Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School and Veterans Affairs Chicago Healthcare System, Chicago, Illinois 60611
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ABSTRACT |
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To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC). Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth. Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity. Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose. The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity. In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity. Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth. In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms. Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells.
NH2-terminal c-Jun kinase; extracellular signal-regulated kinase 5; glucosamine; oxidative stress
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INTRODUCTION |
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CARDIOVASCULAR DISEASE is the leading cause of death in adults with diabetes mellitus (9). Compared with other individuals with equivalent risk factors, people with diabetes have a four- to fivefold increase in mortality from vascular disease (9). The factors that are responsible for this increased risk of cardiovascular disease are not known, but studies in patients with types 1 and 2 diabetes have demonstrated that good metabolic control is able to prevent early changes of atherosclerosis and decrease the risk of coronary heart disease events and deaths (15, 22). These data suggest that hyperglycemia contributes to the increased risk of cardiovascular disease, but whether this is secondary to a direct effect of glucose on vascular cells or an indirect effect secondary to the modification of other risk factors is not known.
Previous studies have demonstrated that glucose has effects on
endothelial cells that are proatherogenic. Prominent among these
effects is the inhibition of endothelial cell growth by hyperglycemia
(7, 10, 17, 26, 34). In human umbilical vein endothelial
cells, the inhibition of cell growth is secondary to delayed passage
through the cell cycle (26). To date, however, the
mechanism for the effect of glucose on endothelial cell growth has not
been clearly defined. One recent study suggested that the inhibition of
proliferation was secondary to increased production of transforming
growth factor-, with a subsequent decrease in hepatocyte growth
factor production, whereas others have suggested that sorbitol
accumulation and/or the generation of reactive oxygen species
contributes to the inhibition of endothelial cell proliferation by
glucose (7, 10, 17).
Regardless of the mechanism, it is likely that the effect of glucose is mediated by proximal signaling events that stimulate downstream effects. To date, the nature of these proximal signaling events remains poorly defined. The mitogen-activated protein kinase (MAPK) pathway consists of a group of interconnecting protein kinase cascades that link signals at the plasma membrane to nuclear events and are important for both stimulating and inhibiting cell proliferation (23, 25, 42). Four different families of MAPKs that are linked to different signals at the plasma membrane and have different substrate specificities have been described, including extracellular signal-regulated kinase (ERK)-1 and -2, c-Jun NH2-terminal kinases (JNKs), p38 kinases, and big MAPK 1 or ERK5 (23, 25, 42). ERK1 and -2 are activated primarily in response to proliferative stimuli (30, 42), whereas the other MAPKs are activated primarily in response to inflammatory and stressful stimuli, including oxidant and osmotic stresses (1, 8, 23, 24). Activation of the JNKs and p38 kinases is associated with inhibition of cell growth in some cell types, although this effect appears to be cell type specific (8, 23, 24). The biological role of the more recently defined ERK5 is less clear.
Given the critical role that the MAPKs play in the regulation of cell growth and the described effects of glucose on endothelial cell growth, the studies described herein were designed to address whether glucose alters MAPK activity in endothelial cells and the metabolic pathways that account for glucose-induced effects on MAPK activity.
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MATERIALS AND METHODS |
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Cell culture. Bovine pulmonary artery endothelial cells (PAECs) were provided by Drs. Mark Yorek and Robert Bar (University of Iowa). The cells were maintained in 75-cm2 flasks in low-glucose (5.5 mM glucose) DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 3.7 gm/l sodium bicarbonate, penicillin (50 U/ml), and streptomycin (50 U/ml) at 37°C in 5% CO2. Upon reaching confluence, the cells were replated at a dilution of 1:4. Cells between passages 5 and 15 were used for all studies.
Cell growth assay. For cell growth assays, PAEC were plated onto 24-well plates at a density of 5 × 103 cells/well and maintained for 24 h in the low-glucose DMEM + 10% FBS described above. At that time, the medium was changed and replaced with either low-glucose DMEM + 10% FBS or low-glucose DMEM + 10% FBS supplemented with increasing concentrations of glucose, fructose, or raffinose. After being maintained in the test media for 24, 48, or 72 h, cells were collected using trypsin and resuspended in Isoton II (Coulter, Miami, FL). Visual inspection of the plates was used to ensure that all cells had been released with trypsin. The number of cells per well was determined using a Coulter counter (Beckman Coulter, Fullerton, CA).
