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(The FASEB Journal. 1999;13:2329-2337.)
© 1999 FASEB

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice

DAISUKE KOYA*, JAMES W. DENNIS{ddagger}, CHARLES E. WARREN{ddagger}, NORIKO TAKAHARA*, FREDERICK J. SCHOEN{dagger}, YOSHIHIKO NISHIO*, TOSHIHIRO NAKAJIMA*, MYRA A. LIPES* and GEORGE L. KING1

* Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02215, USA;
{dagger} Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02215 USA; and
{ddagger} Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5

1Correspondence: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail: Kingg{at}joslab.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP-GlcNAc: Galß1–3GalNAc{alpha}Rß1–6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc-T with severe increase in cardiac core 2 GlcNAc-T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O-glycan and increases of c-fos gene expression and AP-1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc-T also induced c-fos promoter activation, mitogen activated-protein kinase (MAPK) phosphorylation, Trk receptor glycosylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc-T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.—Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.


Key Words: O-linked glycans • diabetes • cardiac hypertrophy • MAP kinase activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CARDIOVASCULAR DISEASES ARE the major cause of mortality and myocardial dysfunction in diabetic patients (1 2 3) . Using mRNA differential display, we have recently identified that the gene expression of the O-glycan-specific UDP-GlcNAc: Galß1–3GalNAc{alpha}Rß1–6N-acetylglucosaminyltransferase (core 2 GlcNAc-T)2 was increased in the hearts of diabetic rats (4) . O-glycan biosynthesis is initiated with the substitution of Ser/Thr by residues GlcNAc-T, then extension by ß1–3Gal-T to produce the disaccharide Galß1–3GalNAc-{alpha}, and followed with substitution by {alpha}2,3SA-T and {alpha}2,6SA-T, which completes the common O-linked tetrasaccharide structure (5) . The action of core 2 GlcNAc-T on the Galß1–3GalNAc-{alpha} intermediate is a key branch point in the pathway for the addition of polylactosamine and terminal sequence, such as the Lewis antigen (6) . Increases in core 2 GlcNAc-T activities have been associated with lymphocyte activation (7 , 8) , which results in the addition of branched O-glycans to a number of lymphocyte surface glycoprotein, including PSGL-1, CD43, CD44, CD45, and RPTP{alpha} (8 9 10) . The replacement of the common tetrasaccharide by core 2 GlcNAc-T dependent O-glycans is required for Slex expression on PSGL-1 and binding to the P-selectin and L-selectin on activated endothelium (9 , 11) . Core 2 GlcNAc-T O-glycan structures on CD43 glycoproteins also regulate cell–cell adhesion and lymphocyte activation (12 , 13) .

Previously, we reported that the expression of core 2 GlcNAc-T was increased in the myocardium of diabetic rats and was normalized by insulin and protein kinase C (PKC) activation (4) . Given that alterations in PKC activation, insulin resistance, and hyperglycemia have all been associated with cardiovascular complications, the increased expression of core 2 GlcNAc-T may contribute to cardiac pathology (14) . The myocardium of diabetic patients frequently exhibits cardiac muscle cell hypertrophy and increased glycoconjugate content detected as PAS-positive material (1 2 3 , 15.) . Thus, we have explored the possibility that hyperglycemia-induced overexpression of core 2 GlcNAc-T may contribute to the cardiac abnormalities similar to those observed in experimental animals and patients with diabetes (1 2 3 , 15) . To achieve this end, transgenic mice overexpressing core 2 GlcNAc-T under control of the {alpha}-myosin heavy chain ({alpha}MHC) gene promoter were made (16) and examined for cardiac pathology and molecular evidence of cardiac stress (17) . The results suggested that elevations of core 2 GlcNAc-T activities contributed to cardiac hypertrophy and increased c-fos/gene expression and AP1 activity, which are molecular markers of cardiac stress (17) . The mechanisms of actions of core 2 GlcNAc-T were explored further in PC 12 cells where core 2 GlcNAc-T overexpression also induced c-fos expression, AP1 activation, altered glycosylation, and signaling threshold of Trk receptors (18) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Anti-rat core 2 GlcNAc-T antibody was raised against GST-rat core 2 GlcNAc-T fusion protein, which was purified after induction with IPTG of a bacterial strain harboring a GST fusion expression vector pGEX-2T (Amersham, Arlington Heights, Ill.) fused to a full rat core 2 GlcNAc-T coding region. The anti-p44/42 MAP kinase and phosphoplus p44/42 antibodies were purchased from New England Biolabs (Beverly, Mass.). Monoclonal and polyclonal anti-c-fos antibodies, polyclonal anti-Trk antibody, and AP-1 consensus oligonucleotides were obtained from Santa Cruz Biotec (Santa Cruz, Calif.), and monoclonal anti-phosphotyrosine antibody were purchased from Upstate Biotechnology (Lake Placid, N.Y.). 125I-NGF (nerve growth factor) were purchased from New England Nuclear (Waltham, Mass.).

