Hexosamine regulation of glucose-mediated laminin synthesis in mesangial cells involves protein kinases A and C

Lalit P. Singh1 and Errol D. Crook1,2

1 Division of Nephrology, Department of Medicine, University of Mississippi Medical Center and the 2 G. V. "Sonny" Montgomery Veterans Affairs Medical Center, Jackson, Mississippi 39216-4505


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by glucose metabolism through the hexosamine biosynthesis pathway (HBP). The HBP converts fructose-6-phosphate to glucosamine-6-phosphate via the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA). We have investigated the effects of high glucose (HG, 25 mM) and glucosamine (GlcN, 1.5 mM) on the synthesis of the ECM protein laminin in a SV-40-transformed rat kidney mesangial (MES) cell line. The roles of protein kinases C (PKC) and A (PKA) in mediating laminin accumulation were also investigated. Treatment of MES cells with HG or GlcN for 48 h increased laminin levels in cellular extracts more than twofold compared with 5 mM glucose (low glucose; LG). The presence of the GFA inhibitor diazo-oxo-norleucine (DON, 10 µM) blocked HG but not GlcN-induced laminin synthesis. HG resulted in a time-dependent increase in total PKC and PKA activities, 57±11.3 (P < 0.01 vs. LG) and 85±17.4% (P < 0.01 vs. LG), respectively. GlcN had no effect on the total PKC activity; however, both glucose and glucosamine increased membrane-associated PKC activity by twofold compared with LG. GlcN stimulated total PKA activity by 47±8.4% (P < 0.01 vs. LG). Similarly, membrane- associated PKA activity was also increased by HG and GlcN ~1.8 and 1.5-fold, respectively. HG and GlcN increased cellular cAMP levels 2.2- and 3.4-fold, respectively. Pharmacological downregulation of PKC by long-term incubation of MES cells with 0.5 µM phorbol 12-myristate 13-acetate (PMA) or inhibition of PKA activity by 2 µM H-8 blocked the effects of HG and GlcN on laminin synthesis. These results demonstrate that glucose-induced laminin synthesis in MES cells is mediated by flux through the HBP and that this stimulation involves PKC and PKA signaling pathways.

diabetic nephropathy; laminin; hexosamine biosynthesis pathway


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INTRODUCTION
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DIABETIC NEPHROPATHY IS ASSOCIATED with the accumulation of extracellular matrix (ECM) proteins in the glomerulus and is represented morphologically by thickening and expansion of the glomerular basement membrane and the mesangium (23, 29). Hyperglycemia is an important contributor to the development of diabetic nephropathy (10). Several laboratories have shown that elevated levels of glucose in mesangial (MES) and tubular cells cause significant increases in the synthesis and accumulation of matrix components such as collagen type IV, laminin, fibronectin, and proteoglycans (9, 11, 26, 31, 45). Numerous autocrine/paracrine growth factors found in renal tissue, namely, transforming growth factor-beta (TGF-beta ), ANG II, and insulin-like growth factor I (IGF-I) have been implicated in ECM accumulation and the development of diabetic nephropathy (3, 17, 32, 34, 43).

High concentrations of glucose lead to increased expression of TGF-beta mRNA and bioactivity in both MES and proximal tubule cells (17, 45) and a neutralizing antibody against TGF-beta blocked the glucose-induced increase in collagen synthesis, suggesting a direct role for TGF-beta in hyperglycemia-induced matrix protein accumulation (33, 41). High glucose also leads to a sustained (days to weeks) increase in protein kinase C (PKC) activity (2, 38, 39, 42). Agents that activate PKC, including high glucose, also enhance TGF-beta mRNA expression and bioactivity (17, 18, 27, 35). cAMP- dependent pathways have been implicated in these events as well. High glucose leads to a twofold increase in intracellular cAMP levels in MES cells, and the addition of the cAMP analog, 8-bromo-cAMP, results in transcriptional activation of type IV collagen (44). These findings suggest that hyperglycemia may lead to diabetic nephropathy through TGF-beta -induced stimulation of ECM synthesis, and that both protein kinase A (PKA) and PKC may be involved in mediating these effects.

