High Concentration of Glucose Increases Mitogenic Responsiveness to Heparin-Binding Epidermal Growth Factor–like Growth Factor in Rat Vascular Smooth Muscle Cells
Abstract The effect of a high extracellular glucose concentration on the mitogenic response of rat vascular smooth muscle cells (SMCs) to heparin-binding epidermal growth factor–like growth factor (HB-EGF) was investigated. The mitogenic effect of HB-EGF was significantly greater in SMCs cultured in high glucose (25 mmol/L) than in cells cultured in low glucose (5.5 mmol/L) or at high osmolarity (5.5 mmol/L glucose plus 19.5 mmol/L mannitol). The mitogenic effect of epidermal growth factor (EGF), which shares the EGF receptor with HB-EGF, was not affected by glucose concentration. The mitogenic effect of HB-EGF was greater when incubated with heparan sulfate (HS) isolated from SMCs cultured in high glucose than with HS from cells cultured in low glucose. HS synthesized by cells in high glucose was of smaller molecular size and less sulfated than HS synthesized by cells in low glucose. The abundance of mRNA encoding HS-N-deacetylase/N-sulfotransferase (HS-NdAc/NST), a regulatory enzyme in the biosynthesis of HS, was decreased by high glucose in a protein kinase C–independent manner. These observations suggest that the enhanced mitogenic response to HB-EGF in SMCs cultured in high glucose may be attributable to changes in cell-associated HS. Downregulation of HS-NdAc/NST gene expression by high glucose may be related to the altered HS biosynthesis.
- vascular smooth muscle cells
- heparan sulfate
- heparan sulfate N-deacetylase/N-sulfotransferase
- Received December 28, 1995.
- Accepted December 12, 1996.
Heparin-binding EGF–like growth factor, first identified from medium conditioned by the human macrophage-like cell line U-937, is a potent mitogenic and migratory factor for vascular SMCs.1 2 Mature, bioactive HB-EGF is a 22-kD glycoprotein that binds to the EGF receptor and stimulates its phosphorylation.1 Previous studies have suggested that HB-EGF may play a regulatory role in the growth of SMCs in vivo.3 4 5 6 7
The biological activities of b-FGF,8 9 10 11 12 13 14 vascular endothelial growth factor,15 and HB-EGF,16 17 all of which are heparin-binding growth factors, depend on a dual receptor system consisting of a high-affinity receptor and a cell-surface HSPG. Responses to acidic and basic FGF in murine neural precursor cells are regulated by cell-surface HSPG, thus allowing for rapid changes in cell signaling during development.18 We have previously shown that the mitogenic response to HB-EGF is increased in SMCs of diabetic rats because of altered cell-associated HS.16 These observations suggest that HSPG may regulate cell proliferation or differentiation by affecting the response to heparin-binding growth factors.
The biosynthesis of HS is altered in the diabetic state. HS from the liver of rats with streptozotocin-induced diabetes is less sulfated than HS from control rat liver.19 Defective formation of glomerular basement membrane HS in the diabetic state perturbs the electrostatic filtration barrier in the kidneys and thus results in diabetic proteinuria.20 21 Because some complications of diabetes are considered to be attributed to altered formation of HS associated with the extracellular matrix and the cell surface, it is important to gain insight into the mechanism by which the biosynthesis of HS is regulated in the diabetic state.
The polysaccharide component initially formed during the biosynthesis of HS comprises GlcA and GlcNAc units that are joined in a (GlcA-GlcNAc)n structure in reactions catalyzed by d-glucuronosyl- and N-acetyl-d-glucosaminyl transferase.22 23 24 HS-NdAc/NST catalyzes both the N-deacetylation and N-sulfation of GlcNAc residues in HS.25 26 27 Both these deacetylation and sulfation reactions are obligatory for further modification of HS; C-5 epimerization of GlcA to l-iduronic acid and various O-sulfation reactions occur only within or adjacent to the N-sulfated residues.12 26 In addition, transfer of a GlcA to a terminal GlcNAc unit, presumably the rate-limiting step of chain elongation, is greatly facilitated by the occurrence of an N-sulfate group on the adjacent GlcNAc residue.22 23 These observations suggest that HS-NdAc/NST may play a regulatory role in the biosynthesis of HS.
We have now investigated the effects of high glucose concentration on cell-associated HS and its relation to the mitogenic response to HB-EGF in rat SMCs. We have further examined regulation of HS-NdAc/NST mRNA expression.