Generation and characterization of transgenic mice
A SalI/EagI fragment of the rat {alpha} myosin heavy chain promoter (-631 to +32, relative to translation initiation), including a heterologous splice cassette consisting of adenoviral and immunoglobulin sequences in Bluescript Ks (gift from Dr. Glenn I. Fishman) (16) , was inserted into SalI/NotI digested pBK-CMV (Stratagene, La Jolla, Calif.) to create pBK-MHC-S. The rat core 2 GlcNAc-T transgene was prepared by inserting a 2.1 kilobase StuI/SpeI fragment of the cDNA in Bluescript Ks into the SmaI site of pBK-MHC-S creating pMHC-rc2-S. pMHC-rc2-S was then digested with MluI/SacI to generate a linear fragment containing the transgene, which was purified and then microinjected into the pronuclei of fertilized FVB mouse eggs (19 , 21) . For Southern blot analysis, tail DNA from wild type and transgenic mice were digested with EcoRI/XhoI, separated on 1% agarose gels, and transferred to nylon membranes in 10x SSC. The filters were hybridized with EcoRI/XhoI fragments of transgene, which was labeled with {alpha}-32p-dCTP New England Nuclear using multiprime DNA labeling system (Amersham). Northern blot analysis was performed by fractionation of total RNA (20 µg) from hearts on a 1% agarose-formaldehyde gel followed by blotting onto nylon membranes in 10x SSC. Following ultraviolet crosslinking of the RNA to the membranes, the blots were hybridized with random priming labeled EcoRI/XhoI fragment of transgene. Northern blot analysis for c-fos was done with end-labeled c-fos oligonucleotide (Oncogene Science, Cambridge, Mass.). The enzymes used were purchased from New England Biolabs and used according to the manufacture’s directions.

Core 2 GlcNAc-T activity assay
Core 2 GlcNAc-T activity in the heart was measured as described (4 , 6) . In brief, PBS-rinsed frozen hearts were homogenized using a Polytron in 0.9% w/v saline, 0.4% v/v TritonX-100, 0.1 mm PMSF, and 0.1% w/v Trasylol on ice. Reaction mixtures contained 50 mm MES (pH 6.5), 1 mm UDP-GlcNAc, 0.5 µCi UDP-6[3H]-N-acetylglucosamine (New England Nuclear), 0.1 mm GlcNAc, 1 mm of Galß1–3GalNAc{alpha}-pNP (Toronto Research Chemicals, Toronto, Canada) as an acceptor, and 16 µl of heart lysate (8–12 mg/ml) for a final volume of 32 µl. Reactions were conducted for 1 h at 37°C, diluted to 5 ml in H2O, and applied to a C18 Sep-Pak (Millipore, Bedford, Mass.) in H2O, and washed with 20 ml H2O. The final products were eluted with 5 ml of methanol and radioactivity quantified in a scintillation counter. Endogenous activity was measured in the absence of an acceptor and subtracted from values determined in the presence of an acceptor.

Western blot, detection, and characterization of glycans
Heart tissues were homogenized in lysis buffer containing 0.1% v/v Tween 20, 50 mm Hepes (pH 7.4), 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 1 mm DTT, 10% v/v glycerol, 1 mm NaF, 2 mm PMSF, 25 mg/ml aprotinin, and 25 mg/ml leupeptin. Nuclear extracts from hearts were prepared as described by Frain et al. (22) . Immunoprecipitates of c-fos were prepared from total heart lysates using monoclonal anti-c-fos antibody. The samples were transferred to PVDF membranes (Novex, San Diego, Calif.) and processed for blotting against polyclonal anti-c-fos antibody, anti-rat core 2 GlcNAc-T antibody, anti-Trk antibody, anti-phosphotyrosine antibody, or digoxigenin-labeled lectins (Boehringer Mannheim, Mannheim, Germany). The immunoreactive bands for c-fos, core 2 GlcNAc-T, Trk, and phosphotyrosine of Trk were visualized by enhanced chemiluminesence (Amersham). The detection of glycans was performed using DIG Glycan differentiation kit (Boehringer Mannheim) according to the manufacturer’s instructions.