Recent studies have demonstrated that some of the effects of high glucose on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA) (as shown in Fig. 1, Ref. 24). This pathway normally accounts for ~2% of the total intracellular glucose flux resulting in the production of UDP-N-acetyl glucosamine (UDP-GlcNAC) and other nucleotide hexosamines that serve as precursors for glycoproteins and glycolipids. Chronic exposure of rat-1 fibroblasts to high glucose decreases both basal and insulin-stimulated glycogen synthase activity, and overexpression of GFA in these cells mimics these effects of high glucose (5, 6). Moreover, transgenic mice overexpressing GFA in skeletal muscle and fat are insulin resistant (15). These results provide evidence that the hexosamine pathway may serve as a cellular glucose sensor.


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Fig. 1.   The hexosamine biosynthesis pathway.

The HBP has also been linked to glucose-mediated changes in cellular growth and growth factor expression. High glucose leads to a doubling of the level of TGF-alpha mRNA in primary cultures of rat aortic smooth muscle whereas glucosamine, at a much lower concentration, stimulates TGF-alpha mRNA levels by six- to sevenfold (7, 25). Glucosamine is also more potent than glucose in stimulating the expression of TGF-beta mRNA in cultured renal glomerular and proximal tubule cells (8, 20). These findings suggest that the HBP mediates some of the metabolic abnormalities associated with hyperglycemia and may be important in the pathogenesis of diabetic nephropathy. The present study was initiated to examine the underlying mechanism(s) by which high glucose and glucosamine induce matrix protein synthesis in MES cells (33, 41). We investigated the effect of long-term (48 h) exposure of cells to high glucose and glucosamine on laminin synthesis as well as on PKC and PKA activities in SV-40 transformed rat kidney MES cells. Our results show that both high glucose and glucosamine increase laminin synthesis in MES cells and that the increase in laminin synthesis is correlated with increases in PKC and PKA activities. Pharmacological inhibition of PKC or PKA activity blocks the high-glucose- and glucosamine-induced increases in laminin levels.


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Materials. Protein kinase inhibitors, recombinant human transforming growth factor-beta 1, and antibodies to laminin B1/B2 chain, PKC type III catalytic domain, cAMP RII regulatory subunit, and actinin were purchased from Upstate Biotechnology, Lake Placid, NY. The enhanced chemiluminescence (ECL) system was obtained from Amersham (Arlington Heights, IL). DMEM and F-12 nutrient mixture (Ham's) were from GIBCO (Grand Island, NY). [gamma -32P]triphosphate and 6-diazo-5-oxo-L-norleucine (DON) and azaserine (AZA) were from ICN (Costa Mesa, CA). P-81 phosphocellulose filter paper was from Millipore. PKC peptide substrate (pseudosubstrate) and PKA peptide substrate (Kemptide) were purchased from Pierce. PKA inhibitor, H-8 dihydrochloride, and anti-rat fibronectin antibodies were purchased from Calbiochem-Novabiochem (San Diego, CA). Biotrak cAMP assay kit was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other reagents were obtained from Sigma (St. Louis, MO).

Cell culture. SV-40-transformed rat kidney mesangial cells (American Type Culture Collection, Rockville, MD) were cultured in medium containing DMEM and Ham's F-12 (3:1 ratio) supplemented with 7% fetal calf serum (FCS) and 0.5 mg/ml gentamicin at 37°C in a humidified chamber with a 5% CO2-95% air mixture. Cells were routinely passaged at confluence every 4 days by using 10-cm dishes. These cells retain many of the important morphological and biochemical features of differentiated MES cells in primary cultures. For studies of laminin synthesis, monolayers at 30-40% confluence were incubated in the above medium supplemented with 2.25% FCS and the desired concentrations of D-glucose (5 mM or 25 mM) or glucosamine (5 mM glucose plus 1.5 mM glucosamine) for 48 h. For downregulation of PKC or inhibition of PKA activity, 0.5 µM PMA or 2 µM H-8 was added to the medium 3-4 h before adding high glucose or glucosamine and they were present throughout the time of culture. To examine for osmolar effects of high glucose in the media, 25 mM L-glucose with 3 mM D-glucose was utilized instead of 25 mM D-glucose. At the end of incubation, culture dishes were rinsed twice with PBS and harvested in 1 ml PBS by using a rubber policeman. The cells were centrifuged at 16,000 g for 5 s in a microcentrifuge and resuspended in 200 µl of extraction buffer A (50 mM beta -glycerophosphate, pH 7.3, 0.1% Tween-20, 1.5 mM EGTA, 1 mM dithiothreitol, 0.2 mM Na orthovanadate, 1 mM benzamidine, 1 mM NaF, 10 µg/ml aprotonin, 20 µg/ml leupeptide, 2 µg/ml pepstatin, and 0.5 µg/ml microcystin LR). Cell pellets were immediately frozen in liquid nitrogen and stored at -80°C until use. Cells were subsequently thawed, sonicated for 20 s and centrifuged at 16,000 g for 10 min at 4°C. To obtain membrane fractions, cells were extracted in buffer A without detergent and centrifuged as above. The pellet was washed and resuspended in buffer A plus 1% (vol/vol) Triton X-100, sonicated for 20 s, and centrifuged at 16,000 g for 10 min. The supernatant was collected as the membrane fraction. Protein concentration in extracts was determined by the Coomassie Protein Assay Reagent from Pierce by using BSA as the standard.