MAPK immune complex assays. For the kinase assays, cells were plated at a density of 5 × 105 cells/75-cm2 plate and maintained for 24 h in low-glucose DMEM + 10% FBS for 24 h. For assays performed in the presence of FBS, medium was changed after 24 h and replaced with either low-glucose DMEM + 10% FBS or low-glucose DMEM + 10% FBS supplemented with 25 mM glucose, fructose, or raffinose. For assays performed in serum-free medium, cells were transferred to low-glucose DMEM + 0.25% BSA for 24 h. This medium was then replaced with either low-glucose DMEM + 0.25% BSA or low-glucose DMEM + 0.25% BSA supplemented with either 25 mM glucose, fructose, or raffinose.
Antibodies directed against JNK1, p38 kinase, ERK5, and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The substrate protein for the JNK immune complex assay was a GST-c-Jun fusion protein. To generate a cDNA for the synthesis of this fusion protein, a fragment of DNA encoding the amino-terminal 81 amino acids of c-Jun was amplified using the PCR with a rat c-jun cDNA as template DNA. The amplified fragment, the identity of which was confirmed by DNA sequencing, was purified and cloned into pGEX4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) in frame with the GST gene. The resulting fusion protein (GST-c-Jun81) was purified from bacterial lysates using glutathione-agarose beads according to the manufacturer's instructions (Amersham Pharmacia Biotech). The substrate for the p38 kinase assay was GST-ATF2. The GST-ATF2 fusion protein was prepared using the plasmid pGST-ATF2 (kindly provided by Dr. Roger Davis, Howard Hughes Medical Institute, University of Massachusetts Medical School), as described above. The substrate for the ERK2 and ERK5 assays was myelin basic protein (Sigma, St. Louis, MO). ERK2 assays were performed using an immune complex assay as described previously (27). The JNK1 immune complex assay was performed using a previously described method (40). Briefly, cells were maintained in the different media for the indicated period of time and solubilized in lysis buffer [20 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mMWestern blot analysis. Western blots were probed with a polyclonal antibody directed against phospho-ERK1/2 (New England Biolabs, Beverly, MA) at a dilution of 1:1,000 or with a polyclonal antibody directed against ERK2 (Santa Cruz Biotechnology) at a dilution of 1:7,500.
For Western blot analyses, cell lysates were prepared in the cell lysis buffer used for the ERK5 assay, as described above, and protein content of the lysate was determined using the Coomassie blue protein assay. Forty micrograms of protein were then diluted 1:4 in sample buffer [62.5 mM Tris · HCl (pH 6.8), 10% glycerol, 2% SDS, 5%Statistical analysis. Values are reported as means ± SE. P values were calculated using the one-way repeated-measures ANOVA with Tukey's pairwise multiple comparision procedure or the Kruskal-Wallis one-way ANOVA on ranks with Dunnett's pairwise multiple comparison procedure, as appropriate, by SigmaStat 2.0 software (Jandel, San Rafael, CA).
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RESULTS |
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Effect of sugars on cell growth.
Initial studies were performed to examine the effect of glucose on PAEC
growth by growing cells in the presence of increasing concentrations of
glucose (Table 1). Supplementation of
normal growth medium (low-glucose DMEM + 10% FBS) with additional
glucose resulted in a concentration-dependent decrease in cell growth. After 1 day of treatment, addition of both 20 and 25 mM glucose resulted in a significant inhibition of cell growth, whereas treatment with additional concentrations of glucose as low as 10 mM (final concentration 15.5 mM) resulted in a significant inhibition of cell
growth after 2 and 3 days of treatment. Supplementation of the medium
with 25 mM glucose decreased PAEC growth by 20, 39, and 45% after
treatment for 1, 2, and 3 days, respectively.
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Effect of sugars on MAPK activity.