Recombinant human HB-EGF was kindly provided by Dr Judith A. Abraham, Scios Nova Inc. Recombinant human EGF and b-FGF were obtained from GIBCO-BRL. The 125I-labeled EGF (3.7 MBq/mL) used in our experiments was obtained from Amersham Life Science.
Culture of SMCs
Rat aortic SMCs were isolated by the explant method as previously described.16 28 In brief, aortic tissue was obtained from the thoracic aorta of male Wistar rats (350 g), and the adventitia was removed. Explants were cultured at 37°C under 5% CO2 in DMEM (glucose, 5.5 mmol/L) supplemented with 15% fetal bovine serum (Irvine Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin. After 2 weeks, cells that had migrated out of the explant were removed by trypsinization and seeded in T-75 flasks. Cells were characterized as SMCs by morphological criteria (spindle shape and hill-and-valley pattern) and by their expression of smooth muscle α-actin. SMCs were subcultured in three different conditions: high glucose (25 mmol/L glucose, HG-SMCs), high osmolarity (5.5 mmol/L glucose plus 19.5 mmol/L mannitol, Mann-SMCs), and low glucose (5.5 mmol/L glucose, LG-SMCs). Each group of SMCs was subcultured at a 1:4 split ratio at weekly intervals. Cells from the third to fourth passages were used for experiments. Three different strains of SMCs were used, with similar results.
Effects of Growth Factors on DNA Synthesis
DNA synthesis in SMCs was assessed by [3H]thymidine incorporation as previously described.16 Subconfluent cells seeded in 96-well plates were incubated for 48 hours in DMEM (high-glucose, high-osmolarity, and low-glucose conditions) supplemented with 0.1% bovine PDS to induce quiescence.16 29 The medium was then replaced with differential DMEM containing 1% or 5% PDS (as noted in figure legends) and various concentrations of growth factors, and after 20 hours, cells were pulse labeled with [3H]thymidine (37 kBq per well, Amersham) for 4 hours. Incorporated [3H]thymidine was quantitated with a β-Plate system (Pharmacia LKB).
EGF Receptor–Binding Assay
Cells were washed twice with binding buffer (DMEM supplemented with 50 mmol/L BES, pH 7.4, and 0.1% BSA) and incubated for 4 hours at 4°C in 24-well plates containing the same buffer with various amounts of 125I-EGF in the absence or presence of a 100-fold excess of unlabeled EGF.30 The cells were then washed with binding buffer and solubilized with 0.75 mL of a lysing buffer (10 mmol/L Tris-HCl, pH 7.4; 0.5% SDS; and 1 mmol/L EDTA). Cell-bound and free 125I radioactivity was measured with a gamma counter. Nonspecific binding to cells was usually <10% of total binding. Data were applied to Scatchard plots using least-squares analysis.
Isolation of Cell-Associated HS
Quiescent SMCs cultured in 10-cm dishes were preincubated at 37°C for 48 hours in the presence of [35S]sulfate (100 kBq/mL; Amersham) in high-glucose or low-glucose conditions. Cell-associated protein-free HS was isolated according to the method of Schmidt and Buddecke31 as previously described.16 Incorporation of 35S radioactivity into HS was determined by scintillation spectroscopy and normalized to cell protein content, which was determined before trypsin digestion with a Bio-Rad protein assay kit. Uronic acid content was determined by the carbazole reaction, with glucuronic acid as a standard.32 For determination of relative molecular mass, isolated HS was subjected to chromatography on a Sephacryl S-200 (Pharmacia) column (1 by 50 cm) that had been equilibrated with 50 mmol/L sodium acetate (pH 6.0), 50 mmol/L NaCl, and 0.2% SDS. After application of the sample, components were eluted with the same buffer at a flow rate of 10 mL/h, and 0.5-mL fractions were collected. 35S radioactivity was determined with a scintillation counter.
Effects of Cell-Associated HS on the Mitogenic Activity of HB-EGF and b-FGF in Chlorate-Treated SMCs
Unlabeled HS was isolated from quiescent HG-SMCs (HG-HS) and LG-SMCs (LG-HS). LG-SMCs were treated with 5 mmol/L sodium chlorate (Aldrich) in low-glucose DMEM containing 0.1% PDS for 48 hours to inhibit HS sulfation and then incubated with various amounts of HS and either HB-EGF or b-FGF for 24 hours. DNA synthesis was then determined.