Histological examination
Sections were cut at 5 µM from hearts fixed in 2.0% paraformaldehyde and 2.5% glutaraldehyde and embedded in para-fin. The sections were stained with hematoxylin and eosin and examined by light microscope by cardiac pathologist (F. J. S) for hypertrophy, necrosis, inflammation, fibrosis, and vascular changes.

Gel mobility shift assay
Aliquots of cardiac nuclear extract (2–5 µg protein) in a 20 ml reaction mixture containing 10 mm Tris-Cl (pH 7.5), 5 mm DTT, 1 mm EDTA, 4% v/v glycerol, 1 mm MgCl2, and 10 mg/ml poly(dI-dC) were incubated for 10 min at room temperature before and after the addition of the end-labeled DNA probes. When competitor oligonucleotide was used, it was added before addition of the end-labeled DNA assay fragment and incubated for 10 min at room temperature. After 10 min, the samples were resolved by electrophoresis in 0.5 X Tris borate-EDTA on 4% polyacrylamide gels. The dried gels were developed with a Phosphor Imager.

Transient transfection assay
PC 12 cells (1x105) were plated in six-well dishes (Coster, Cambridge, Mass.) and allowed to attach overnight in Dulbecco’s modified Eagle medium supplemented with 20% fetal bovine serum. All transfections were performed using Lipofectamine (Gibco, Gaithersburg, Maryland) according to the manufacturer’s instructions. Transfections were carried out with 0.5 µg of CAT reporter plasmid (c-fos promoter containing -404 or -72) (22) , 0.5 µg of RSV-ß gal expression plasmid, and 0.5 or 1.0 µg of pCI-neo expression vector (Promega, Madison, Wisc.) CAT assay was done described elsewhere (26) . The results were quantified with a Phosphor Imager and normalized to ß galactosidase activity measured in the extract. In all cases, results were expressed as percent conversion of chloramphenicol to acetylated forms ± SD on the basis of three independent transfections.

Stable transfection of core 2 GlcNAc-T
PC 12 cells (1.0x105 cells/ml) were plated in 10 cm culture dishes 1 day before the transfection. Transfection was carried out with 1.0 µg/ml pCI-neo expression vector containing core 2 GlcNAc-T gene or control pCI-neo plasmid alone using Lipofectamine. Selection of cells transfected with or without core 2 GlcNAc-T was performed using culture media containing G418 at 300 µg/ml. After successful selection of neoresistant cells, cells were cloned by limited-dilution procedure.

125I-NGF binding assays
Competitive binding assays were performed using 12-well plates and 125I-NGF and serially diluted unlabeled NGF using a modification described by Urfer et al. (20) . After the addition of binding buffer (DMEM containing 20 mm Hepes and 0.5% BSA), unlabeled NGF was serially diluted in binding buffer to a concentration range of 0.1–1000 pM, and 5 µl labeled NGF was added at the final concentration in each well of 30 pM. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ligand. After 3 h incubation at 15°C, the wells were washed with cold PBS containing 0.5% BSA, and radioactivity was counted after harvesting the cells with 0.5 M NaOH.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transgenic mice expressing core 2 GlcNAc-T in the heart
The transgene contained the cardiac-specific rat {alpha} myosin heavy chain promoter (23) , a heterologous adenoviral-immunoglobulin splice cassette (17) , 2.2 kilobase of rat core 2 GlcNAc-T including the full coding region (4) , and SV40 poly(A) signal in the 3' flanking sequence (Fig. 1A ). Southern and Northern blot analysis using EcoRI/XhoI fragment of rat core 2 GlcNAc-T as probes showed the 2.1 kilobase genomic transgenic fragment and the expression of the 5.0 kilobase mRNA in the heart of transgenic mice, respectively (Fig. 1B , 1C ). Two transgenic mouse lines (low expression in line 3 and high expression in line 32) were established. Core 2 GlcNAc-T activity in the heart increased 5- and 20-fold in transgenic lines 3 and 32, respectively, as compared with wild type and correlated with the relative increases in core 2 GlcNAc-T protein contents (Fig. 1D , 1E ).