Subsequent studies examined the effect of glucose, raffinose, and
fructose on MAPK activity. For these studies, PAEC were treated for
6-48 h with normal growth medium supplemented with 25 mM of
glucose, fructose, or raffinose, and the activity of the different
MAPKs was measured using an immune complex assay. Initial studies
examined the effect of the sugars on JNK1 and ERK5 activity. In cells
maintained for 6-48 h in normal growth medium in the absence of
added sugars, there was no change over time in either JNK1 or ERK5
activity (data not shown). In contrast, JNK1 activity was increased by
glucose, fructose, and raffinose, with an initial increase in JNK1
activity apparent after 12 h of treatment (Fig.
1). A peak approximately twofold increase
in JNK1 activity was present after 24 h of incubation, with a
return to basal kinase activity by 48 h. In contrast to this
effect on JNK1 activity, only glucose had a marked effect on ERK5
activity (Fig. 2). The effect of glucose
on ERK5 activity was delayed compared with its effect on JNK1 activity,
with a significant increase in activity first noted after 24 h of
incubation in high-glucose medium, and a maximal 2.0-fold increase
present at 48 h. Raffinose stimulated a smaller, but significant,
52% increase in ERK5 activity after 48 h of treatment.
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Metabolic pathways that mediate the effect of glucose on MAPK activity. Having determined that glucose increases JNK1 and ERK5 activity, the contribution of different metabolic pathway(s) to the effect of glucose was examined. A well-described pathway of glucose metabolism that is active in hyperglycemic conditions is the polyol pathway, i.e., the reduction of glucose to sorbitol by aldose reductase (39). Treatment of cells with 0.4 mM sorbinil, an inhibitor of aldose reductase, had no effect on the induction of either JNK1 or ERK5 activity by the addition of 25 mM glucose (data not shown).
Previous studies have demonstrated that incubation of endothelial cells in the presence of high glucose increases the generation of reactive oxygen species, which may alter endothelial cell growth (6, 7, 10). Moreover, reactive oxygen species have also been shown to activate JNK1 and ERK5 in some cell types (1, 16, 23, 24); thus studies were performed to determine whether the generation of reactive oxygen species by PAEC incubated in hyperglycemic medium contributed to the glucose-induced increase in JNK1 and ERK5 activity. For these studies, cells were incubated for 24 h in growth medium supplemented with 25 mM glucose in the absence or presence of 10 mM N-acetylcysteine, and the activity of JNK1 and ERK5 was measured. N-acetylcysteine is an antioxidant that is a precursor of glutathione synthesis and is a free radical scavenger. Coincubation of PAEC with N-acetylcysteine had no effect on glucose-induced JNK1 activity but significantly decreased glucose-induced ERK5 activity by 64% (Fig. 3). These data suggest that the glucose-induced increase in ERK5 activity occurred, at least in part, secondary to increased oxidative stress.
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Effect of glucosamine on PAEC.
If increased flux through the hexosamine biosynthesis pathway and
increased generation of glucosamine 6-phosphate account, in part, for
the effects of glucose on PAEC, incubation of PAEC with glucosamine
should reproduce those effects of glucose that are inhibited by
azaserine, because the majority of exogenously added glucosamine is
phosphorylated by hexokinase and enters the hexosamine biosynthesis
pathway (31). Initial studies examined the effect of
glucosamine on JNK1 activity. For these studies, PAEC were incubated
for 6-48 h with normal growth medium supplemented with either 2 or
5 mM glucosamine. Treatment with 5 mM glucosamine increased JNK1
activity, with an increase in activity noted as early as 12 h and
a peak 2.4-fold increase present after 24 h of incubation (Fig.
5). Two mM glucosamine had no effect on
JNK1 activity at 12 and 24 h and stimulated only a modest 1.5-fold increase in activity after 48 h of incubation. In contrast to its
effect on JNK1 activity, glucosamine had no effect on ERK5 activity
(data not shown). Subsequent studies examined the effect of glucosamine
on JNK1 activity in the presence of the GFAT inhibitor azaserine.