Electrophoresis and immunoblotting. The soluble cell lysates (30 µg protein in 20 µl) were mixed with 5 µl of a fivefold concentrated SDS sample buffer and heated for 5 min at 100°C. The proteins were then subjected to SDS-polyacrylamide gel electrophoresis by using 5% stacking and 10% resolving gels. Proteins were electroblotted on to a polyvinyledene difluoride filter (PVDF) membrane. The filter was blocked with 5% nonfat dry milk in buffer B containing 10 mM Tris · HCl, pH 7.8, 150 mM NaCl, and 0.05% Tween-20 for 30 min. The membrane was washed twice with buffer B and incubated overnight with appropriate antibodies at 4°C with continuous shaking. Antibodies in buffer B plus 5% dry milk were used at a 1:3,000 dilution for anti-laminin, 1 µg/ml for anti-PKC and 0.5 µg/ml for anti-alpha actinin. After incubation with the primary antibodies, the PVDF membrane was rinsed and washed thrice (10 min each) with water or buffer B and then incubated for 1.5 h at room temperature with horseradish peroxidase-conjugated anti-rabbit [for laminin and alpha -actinin or anti-mouse (for PKC)] IgG (at a 1:3,000 dilution) in buffer B plus dry milk. After extensive washing (15 min × 1, 5 min × 2), immunoreactive bands on the membrane were detected by ECL. For quantitative studies, the intensities of the bands were measured by a Bio-Rad GS-700 imaging densitometer.

Determination of PKC activity. PKC peptide substrate (pseudosubstrate), RFARKGSLRQKNV, was used to measure PKC activity in MES cell extracts. The reaction was carried out in 30 µl containing 20 mM Tris · HCl, pH 7.5, 10 mM Mg-acetate, 0.9 mM CaCl2, 0.4 mM EGTA, 30 mM beta -mercaptoethanol, 25 µg/ml micellar phosphatidylserine, 0.4 µM PKA inhibitor peptide (PKI), 4 µM compound R24571 (an inhibitor of Ca2+/calmodulin-dependent protein kinases), 100 µM pseudosubstrate, 5 µg protein of cell extract, and 250 µM [gamma -32P]ATP (800-1,000 counts · min-1 · pmol-1). After 15 min at 30°C, 25 µl of the reaction mixture were spotted on P-81 phosphocellulose filters. The filters were washed 4 times (5 min each wash) with 0.5% (wt/vol) phosphoric acid, and 32P incorporated into peptides was determined by counting radioactivity in a liquid-based scintillation counter. The amount of radioactivity associated with cell extracts in the absence of pseudosubstrate was subtracted to obtain PKC activity. 32P incorporated into PKC pseudosubstrate without adding cell extracts was negligible. The protocol for determining the membrane fraction PKC activity is identical to that of the total PKC activity described above.