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  		Figure 1. Generation and characterization of transgenic mice. A) Schematic drawing of rat core 2 GlcNAc-T transgene. Transgene includes 5' regulatory elements from the cardiac-specific rat {alpha} MHC promoter, a heterologous splice cassette (cross-hatched box, open box, cross-hatched box), rat core 2 GlcNAc-T coding cDNA (DH-1, black box), and simian virus 40 poly(A) in 3' flanking region, as indicated in the scheme. EcoRI/XhoI fragment of transgene was used as a probe for Southern and Northern blot analysis, as shown below. B) Southern blot analysis of genomic DNA treated with EcoRI/XhoI fragment using the transgene as a probe. DNA from wild type and transgenic (lines 3 and 32) mice. Immobilized DNAs were hybridized with 32P-labeled probe. C) Northern blot analysis of total RNA. Samples from wild type and transgenic (lines 3 and 32) mice were indicated below each lane. Immobilized RNAs were hybridized with 32P-labeled probe. Relative positions of 18S and 28S ribosomal RNAs were shown on right. D) Western blot analysis of total heart lysates. Samples from wild type and transgenic (lines 3 and 32) mice were indicated below each lane. Relative mass, in kilodaltons, was indicated alongside. Arrowhead indicated the position of immunoreactive 53 kDa rat core 2 GlcNAc-T. E) Core 2 GlcNAc-T activities in the hearts. Core 2 GlcNAc-T activity assays were performed as described in Experimental Procedures. PBS-washed frozen hearts from wild type and transgenic (lines 3 and 32) mice were homogenized to obtain lysates. The radiolabeled core 2 GlcNAc-T reaction products in reaction mixture containing lysates were purified by C18 Sep-Pax column chromatography and counted in liqiud scintillation counter as described in Experimental Procedures. All data were shown as means ± SD for four different mice from each group. *P < 0.01 vs. wild type, **P < 0.01 vs. wild type and transgenic (line 3).

O-linked glycans in core 2 GlcNAc-T transgenic heart
Total heart lysates from wild type and transgenic mice were analyzed by lectin blotting analysis. Maackia amurensis agglutinin (MAA), which reacts with SA{alpha}2–3Gal sequences, stained more intensely the glycoprotein in the hearts of transgenic mice compared with wild type (Fig. 2A ). This is consistent with the specificity of core 2 GlcNAc-T that initiates a second branch per O-glycans and thereby potentially doubles the MAA-reactive SA{alpha}2–3Gal termini (24) . The levels of MAA reactivity in the liver and kidney were similar in transgenic and wild type mice and demonstrated that the transgene overexpression and core 2 branched O-glycans were increased specifically in the hearts (Fig. 2B ). Reactivity of cardiac glycoprotein with SA{alpha}2–6Gal specific lectins Galanthus nivalis agglutinin (GNA) or Sambucus nigra agglutinin (SNA) were unaffected (data not shown).



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  		Figure 2. Increased quantity of O-glycosylated proteins in transgenic mice. A) Hearts. B) Liver and kidney. Aliquots of total lysates from wild type and transgenic (lines 3 and 32) mice as indicated were heated in a sample buffer and fractioned on SDS-PAGE. After transfer to PVDF membranes, blots were stained with lectin MAA. Molecular mass, in kilodaltons, is indicated on the left.

Histological studies of the myocardium in core2 GlcNAc-T transgenic heart
Phenotypic changes were observed in the hearts of core 2 GlcNAc-T transgenic mice compared with wild type. The ratio of heart weight to body weight increased in the transgenic mice by as much as 24% and correlated with the amount of core 2 GlcNAc-T expression in the two transgenic lines (Fig. 3A ). Histological examination showed myocardial hypertrophy with increased wall thickness and myocyte enlargement at 6 months of age in transgenic mice line 32 compared with wild type (Fig. 3B , 3C ). There was no evidence of myocyte disarray, necrosis, or fibrosis. Moreover, medial thickening in myocardial arterioles and coronary arteries was not observed.



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  		Figure 3. Myocardial histology of transgenic mice. A) Heart weight to body weight ratio. Ventricular myocardium of wild type (B) and of transgenic mice (line 32) (C). Tissues were stained with hemotoxylin and eosin, 200x. All data are shown as means ± SD derived from 13 mice of each group. *P < 0.05 vs. wild type, ** P < 0.01 vs. wild type and transgenic mice (line 3).

c-fos expression and AP-1 binding in core 2 GlcNAc-T transgenic heart
The expression levels of c-fos protein and mRNA in the transgenic heart were increased ~10-fold in line 32 and 5-fold in line 3 (Fig. 4A, B ), whereas the expression of c-jun did not differ between transgenic and wild type. To determine whether c-fos protein expression could be increased in the myocardium of diabetic rats, as in the transgenic mouse, we also examined the expression of c-fos protein in heart lysates of NOD mice. This is a model of autoimmune-induced diabetes, which we had previously reported to have a twofold increase in core 2 GlcNAc-T activities in the heart (21) . c-fos protein in the hearts of diabetic NOD mice was increased by approximately fivefold compared with nondiabetic NOD mice (Fig. 4C ). The ratio of heart to body weight in diabetic NOD mice was greater than that in nondiabetic mice by 18% (4.72±0.20x10-3 vs. 4.01±0.12x10-3, mean ± SD, n=3, P<0.05), comparable to those in the core 2 GlcNAc-T transgenic mice.