Northern Blot Analysis of HS-NdAc/NST mRNA
Total RNA was extracted from quiescent SMCs as described.33 RNA samples (20 μg) were subjected to electrophoresis through 1% agarose gels and transferred to nylon membranes (Hybond-N; Amersham). The membranes were incubated with a random-primed, 32P-labeled rat HS-NdAc/NST cDNA, kindly provided by Dr Carlos B. Hirschberg.34 The membranes were then washed and subjected to autoradiography. As the expression of actin or GAPDH mRNA was affected by glucose or DOG, the expression of 18S rRNA was used as an internal standard. mRNA abundance was calculated by scanning laser densitometry.
Effect of Glucose on HS-NdAc/NST mRNA Abundance
Quiescent SMCs that had been cultured under the low-glucose condition were incubated for various times with various concentrations of glucose and HS-NdAc/NST. mRNA abundance was then determined by Northern blot analysis. The effects of fructose, galactose, mannitol, and l-glucose (Wako) were also determined. The role of PKC in the regulation of HS-NdAc/NST gene expression by glucose was examined by investigating the effects of a specific PKC inhibitor, calphostin C35 (Biomol), and a PKC stimulator, DOG36 (Sigma).
Differences between groups were analyzed by Student’s t test. A value of P<.05 was considered statistically significant.
Effects of Growth Factors on DNA Synthesis in SMCs
When SMCs were stimulated by 10% fetal calf serum, [3H]thymidine incorporation increased about fivefold to sixfold in HG-SMCs and threefold to fourfold in Mann-SMCs or LG-SMCs (data not shown). At concentrations of 0.63 to 10 nmol/L HB-EGF, the mitogenic response of HG-SMCs to HB-EGF was greater than that of Mann-SMCs or LG-SMCs (Fig 1A⇓). The mitogenic effect of HB-EGF was greater than that of EGF (Fig 1B⇓) in HG-SMCs; the effects of HB-EGF were similar to those of EGF for Mann-SMCs and LG-SMCs.
EGF Receptor Analysis
Scatchard analysis demonstrated that the affinity of EGF for the EGF receptor was similar in HG-SMCs and LG-SMCs (dissociation constant of 870 and 990 pmol/L, respectively). However, the number of receptors on the surface of HG-SMCs was 40% of the number on LG-SMCs (0.10 versus 0.25 fmol/104 cells; Fig 2⇓).
Incorporation of [35S]sulfate into HS in HG-SMCs was 85% of that in LG-SMCs (183±6 versus 216±25 cpm/μg protein, P<.05). Uronic acid contents in HS were similar between HG-SMCs and LG-SMCs (3.9±0.5 versus 3.7±0.5 ng/μg protein). Therefore, [35S]sulfate/uronic acid ratio in HS was significantly lower in HG-SMCs (46±2 versus 58±6 cpm/ng uronic acid, P<.05). The kaV value of the elution maximum of HG-HS from a Sephacryl S-200 column was higher than that of LG-HS (0.15 versus 0.10), indicating smaller molecular size of HG-HS (Fig 3⇓).
Effect of HS on the Mitogenic Response to HB-EGF and b-FGF in Chlorate-Treated SMCs
HS isolated from SMCs restored the mitogenic response to HB-EGF (Fig 4A⇓) and b-FGF (Fig 4B⇓) in LG-SMCs treated with chlorate. HG-HS was significantly more effective than LG-HS in enhancing the mitogenic response to HB-EGF; the effects of HG-HS and LG-HS were similar for response to b-FGF. HS did not affect the basal [3H]thymidine incorporation. Addition of 1 μg/mL heparin, in contrast, reduced basal [3H]thymidine incorporation by 80% in SMCs and did not restore the responses to HB-EGF in chlorate-treated cells (data not shown).
HS-NdAc/NST mRNA Abundance in SMCs
The expression of HS-NdAc/NST mRNA was examined in SMCs subcultured for three passages in high-glucose, high-osmolarity, or low-glucose condition. The abundance of HS-NdAc/NST mRNA in HG-SMCs was 50% of that in LG-SMCs (Fig 5⇓). The amount of HS-NdAc/NST mRNA in Mann-SMCs was also reduced to 80% of that in LG-SMCs.
Regulation of HS-NdAc/NST mRNA Abundance
The addition of glucose to a final concentration of 25 mmol/L to quiescent SMCs that had been cultured under the low-glucose condition resulted in a decrease in HS-NdAc/NST mRNA abundance that was apparent at 30 minutes and sustained after 2 hours (Fig 6⇓). The addition of 19.5 mmol/L mannitol to cells that had been cultured in 5.5 mmol/L glucose also reduced the amount of HS-NdAc/NST mRNA at 30 minutes; however, the effect was less marked at 1 and 2 hours.