Because glucosamine enters the hexosamine biosynthesis pathway
downstream of GFAT, azaserine should have no effect on glucosamine-induced effects. In PAEC incubated for 24 h in the presence of 5 mM glucosamine, JNK1 activity increased 1.8 ± 0.1 and 1.8 ± 0.2-fold (means ± SE, n = 5) in
the presence and absence of 5 µM azaserine, respectively. As
expected, these data demonstrate no effect of azaserine on
glucosamine-induced JNK1 activity and are consistent with the effect of
azaserine on glucose-induced JNK1 activity being mediated by its effect
on GFAT activity.
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DISCUSSION |
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Diabetes mellitus is a risk factor for premature atherosclerosis
(9), and although the mechanisms responsible for this diabetes-induced risk have not been completely defined, hyperglycemia appears to contribute to the increased risk (15, 22).
Previous studies have documented a variety of glucose-induced effects
on endothelial cells that would promote atherogenesis, including decreased cell proliferation and apoptosis, increased production of the
cytokines interleukin-1 and interleukin-8, and enhanced monocyte and
leukocyte binding (2, 7, 10, 13, 17, 20, 26, 33, 34, 43).
In the present study, we have confirmed the finding that incubating
endothelial cells in high glucose inhibits their growth. Interestingly,
unlike other studies in which either mannitol or L-glucose
did not reproduce the effect of glucose (10, 17, 34), in
our studies treatment of cells with either raffinose or fructose was
able to reproduce the effect of glucose on cell growth. The molecular
mechanisms for this growth inhibitory effect of glucose and other
sugars remain obscure. A previous study suggested that an induction of
transforming growth factor-
production by glucose with a secondary
decrease in hepatocyte growth factor production accounted for the
effect of glucose (34), whereas other studies have
implicated the increased production of reactive oxygen species in the
effect of glucose (7, 10). Given the fundamental role of
the MAPKs in the regulation of cell growth, the present study attempted
to correlate glucose-induced changes in endothelial cell growth with
glucose-induced activation of the MAPKs. As expected, given the
association of ERK1/2 activation with proliferative stimuli (30,
42), neither glucose nor the other sugars had an effect on
ERK1/2 phosphorylation and activity in PAEC. Only glucose had a marked
effect on ERK5 activity, whereas glucose, fructose, and raffinose
stimulated JNK1 activity to a similar degree, suggesting that JNK1
activation may contribute to the growth inhibitory effect of these
sugars on endothelial cell growth.
The JNKs typically are activated in response to inflammation and
cellular stresses, e.g., ultraviolet light, hyperosmolarity, and
inhibitors of protein synthesis (23-25). The effects
of the JNKs on cell growth are complex and cell type specific. In
general, however, activation of the JNKs has been associated with
inhibition of cell growth and apoptosis, although association of JNK
activation with cell growth also has been reported (5, 36,
40). The effect of JNK activation on endothelial cell growth
also appears to be complex. For example, an important role for JNK
activation in vascular endothelial cell growth factor-induced growth of
endothelial cells has been reported (37), but a
contribution of JNK activation to the induction of an inflammatory
response by tumor necrosis factor- in endothelial cells and to
apoptosis in mannitol- and ceramide-treated as well as serum-starved
endothelial cells also has been reported (11, 29, 38).
Interestingly, in the present study, the temporal sequences of JNK1
activation and the inhibition of cell growth by glucose, fructose, and
raffinose differed, i.e., the increase in JNK1 activity had dissipated
by 48 h, whereas the inhibition of cell growth was most prominent
after 48 and 72 h of treatment with these sugars. These data
suggest that JNK activation may be important early in the inhibition of
cell growth and/or that JNK activation is an early part of or initiates
a program of events associated with the inhibition of cell growth. In
the absence of specific inhibitors of the JNK pathway, the role of JNK
activation in endothelial cell growth cannot be directly assessed.