PKA activity assay and cAMP determination. Phosphorylation of PKA peptide substrate (Kemptide), LRRASLG was carried out in a 30-µl reaction containing 20 mM Tris · HCl, pH 7.5, 100 mM KCl, 1 mM DTT, 15 mM Mg-acetate, 4 µM PKC inhibitor peptide, 4 µM compound R24571, 250 µM Kemptide, 250 µM [gamma -32P]ATP, and 5-µg cell extract. After incubation at 30°C for 15 min, the amount of 32P incorporated into Kemptide was determined by binding to a P-81 filter as described above for PKC. The addition of 0.5 µM PKA inhibitor, PKI, to the reaction mixture completely blocked the phosphorylation of Kemptide, indicating a specific phosphorylation of the peptides by PKA. The activity of PKA in the membrane fraction was also determined by the same reaction condition described above. Total cellular cAMP level was determined after cells were exposed to low glucose, high glucose, or glucosamine by using the cAMP [125I] scintillation proximity assay (SPA) kit from Amersham according to the manufacturer's instructions.

Statistical analysis. Results are expressed as means ± SE of the indicated number of experiments. Student's t-test was used to compare differences between cultures. A P value of <0.05 was considered statistically significant.


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Glucose and glucosamine increase laminin synthesis. Laminin is an ECM component found in virtually all basement membranes, and the expression of renal laminin B2 mRNA and protein has been reported to be elevated in diabetic nephropathy (1, 11). To determine whether cellular laminin expression is increased by high glucose or glucosamine, MES cells were cultured for 48 h in DMEM/Ham's medium with 2.25% FCS in the presence of low glucose (5 mM), high glucose (25 mM), or glucosamine (5 mM glucose plus 1.5 mM glucosamine). Immunoblotting showed that the amount of laminin in cellular extracts was increased 2.0-and 1.7-fold (P < 0.05) by high glucose or glucosamine, respectively, compared with low glucose (Fig. 2, A and B). The effects of high glucose and glucosamine on laminin only began to be observed after 24 h of incubation (data not shown). Culturing the cells in DON (10 µM), an inhibitor of GFA, blocked the stimulation of laminin synthesis by high glucose but not glucosamine (Fig. 2, C and D), consistent with the involvement of the HBP in hyperglycemia-mediated laminin production in MES cells. High glucose and glucosamine had no effect on alpha-actinin, a structural protein (Fig. 2E). The effects of glucose on laminin synthesis were not due to osmolar changes as 25 mM L-glucose had no effect on laminin levels (Fig. 3).


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Fig. 2.   Stimulation of laminin synthesis by high glucose (HG) and glucosamine. SV-40-transformed rat kidney mesangial (MES) cells were cultured for 48 h at low glucose (LG; 5 mM), HG (25 mM, HG), or glucosamine (1.5 mM, LG/GlcN) and harvested as described under MATERIALS AND METHODS. Cell extracts (30 µg protein) were separated on SDS-polyacrylamide gels, transferred to polyvinyledene difluoride filter membrane and probed with anti-laminin B1/B2 antibodies. Immunoreactive bands were detected by ECL system. The intensity of the bands was measured by a Bio-Rad GS-700 Imaging densitometer. The values are expressed as % of control at LG and are means ± SE for n = 9. A: a representative Western blot. Lane 1, LG; lane 2, HG and lane 3, GlcN. B: densitometric analysis data. Lane 1, LG (100%); lane 2, HG (185.7 ± 19.9% vs. LG, P < 0.02) and lane 3, GlcN (168.6 ± 16.7% vs. LG, P < 0.02). C: effect of diayo-oxo-norleucine (DON) on laminin synthesis. D: densitometric analysis of data. Lane 1, LG + DON (100%); HG + DON (141.2 ± 15.2% vs. LG + DON, P = 0.35, n = 3), and lane 3, GlcN + DON (196.8 + 63.7% vs. LG + DON, P < 0.03, n = 3). DON did not have a significant influence on the basal laminin level at LG. E: Western blot analysis of alpha-actinin. Lane 1, LG; lane 2, HG; and lane 3, GlcN.



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Fig. 3.   The effects of glucose on laminin were not due to changes in osmolarity. MES cells were cultured in the indicated D-glucose or L-glucose concentration for 48 h. Laminin levels were determined as above. The results are for n = 4 experiments. OD, optical density. *P < 0.05 compared with 5 mM D-glucose.