The effect of glucose on HS-NdAc/NST mRNA was dose dependent up to a concentration of 25 mmol/L (Fig 7⇓). No difference between the effects of mannitol at 9.5 mmol/L and 19.5 mmol/L was apparent. At equivalent osmotic pressure, 25 mmol/L glucose reduced HS-NdAc/NST mRNA abundance to a greater extent than mannitol.
l-Glucose decreased HS-NdAc/NST mRNA abundance to the same extent as d-glucose (Fig 8⇓). Fructose and galactose showed greater effects than mannitol on the gene expression, although the effects were less marked than that of glucose. Alterations of HS-NdAc/NST mRNA observed in these conditions returned to almost control level at 24 hours or longer (data not shown).
Finally, DOG had no effect on the abundance of HS-NdAc/NST mRNA (Fig 9⇓), and the inhibitory effect of glucose on HS-NdAc/NST gene expression was not influenced by calphostin C (data not shown).
Addition of heparin or HS isolated from SMCs did not affect HS-NdAc/NST mRNA abundance in SMCs (data not shown).
We have demonstrated that the mitogenic response of SMCs to HB-EGF was increased when the cells were cultured at a high glucose concentration. High osmolarity did not affect the mitogenic response to HB-EGF. The mitogenic effect of EGF, which shares the EGF receptor with HB-EGF, was not affected by glucose concentration. The number of EGF receptors in SMCs cultured at a high glucose concentration was decreased to 40% of that in SMCs cultured in low glucose. HS isolated from SMCs cultured at a high glucose concentration enhanced the mitogenic effect of HB-EGF to a greater extent than HS from SMCs cultured in low glucose. These observations suggest that the increased mitogenic responsiveness to HB-EGF in SMCs cultured under high-glucose conditions may be related to changes in cell-surface HS rather than to changes in the EGF receptor itself.
The incorporation of [35S]sulfate into cell-associated HS was decreased in HG-SMCs. The [35S]sulfate/uronic acid ratio in HS was significantly lower in HG-SMCs. The relative molecular mass of HS isolated from HG-SMCs was smaller than that of HS from LG-SMCs. These observations suggest that high glucose concentrations may modify HS synthesis in SMCs, as previously demonstrated in hepatocytes19 and renal mesangial cells.20 21
We have previously shown that cell-associated HS is altered in SMCs of streptozotocin-induced diabetic rats and that the alteration is associated with an increased mitogenic response to HB-EGF.16 The increased mitogenic response to HB-EGF, as well as the difference in cell-associated HS in SMCs of diabetic rats, persists for at least six passages of the cells in culture, suggesting a clonal difference between SMCs of diabetic rats and controls. In this study, however, relatively short-term exposure of SMCs to a high concentration of glucose induced similar changes to those observed in SMCs of diabetic rats. This finding indicates that among various metabolic alterations observed in diabetic rats, hyperglycemia might be an important factor in inducing these changes. Thus, SMCs of diabetic rats may be transformed as a result of prolonged exposure to high glucose concentrations in vivo.
The abundance of mRNA encoding HS-NdAc/NST, a regulatory enzyme in the biosynthesis of HS, was decreased in HG-SMCs. HS-NdAc/NST plays an important role in the regulation of HS sulfation.13 26 Chain elongation of HS might also be related to HS-NdAc/NST activity.22 23 Both HS-NdAc/NST activity and sulfate incorporation into HS are decreased in hepatocytes of diabetic rats.19 The reduced abundance of HS-NdAc/NST mRNA in HG-SMCs may be related to altered HS synthesis, lower sulfation, and shorter HS chains in these cells.
The inhibitory effect of high glucose on HS-NdAc/NST gene expression in SMCs was partially attributable to hyperosmolarity, because mannitol also reduced HS-NdAc/NST mRNA abundance. However, the effect of high glucose (25 mmol/L) was more marked than that of mannitol at identical osmolarity. Also, unlike the effect of glucose, the inhibitory effect of mannitol did not appear to be dose dependent. Fructose and galactose also did not show an inhibitory effect to the same extent as glucose. These observations suggest that glucose exerts a specific inhibitory effect on HS-NdAc/NST gene expression.
It is not clear why l-glucose exerted an effect similar to that of d-glucose on HS-NdAc/NST mRNA. The effect of l-glucose is not likely attributable to nonenzymatic glycation, because the response was too rapid. Addition of l-glucose increases consumption of d-glucose and shows a similar stimulatory effect to that of d-glucose on the synthesis of collagens type IV and VI in rat mesangial cells.37 l-Glucose may also affect the metabolism of d-glucose in SMCs and thereby modulate HS-NdAc/NST gene expression.