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  		Figure 4. Expression of c-fos and AP-1 binding in the hearts of transgenic mice. A) Northern blot analysis of c-fos in the hearts. Blots of 20 µg total RNAs from wild type and transgenic (lines 3 and 32) as indicated below each lane were fractioned on 1% agarose gel, blotted on nylon membranes, and hybridized with either c-fos or 36B4 probe. Upper panel and lower panel arrowheads in Fig. 4A point to c-fos and 36B4 mRNAs, respectively. B) Western blot analysis of c-fos after immunoprecipitation with monoclonal anti-c-fos antibody. Samples from wild and transgenic (lines 3 and 32) mice were indicated below each lane. Relative mass, in kilodaltons, is indicated on the left. Line points to c-fos protein. C) Western blot analysis of c-fos after immunoprecipitation with monoclonal anti-c-fos antibody. Immunoprecipitated complexes were obtained using either 1,000 or 3,000 µg of total heart lysates from mice, as indicated above each lane. Samples were from nondiabetic (control) and diabetic NOD mice as indicated below each lane. Molecular mass, in kilodaltons, is indicated on left. Arrowhead points to c-fos protein. D) Enhancement of heart nuclear extract binding to the AP-1 site in transgenic mice. Representative result of gel mobility shift assay of heart nuclear extracts from wild type and transgenic mice (line 32) as indicated below each lane using the AP-1 oligonucleotide were shown. Presence or absence of competitor oligonucleotide (AP-1) at 50-fold molar excess is shown as plus or minus above each lane.

c-fos protein heterodimerizes with c-jun, and the resulting transcription factor complex binds DNA at AP-1 sites (also called TPA-response element, TRE) to stimulate the expression of a large and diverse groups of genes (25 26 27) . Therefore, we tested nuclear extracts from hearts of wild type and transgenic mice line 32 for binding activities to AP-1 consensus sequences using a gel mobility shift assay. AP-1 binding activities of the nuclear extracts from transgenic mouse hearts were double that of the wild type.

Transactivation of c-fos in core 2 GlcNAc-T transfected PC 12 cells
To study the role of branched O-glycans in the trans-activation of the c-fos gene, PC 12 cells, a well characterized cell link for studying c-fos expression, were transiently cotransfected with a combination of core 2 GlcNAc-T expression vector, a c-fos promoter fusion to CAT as a reporter (25 , 28) , and a RSV-ß Gal. Transfection of the PC 12 cells with core 2 GlcNAc-T vector increased the amount of MAA-lectin staining glycoprotein (Fig. 5A ) and core 2 GlcNAc-T enzyme (Fig. 5B ) in a dose-dependent manner. In addition, expression of c-fos protein (Fig. 5C ) was elevated in the core 2 GlcNAc-T transfected cells. Transfection of PC12 cells with the FC 4 constructs, which contained 404 bases of the c-fos promoter linked to CAT (25 , 28) , showed a sixfold induction of CAT activity on the addition of 50 ng/ml NGF (Fig. 5D ). This confirmed the inducibility of the c-fos expression by a receptor signaling pathway using SRE, as previously studied using NGF in PC12 cells (25 , 28) . PC12 cells cotransfected with c-fos-CAT vector and the core 2 GlcNAc-T exhibited higher CAT activity than cells cotransfected with control plasmid lacking core 2 GlcNAc-T cDNA in the presence or absence of NGF (Fig. 5D ). To exclude the possibility that increased CAT activity associated with core 2 GlcNAc-T expression arose because of the presence of cAMP responsive element (CRE), CAT construct driven by only 72 bases of c-fos promoter that contained only the CRE sites but not SRE was examined (22 , 28 , 29) . CAT activity was induced by vehicle or forskolin, a cAMP phosphodiesterase inhibitor, but cotransfection with core 2 GlcNAc-T had no effect on CAT activity in the presence or absence of forskolin (data not shown), indicating an effect of O-linked glycans required the presence of SRE.



