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* Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts 02215, USA;
Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02215 USA; and
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Using mRNA differential display, we have recently
identified that the gene expression of the O-glycan-specific
UDP-GlcNAc: Galß1–3GalNAcRß1–6N-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-, and
followed with substitution by 2,3SA-T and 2,6SA-T, which
completes the common O-linked tetrasaccharide structure
(5)
. The action of core 2 GlcNAc-T on the
Galß1–3GalNAc- 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 (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 -myosin heavy chain (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)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation and characterization of transgenic mice
A SalI/EagI fragment of the
rat 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 -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-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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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 SA2–3Gal 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 SA2–3Gal 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 SA2–6Gal specific lectins Galanthus nivalis
agglutinin (GNA) or Sambucus nigra agglutinin (SNA) were unaffected
(data not shown).
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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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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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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MHC, -myosin heavy chain;
core 2 GlcNAc-T, UDP-GlcNAc:
Galß1–3GalNAcRß1–6-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.
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