The effects of glucose and glucosamine on the ECM are not unique to laminin. We sought to see whether the effects of the HBP were limited to laminin or whether it affected other ECM components. Exposure of MES cells to high glucose and glucosamine increased fibronectin synthesis, another ECM protein, by 2.4- and 1.9-fold, respectively, (Figs. 4A). The addition of AZA, another inhibitor of GFA, resulted in a 68% decrease in the high-glucose-induced increase in fibronectin synthesis. Again, the GFA inhibitor had no effect on glucosamine-mediated fibronectin synthesis, supporting further the role of the HBP in mediating the effects of glucose on the ECM (Fig. 4B).


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Fig. 4.   Regulation of the extracellular matrix by the hexosamine biosynthesis pathway is not specific to laminin. MES cells were cultured in LG, HG, or GlcN for 48 h in the presence or absence of 0.5 µM azaserine (AZA), an inhibitor of GFA. Western blots were performed for fibronectin in cell extracts similar to those described for laminin in Fig. 2. The values are expressed as percent of control at low glucose (LG) and are means ± SE for n = 4. A: shows a representative fibronectin blot: lane 1, LG; lane 2, HG; and lane 3, GlcN. B: shows fibronectin blots in extracts from MES cells cultured as follows: lane 1, LG + AZA (100%), lane 2, HG + AZA; and lane 3, GlcN + AZA. AZA resulted in a 68% decrease in the HG-induced increase fibronectin levels. There was no significant difference in fibronectin levels at LG with or without AZA. *P < 0.03 compared with LG without AZA. **P < 0.04 compared with LG + AZA. ***P = 0.01 compared with HG without AZA.

Effect of high glucose and glucosamine on PKC and PKA activities. Agents that stimulate PKC and PKA activities have been shown to increase extracellular matrix protein synthesis in glomerular MES cells (27, 35, 37, 44). The effects of high glucose and glucosamine on PKC and PKA activities in MES cells were therefore determined by using specific peptide substrates. Figure 5A shows that high glucose stimulated total cellular PKC activity by 57.8 ± 11.3 (P < 0.01 vs. low glucose) while glucosamine had no effect on the total PKC activity. However, in isolated membrane fractions, both high glucose and glucosamine-stimulated PKC activity by 109.3 ± 25.7% (P < 0.006, n = 5) and 134.9 ± 51.5% (P < 0.03, n = 5), respectively (Fig. 5B). Neither high glucose nor glucosamine had any influence on total cellular PKC protein content on Western blots with anti-type III PKC catalytic domain antibodies that detect the 82-kDa alpha -, beta 1-, beta 2-, and gamma -isoforms (21, 38) (Fig. 5C). Similarly, glucose and glucosamine had no effect on PKC protein content in the membrane fraction (data not shown).


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Fig. 5.   Effect of HG on PKC activity. MES cells were cultured for 48 h at LG, HG, or glucosamine and harvested. Cell extracts were prepared in buffer A. Total PKC activity in extracts (A) or PKC activity in the membrane fraction (B) was measured by using specific peptide substrates as described under MATERIALS AND METHODS. The basal activity for total PKC LG was ~0.3 pmol 32Pi incorporated into peptide substrates · mg protein extract-1 · min-1. The basal PKC activity in membrane fraction was ~0.1 pmol · µg-1 · min-1. Results are normalized to activity determined at LG (100%) and are expressed as means ± SE for n = 5. *P < 0.01 compared with control (LG). C: Western blot analysis of PKC. MES cells were treated with either 25 mM glucose or 1.5 mM glucosamine as described under MATERIALS AND METHODS. Cell extracts (30 µg each) were separated on SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane, and PKC protein level was detected by Western immunoblotting with anti-type III PKC antibodies and analyzed with densitometry. The relative ODs (compared with LG) are shown for n = 4 experiments. There were no significant differences between conditions.