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  		Figure 5. Transactivation of c-fos gene in core 2 GlcNAc-T transfected PC 12 cells. A, B) Increased MAA binding protein in parallel with increases in protein expression of core 2 GlcNAc-T. Aliquots of total cell lysates prepared from PC 12 cells transfected with 0.5 µg of control plasmid or 0.5–1.0 µg of core 2 GlcNAc-T expression plasmid, as indicated below each lane, were heated in a sample buffer and followed by SDS-PAGE. Blots were probed with lectin MAA A) Anti-rat core 2 GlcNAc-T antibody. B) Arrowheads point to core 2 GlcNAc-T (DH-1) protein. Molecular mass, in kilodaltons, is indicated on the left in panel A. C) Western blot analysis of c-fos after immunoprecipitation of cell lysates, as indicated below each lane, using monoclonal anti-c-fos antibody. D) Enhancement of c-fos-CAT reporter gene by core 2 GlcNAc-T transfection in PC 12 cells. Representative CAT assay of PC 12 cells transfected with c-fos-CAT reporter plasmid. 1.0 µg of control or core 2 GlcNAc-T expression plasmid were cotransfected with c-fos-CAT reporter plasmid into PC 12 cells as indicated below each lane in the upper panel or bar in the graph. The presence of vehicle or NGF (50 ng/ml) are indicated below lanes or bars as plus or minus. Bars indicate (14C) chloramphenicol converted to acetylated forms in each assay. Three independent CAT assays were performed.

Given that the activation of c-fos gene transcription can be regulated by the binding of SRF/TCFs complexes to SRE after the phosphorylation of Elk-1 by MAP kinase (30) , the activation of mitogen-activated protein kinase (MAPK) in control or PC12 cells transfected with core 2 GlcNAc-T were examined. The levels of phosphorylation in MAPK proteins were significantly increased by NGF in core 2 GlcNAc-T transfected cells compared with wild type cells (Fig. 6A ), whereas the protein levels of MAPK were unaffected, suggesting that increases in core 2 GlcNAc-T activities enhanced intracellular signaling of MAPK. To examine this possibility, the expression and the tyrosine autophosphorylation of Trk protein were studied. In cells stably transfected with core 2 GlcNAc-T, NGF-dependent tyrosine phosphorylation of Trk protein was also increased twofold in comparison to wild type (Fig. 6B ). The total protein expression of Trk was also increased in core 2 GlcNAc-T transfected cells, but NGF binding to PC 12 cells was unchanged. The total binding and Kdof NGF to core 2 GlcNAc-T transfected cells were 5.7 ± 0.3%, (mean ± SD, n=4) and Kd=2.8 x±0.8x10-9M), which were not different from its binding to wild type cells (5.6±0.3%, mean ± SD, n=4, Kd=3.0±1.1x10-9M). Trk from core 2 GlcNAc-T transfected cells showed increased MAA reactivity, characteristics of the core 2GlcNAc-T dependent change in O-glycans (Fig. 6C ). These results suggested that increase in branching of O-glycans of Trk by core-2 GlcNAc-T in the PC-12 cells enhanced post-receptor signaling without altering the binding properties of Trk to NGF (20) . Consistent with this interpretation, the core 2 GlcNAc-T transfected cells appeared phase-built and contained extended neurites even in the absence of NGF (Fig. 6D ). (9.7±1.9, n=3 in the wild type; 21.3±1.9, n=3 in core 2 GlcNAc-T transfected cells). These results suggested that core 2 GlcNAc-T dependent glycosyl of receptors (e.g., Trk) can enhance the down stream intracellular signal transduction pathways to effect changes in cellular responses through c-fos and MAPK activation. (Figs. 5D and 6A ).



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  		Figure 6. A) Phosphorylated MAPK was increased in PC 12 cells stably transfected with core 2 GlcNAc-T in response to NGF. After treatment of PC 12 cells with 25 ng/ml NGF for 5 min, cells were lysed and proteins were resolved by SDS-PAGE followed by immunoblot analysis. The membrane was probed with anti-phospho-specific MAPK antibody and anti-MAPK antibody. B) Trk protein expression was increased and was highly tyrosine phosphorylated in PC 12 cells stably transfected with core 2 GlcNAc-T. After treatment of PC 12 cells with 25 ng/ml NGF for 5 min, cells were lysed and processed to immunoprecipitate Trk followed by immunoblot analysis. The membranes were probed with monoclonal anti-phosphotyrosine antibody and anti-Trk antibody. C) Trk was highly O-glycosylated. After immunoprecipitation of total cell lysates with anti-Trk antibody, the samples were processed for immunoblot analysis and probed with specific lectins MAA and PNA. D) Core 2 GlcNAc-T induced differentiation of PC 12 cells. Light photomicrographs of PC 12 cells transfected either control plasmid or core 2 GlcNAc-T in the absence of NGF.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cardiac pathology in diabetic patients included atherosclerotic coronary artery disease and myocardial hypertrophy, excessive extracellular proteins, including glycoconjugates and fibrosis (1 2 3) . Myocardial changes may also occur in diabetic patients without significant coronary artery disease (1 2 3 , 31) .