Interestingly, despite the ability of glucose, fructose, and raffinose to activate JNK1, the mechanism of action of the sugars differed, at least in part. Azaserine, a GFAT inhibitor that inhibits glucose flux through the hexosamine biosynthesis pathway, decreased glucose-induced JNK1 activity by 50% but had no effect on JNK1 activation by fructose or raffinose. This result suggested that increased flux through the hexosamine pathway contributes to glucose-induced JNK1 activation. Consistent with this observation, glucosamine, which is a direct precursor of the downstream product of the hexosamine biosynthesis pathway, glucosamine 6-phosphate, also increased JNK1 activity in the PAEC. Like the other sugars that activated JNK1, glucosamine was able to inhibit cell growth. Together, these observations suggest that increased flux through the hexosamine biosynthesis pathway accounts, in part, for glucose-induced JNK1 activity and, possibly, inhibition of cell growth, although a contribution of osmotic effects cannot be ruled out.
Besides JNK1, glucose activated an additional member of the MAPK family, ERK5. ERK5 is a recently identified novel member of the MAPK family that is activated by stresses, such as oxidative stress and hyperosmolarity, but is also activated by receptor tyrosine kinases (1, 16). In HeLa cells, ERK5 was shown to be important for the growth-promoting effects of epidermal growth factor (18). In contrast to JNK1, a marked increase in ERK5 activity was stimulated only by high glucose, and its activation by glucose was inhibited by an antioxidant, N-acetylcysteine. Together with the lack of effect of glucosamine on ERK5 activity, these data suggest that ERK5 activation occurs via a metabolic pathway distinct from that which stimulates JNK1 activity. The inhibition of glucose-induced ERK5 activation by an antioxidant is consistent with the previous observation that H2O2 activates ERK5 in endothelial cells (1). In addition to H2O2, shear stress also has been shown to increase ERK5 activity in endothelial cells (44). The functional consequence of ERK5 activation by oxidative and shear stress in endothelial cells is not known. Similarly, the functional consequences of the ERK5 activation by high glucose in endothelial cells are not known; however, again, because only high glucose increased ERK5 activity, it is likely that it mediates glucose-induced effects other than inhibition of cell growth.
The effect of glucose on MAPK activity has been examined previously in
two different cell types, pancreatic -cells, which are programmed to
respond to glucose, and vascular smooth muscle cells (VSMC). In
pancreatic
-cells, glucose activates primarily ERK1 and -2 but also
has a modest effect on p38 kinase activity (4, 19, 28).
Activation of the ERKs and, possibly, p38 kinase in
-cells appears
to be important for glucose-induced expression of the insulin gene
(3, 28). In VSMC, high glucose stimulates a modest
increase in ERK1 and -2 activity and has a more marked effect on p38
kinase activity via a pathway dependent on activation of protein kinase
C-
(14). In contrast, glucose has no effect on JNK
activity in VSMC (14). The present study is the first to
examine the effect of glucose on MAPK activity in endothelial cells.
Interestingly, the pattern of MAPK activation by glucose differed
markedly in endothelial cells and VSMC. In endothelial cells, glucose
increased JNK and ERK5 activity but had no effect on either ERK or p38
kinase activity. The activation of different MAPK isoforms by high
glucose in VSMC and endothelial cells is consistent with the disparate
effect of glucose on the growth of these two cell types. In contrast to
endothelial cells, glucose stimulates the growth of VSMC (10, 12,
34, 35, 45, 46).
In summary, the present studies have confirmed previous findings demonstrating an inhibitory effect of glucose on endothelial cell growth and, for the first time, have correlated these changes in cell growth with activation of the MAPKs by glucose. The marked correlation between the ability of several sugars to inhibit endothelial cell growth and activate JNK1 suggests that JNK activation may contribute to the modulation of endothelial cell growth by different sugars, whereas the more specific activation of ERK5 by high glucose suggests that it may mediate other effects of glucose in endothelial cells. Future studies will be needed to define the role of JNK activation in glucose-mediated inhibition of endothelial cell growth and the functional consequences of increased ERK5 activity.
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ACKNOWLEDGEMENTS |
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We thank Drs. Mark Yorek and Robert Bar for providing pulmonary artery endothelial cells, Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School) for providing pGST-ATF2, and Dr. William Schnaper for helpful discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-58832.
Address for reprint requests and other correspondence: W. L. Lowe, Jr., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15-703, Northwestern Univ. Medical School, 303 East Chicago Ave., Chicago, IL 60611 (E-mail: wlowe{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 December 1999; accepted in final form 16 May 2000.
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