Although high concentration of galactose, fructose, or l-glucose decreased the expression of HS-NdAc/NST mRNA to some extent, SMCs cultured in these conditions did not show increased mitogenic response to HB-EGF as did SMCs cultured in high glucose (data not shown). The possible reasons may be as follows: (1) SMCs cultured in a high concentration of galactose, fructose, or l-glucose showed reduced growth rate and [3H]thymidine incorporation, suggesting that these conditions may be inappropriate for SMCs cultured for longer periods. (2) Alterations of HS-NdAc/NST mRNA levels observed at 2 hours in these conditions returned to almost control level at 24 hours or longer.
Increased extracellular glucose concentrations activate PKC in many tissues both in vivo and in vitro.38 39 40 High extracellular glucose concentrations induce a sustained increase in PKC activity in SMCs that is apparent within 10 minutes.41 It has been suggested that a product of glucose metabolism, perhaps diacylglycerol, might be required for glucose-induced activation of PKC in SMCs.6 39 40 41 Activated PKC may influence many aspects of cellular function, inducing proliferation and both catabolism and biosynthesis. The inhibitory effect of glucose on HS-NdAc/NST gene expression was not affected by calphostin C, a specific inhibitor of PKC. DOG, a potent stimulator of PKC, also had no effect on HS-NdAc/NST mRNA abundance. These observations suggest that downregulation of HS-NdAc/NST mRNA by glucose is not mediated by PKC.
A synthetic peptide corresponding to the heparin-binding domain of HB-EGF has been shown to inhibit the bioactivity of HB-EGF but not that of b-FGF,16 17 suggesting that HSPG species that interact with the heparin-binding domain of HB-EGF differ from those that associate with the corresponding domain of b-FGF. In this study, whereas HS isolated from HG-SMCs enhanced the mitogenic effect of HB-EGF to a greater extent than LG-HS, both types of HS showed similar effect on the activity of b-FGF. Thus, changes in HS induced by incubation of SMCs in high glucose may selectively affect species that interact with the heparin-binding domain of HB-EGF and potentiate the bioactivity of this growth factor.
A specific HSPG appears to regulate receptor binding and the biological activity of b-FGF.8 10 11 13 A recent study has identified perlecan as a major candidate for a low-affinity b-FGF receptor.9 Specific structural features of the carbohydrate backbone of HS for b-FGF have also been demonstrated.42 HS-NdAc/NST regulates biosynthesis of high-affinity b-FGF binding domains of HS,12 whereas HS isolated from HG-SMCs and LG-SMCs was equally effective at restoring b-FGF mitogenicity to chlorate-treated SMCs. It seems that a relatively low degree of decrease in HS-NdAc/NST mRNA in HG-SMCs might not affect b-FGF bioactivity, whereas it could alter HB-EGF mitogenicity. Although it is not clear which species of HSPG stimulates receptor interaction and thus regulates the bioactivity of HB-EGF, HS-NdAc/NST may also affect the synthesis of HB-EGF binding domains of HS, thus regulating response to HB-EGF. The fact that heparin did not restore the response to HB-EGF in chlorate-treated SMCs suggests that highly sulfated glycosaminoglycans may not be appropriate for HB-EGF bioactivity.
In conclusion, we have shown that the mitogenic response to HB-EGF was significantly increased in SMCs cultured in a high glucose concentration, possibly due to alteration of cell-associated HS. Downregulation of HS-NdAc/NST gene expression by glucose may be responsible for the changes in HS synthesis.
Selected Abbreviations and Acronyms
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|EGF||=||epidermal growth factor|
|b-FGF||=||basic fibroblast growth factor|
|HB-EGF||=||heparin-binding EGF-like growth factor|
|PKC||=||protein kinase C|
|SMC||=||vascular smooth muscle cell|
This work was supported in part by a grant-in-aid for cancer research for Drs Higashiyama and Taniguchi (05151047), a grant-in-aid for Dr Matsuzawa (04404085), and a grant-in-aid for Dr Kawata (06557058) from the Ministry of Education, Science, and Culture of Japan. Dr Higashiyama is the recipient of a Searl Scientific Research Fellowship and a Suzuken Memorial Foundation award. We thank Dr Judith A. Abraham for recombinant human HB-EGF and Dr Carlos B. Hirschberg for rat HS-NdAc/NST cDNA.
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