The present study suggests that cardiac hypertrophy could partially be a result of the varied expression of core 2 GlcNAc-T. Previously, we reported that core 2 GlcNAc-T was specifically increased in cardiac tissues from streptozotocin-induced or spontaneously autoimmune induced diabetic animals compared with control rats (4) . The specificity for cardiac overexpression of the gene was confirmed by the increased core GlcNAc-T enzyme activities and mRNA levels observed in the hearts of diabetic rodents, which were prevented by insulin treatments. The effects of insulin could be indirect because it reduced plasma glucose levels from 5 to 22 mm, which we have shown can also increase core 2 GlcNAc-T transcripts in cardiomyocytes (4) . These observations prompted the present investigation to examine the contribution of core 2 GlcNAc-T overexpression on myocardial biochemistry and pathology.

In this study, two transgenic lines were made and shown to express 5- or 40-fold increases in cardiac core 2 GlcNAc-T activity. Both lines were grossly normal at birth but developed progressive myocardial hypertrophy observed at 4–6 months of age. Core 2 GlcNAc-T expression by immunohistochemistry was confined to cardiac myocytes beginning at birth and continued to increase through the development of hypertrophy. Increased heart-to-body-weight ratio was significant in both transgenic lines (i.e., 12 and 24%) and was transgene dose-dependent. The diabetic NOD mice at 8 wk of age showed a comparable increase of 18% in heart size, c-fos expressions, and, as reported earlier, an fivefold increase in core 2 GlcNAc-T transcripts (4) . These transgenic mice developed myocardial hypertrophy more slowly than the NOD mice, even though the increases in core 2 GlcNAc-T activities were greater, suggesting that factors associated with hyperglycemia and diabetes besides core 2 GlcNAc-T also contribute to the rapid development of hypertrophy. In this regard, other morphological abnormalities, commonly associated with late stage diabetic myocardial hypertrophy such as myocyte disarray, fibrosis, calcification, and vascular sclerosis, were not observed in the core 2 GlcNAc-T mouse (2 , 3 , 15 , 31) . Thus, core 2 GlcNAc-T and O-glycan products may contribute to a subset of these pathologies, and alternatively more time is required to develop additional histological abnormalities in the core 2GlcNAc-T transgenic mice. Elevated PKC-ß1 isoform activity has been associated with cardiac hypertrophy (30) , and PKC-ß2 transgene expression in myocardium caused pathology at 3–6 wk of age, a more rapid onset than the core 2 GlcNAc-T transgenic or diabetic mouse (30) . The PKC-ß2 transgenic mouse also showed necrosis, of cardiac myocytes, inflammation, fibrosis, and calcification, with lesions involving both the right and left ventricles. In the presence of diabetes alone, cardiomyopathy is usually mild, however, with the addition of hypertension or ischemic heart disease, cardiomyopathy could be severe. Either alone or in combination with other cardiac stresses, the core 2 GlcNAc-T transgenic mouse may provide a model of cardiomyopathy with a slower onset, similar in this regard to human disease progression.

Our results have suggested that the expression of core 2GlcNAc-T gene in cardiomyocytes can be regulated by plasma glucose levels (4) . The mouse core 2 GlcNAc-T gene is known to have at least two transcription start sites and is regulated in a tissue specific manner, with the distal promoter highly active in kidney, and the proximal promoter responsible for transcripts in other tissues (31) . The proximal promoter contains NF-IL6, GATA-3, TCF-1, SP1, and HNF-5 binding sequence in the 1st –560 bp. The mechanisms by which hypoglycemia and possibly diabetes enhances core 2 GlcNAc-T expression in cardiomyocytes could be related to PKC activation because PKC agonist, phorbol myristate acetate, also increased core 2 GlcNAc-T mRNA levels. Recently, PKC activation has been demonstrated in the myocardium of diabetic animals and patients (14 , 21) .

The transcription and protein levels of c-fos and AP-1 activity were increased in the myocardium of core 2 GlcNAc-T transgenic mice. Similar increases in c-fos have been reported in myocardial hypertrophy caused by diabetes in the NOD and streptozotocin-treated rats, pressure-induced hypertrophy, and PKC-ß2 transgenic mouse (4 , 17 , 21 , 33) . The increased expression of c-fos in the transgenic mice and in PC12 cells transfected with core 2 GlcNAc-T could be because of altered O-linked glycosylation of cell surface that resulted in enhanced cytokine signaling. Our results showed that Trk, the NGF receptor, was subjected to core 2 GlcNAc-T dependent glycosylation in PC12 cells, and this resulted in increased Trk levels and phosphorylation and increased PC12 differentiation in the absence of added NGF. In PC12 cells transfected with GlcNAc-TIII, an enzyme in the N-glycan pathway, Trk activity was also modified. GlcNAc-TIII glycosylated Trk receptor showed reduced dimerization and phosphorylation, as well as reduced NGF-dependent receptor phosphorylation and cell differentiation (18) .

