppGaNTases may be involved in sensing the intracellular concentration of hexosamines. It has been shown that excessive hexosamines can induce apoptosis in retinal neurons (26) or Jurkat cells (27). In the absence of functional ppGaNTases, UDP-GalNAc concentration may increase, ultimately leading to a rise in the intracellular hexosamine pool. However, one would have to argue that ppGaNTase-T4 lacks the ability to restore this pool, whereas ppGaNTase-T1, -T2, -T5, and -T10 are able to restore appropriate levels and avert programmed cell death.

Another possible explanation for the rescue seen with ppGaNTase overexpression may involve processes unrelated to O-linked glycosylation. Overexpressed transferases may bind to the inhibitor, reducing its concentration within the cell such that it is unable to affect other cellular processes that are responsible for the induction of apoptosis. To test this latter possibility, we also overexpressed mutant T1 isoforms (ppGaNTase-T1E213Q and -T1E322Q) whose in vitro activity is substantially below wild type (<0.3% and <1%, respectively (21)). Based on structural predictions, the mutations present within these compromised isoforms are not believed to lie within or near the putative binding site of UDP-GalNAc; by inference, they would also not be expected to affect 1–68A binding. Neither of these mutants restored cell surface glycosylation nor rescued the inhibitor-induced apoptosis, suggesting that the rescue seen with the wild type isoforms was a function of enzymatic activity as opposed to ability to bind 1–68A. However, direct binding measurements to 1–68A are required to demonstrate that binding affinities of wild type and mutant ppGaNTase-T1s are similar before this suggestion can be confirmed. Additional support comes from the binding affinities of the wild type isoforms; the Km for UDP-GalNAc as well as IC50 values for 1–68A are very similar for ppGaNTase-T1, -T2, -T4, -T5, and -T10 (15), yet ppGaNTase-T4 failed to significantly reduce the amount of apoptosis observed. This result again suggests that isoform-specific differences in ability to rescue apoptosis may be related to differences in substrate specificity among the isoforms rather than binding affinities to the inhibitor.

The rescue of apoptosis and restoration of lectin staining by the glycopeptide transferase ppGaNTase-T10 is somewhat surprising as this enzyme only adds GalNAc to previously glycosylated protein substrates in vitro (28). However, it remains possible that ppGaNTase-T10 may display different substrate specificities in the context of the Golgi apparatus of the cell, where it has access to factors that may alter enzyme activity or substrate accessibility. Additionally, it is also possible that there are yet uncharacterized ppGaNTases within the cell that are differentially susceptible to 1–68A inhibition and continue to add GalNAc on to certain proteins, creating substrates for ppGaNTase-T10.

Organ cultures were also susceptible to the apoptotic effects caused by 1–68A exposure. Stage embryonic day 10-derived mandibles showed reduction in O-linked glycosylation followed by significant apoptosis and structural effects in the presence of the inhibitor. These results indicate that this phenomenon is not confined to cell culture systems.

Microarray analysis of NIH3T3 cells after 1–68A exposure revealed that genes involved in several different cellular pathways were affected. Although many of the genes showing significant changes in expression levels do not have an obvious association with apoptosis, there are a number that have previously been shown to be involved in cellular stress responses and the modulation of apoptotic processes. For example, annexin A5, which is up-regulated 2-fold after 3 h, is known to be involved in and is used as a marker for apoptosis (29). Heat shock 70-kD protein 5 is also up-regulated almost 4-fold after 3 h; heat shock proteins are known to be synthesized in response to stress as cells attempt to protect themselves and reduce the incidence of apoptosis (30, 31). Ubiquitin, which is responsible for targeting proteins to the proteosome and is associated with many molecules involved in apoptosis (32–34), is up-regulated 2-fold after 3 h of exposure. Peroxiredoxin, a member of a family known to be involved in response to oxidative stress and regulation of apoptosis (35), shows decreased expression after 1 h of 1–68A exposure. And finally, TGF-{beta}, which is pivotal in cellular processes regulating apoptosis and cell growth, is down-regulated upon exposure to 1–68A (36). Although the significance of all the gene expression changes remains to be determined, there is clearly a noticeable effect on the expression of many genes involved in stress response and apoptosis.

In summary, we have found that an inhibitor of mucin-type O-linked glycosylation causes a reduction of O-glycans followed by apoptosis in both cell culture and organ culture systems. Additionally, over-expression of some ppGaNTase isoforms rescues inhibitor-induced apoptosis in cell culture, suggesting a potential role for O-glycosylation in the regulation of apoptosis. Future studies will be directed at elucidating the in vivo substrates of the glycosyltransferases in these systems and further investigating the connection between O-glycosylation and apoptosis.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant GM66047 (to C. R. B.) and EY015134 (to W. W. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a National Institutes of Health Visiting Fellowship grant. Back

¶¶ To whom correspondence should be addressed: NIDDK, National Institutes of Health, Bldg. 31, Rm. 2C39, 31 Center Dr., MSC 2290, Bethesda, MD 20892. Tel.: 301-496-3571; Fax: 301-402-2185; E-mail: tabakl{at}mail.nih.gov.

1 The abbreviations used are: ppGaNTase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase; PBS, phosphate-buffered saline; PNA, peanut agglutinin; EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site. Back


   ACKNOWLEDGMENTS
 
We thank current and past members of the Tabak laboratory for their many contributions. We appreciate the kind assistance provided by the members of the Shum group with the mandibular cultures. We also thank Dr. Matthew P. Hoffman in the National Institute of Dental and Craniofacial Research for help on BD AtlasTM mouse 5K plastic array software analysis, Dr. Bruce Raaka in the National Institute of Diabetes and Digestive and Kidney Diseases for support on flow cytometry analysis, and Tanya Leavy for performing in vitro assays of O-GlcNAc transferase.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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