Increases in core 2 GlcNAc-T transgene expression resulted in increased MAA lectin binding to several major glycoproteins separated by SDS-PAGE. Core 2 GlcNAc-T dependent glycosylation occurred on lumenal and secreted glycoprotein, which may alter receptor–ligand, cell–cell, or cell–substratum interactions. For example, the GlcNAc-residue transferred by core 2 GlcNAc-T formed the primary scaffold for polylactosamine and SLex on O-glycans. These structures on PSGL-1, a cell surface glycoprotein of neutrophils, formed the ligand (SLex) for P- and L-selectin receptors on activated endothelium (9 , 11) . This receptor–sugar ligand interaction resulted in neutrophil rolling and invasion at sites of inflammation.

Core 2 GlcNAc-T enzymatic activity and its product on CD43 are upregulated in peripheral T and B cell following activation by antigens (7 , 8) . CD43 is a major cell surface transmembrane glycoprotein on T lymphocytes, present at the leading edge of migrating T cells, and facilitates T cell extravasation from the blood into secondary lymphoid tissue (33) . T cells from CD43 null mice showed enhanced T cell activation and increased adhesion to ICAM and fibronectin in vitro. Ectopic expression of core 2 GlcNAc-T in T cells of transgenic mice resulted in decreased responsiveness to T cell mitogens, reduced DTH reactions, and decreased T cell adhesions to ICAM and fibronectin (13) . The expression of core 2 GlcNAc-T branched O-glycans on myocardial glycoprotein may, in a similar manner, alter the myocardial cell–cell or cell–substratum interactions, resulting in progressive cardiac stress and associated hypertrophy.

Core 2 GlcNAc-T O-glycans on specific myocardial glycoprotein may alter myocardial cell–cell or cell–substratum interactions, resulting in the activation of stress signaling pathways, elevation of c-fos, and, subsequently, the development of cardiac hypertrophy. The slower onset of the pathology in this mouse may be because of an accumulation of core 2 GlcNAc-T glycoprotein with age of the mouse, unlike the rapid onset of disease in the diabetic and PKC-ß2 transgenic mice (21) . Hyperglycemia induces PKC-ß2, c-fos, and core 2 GlcNAc-T gene expression in the cardiac tissue. Therefore, it is possible that diabetes upregulates c-fos gene expression via several reinforcing pathways including PKC-ß2 and a core 2 GlcNAc-T related pathway. Further analysis of the core GlcNAc-T modified glycoprotein in the hearts of transgenic mice may reveal candidates with potential to regulate c-fos gene expression through differential O-linked glycosylation.

In summary, the phenotype of core 2 GlcNAc-T transgenic mice suggested a direct association between hyperglycemia-induced core 2GlcNAc-T gene expression, c-fos gene expression, and myocardial hypertrophy. In addition, our results suggested that c-fos induction may be a result of core 2GlcNAc-T dependent glycosylation of cell surface glycoproteins offering intracellular signaling. Further analysis of specific cardiac myocyte glycoproteins and intracellular signaling events are required to delineate the relationship between core 2 GlcNAc-T dependent glycosylation and myocardial hypertrophy. Detailed cardiac functional studies will be necessary to determine which cardiac functions are being effected.


   ACKNOWLEDGMENTS
 
The authors wish to express their appreciation to Mr. Ed Boschetti for his excellent technical assistance and Ms. Luisa Dello Iacono for her secretarial contribution in the preparation of this manuscript. These studies were supported by NIH EY05110 (G. L. K., D. K., Y. N., N. T.), NIH DK53281(M. A. L.), and Diabetes Endocrinology Research Center grants NIH DK 36836, and NIC of Canada. (J. W. D., C. E. W.)


   FOOTNOTES
 
2 Abbreviations: {alpha}MHC, {alpha}-myosin heavy chain; core 2 GlcNAc-T, UDP-GlcNAc: Galß1–3GalNAc{alpha}Rß1–6-N-acetylglucosaminyltransferase; CRE, cAMP responsive element; GNA, Galanthus nivalis agglutinin; MAA, Maackia amurensis agglutinin; MAPK, mitogen activated-protein kinase; NGF, nerve growth factor; PKC, protein kinase C; SNA, Sambucus nigra agglutinin

Received for publication March 31, 1999. Accepted for publication September 3, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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