High glucose and glucosamine increased total PKA activity by 85 ± 17.4 (P < 0.01 vs. low glucose) and 47 ± 8.4% (P < 0.01 vs. low glucose), respectively (Fig. 6A). Similarly, high glucose and GlcN (Fig. 6B) also increased PKA activity in the membrane fraction. The effects of high glucose and glucosamine on PKC and PKA activity were time dependent and required exposure of cells to the sugars for at least 24 h (data not shown). In addition, high glucose and glucosamine also led to ~1.2-1.4-fold increase (P < 0.05 vs. low glucose) in the expression of the PKA RII regulatory subunit in total cellular extracts on Western blots by using anti-PKA RII antibodies (Fig. 6C).


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Fig. 6.   Effect of HG on PKA activity. MES cells were cultured for 48 h at LG, HG, or glucosamine, harvested and extracts were prepared as described under MATERIALS AND METHODS. Total PKA activity in extracts (A) and the membrane PKA activity (B) were measured by using specific PKA peptide substrates as described under MATERIALS AND METHODS. The basal activity for total PKA LG were ~0.4 pmol incorporated into substrates · µg protein extract-1 · min-1. The basal PKA activity in membrane fraction was ~0.1 pmol · µg-1 · min-1. Results are normalized to activity determined at LG (100%) and are expressed as means ± SE for n = 5. *P < 0.01 compared with their respective controls. C: PKA RII subunit protein content was determined as described and ODs were determined. Both HG and GlcN resulted in a statistically significant increase in PKA RII subunit protein content. + P < 0.05 compared with LG.

Given the increase in PKA activity with high glucose and glucosamine we also examined the effect of high glucose and glucosamine on cellular cAMP levels. As shown in Fig. 7, both high glucose and glucosamine increased cAMP levels by 2.2 (P < 0.001) and 3.4 (P < 0.004)-fold respectively compared with low glucose. The basal cAMP level at low glucose was 1.78. ± 0.29 pmol/0.5 µg extract.


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Fig. 7.   Analysis of cAMP levels. MES cells were treated with either 25 mM glucose or 1.5 mM glucosamine as described under MATERIALS AND METHODS. Cell extracts were prepared from each 10-cm culture dish in 250 µl of cAMP extraction reagents and supplied for Biotrak cAMP assay kit by Amersham Pharmacia Biotech, according to the manufacturer's instructions. One hundred microliters of cell extract were used in the assay. A standard curved for nonacetylated cAMP ranging from 0.2 to 12.6 pmol was constructed to calculate the cAMP levels in extracts. All assays were performed in duplicate and the results are plotted as means ± SE from four separate experiments. The basal cAMP level was 1.87 ± 0.29 pmol/0.5 mg protein cellular extract. *P < 0.05 compared with control.

The effects of glucose on PKC and PKA activity were not due to osmolar effects. MES cells were cultured in 25 mM L-glucose for 48 h and PKC and PKA activity were determined in the membrane fraction. There was no significant difference in PKC or PKA activity with L-glucose compared with cells cultured in 5 mM D-glucose. Twenty-five millimolar L-glucose resulted in a 0.69 ± 037-fold change in PKC activity (P = ns, n = 6) and a 0.617 ± 0.29-fold change in PKA activity (P = ns, n = 6). In these experiments the effects of 25 mM D-glucose and GlcN were similar to those mentioned above.

Hexosamines regulate laminin via PKC and PKA pathways. We investigated further the roles of PKC or PKA on high glucose and glucosamine-induced laminin synthesis. We utilized inhibitors of PKC (long-term exposure of MES to PMA) and PKA (PKA inhibitor, H-8) activity. As shown in Fig. 8A, downregulation of PKC by long-term incubation of cells with 0.5 µM PMA blocked the effect of high glucose on laminin synthesis completely. PKA inhibition with 2.0 µM H-8 led to a 44% decrease in glucose-induced laminin synthesis (P < 0.05 compared with high glucose alone). Both PMA and H-8 also blocked glucosamine-induced laminin synthesis (1.6 ± 0.1-fold increase by GlcN alone); 1.1 ± 0.01 by GlcN plus PMA and 1.1 ± 0.1 by GlcN plus H-8 (P < 0.03) (Fig. 8B).


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Fig. 8.   Effect of inhibition of PKC and PKA on HG and glucosamine-induced laminin production. MES cells were cultured at LG, HG, or glucosamine for 48 h. PMA (0.5 µM) or H-8 (2 µM) was also added with glucose or glucosamine. Cell extracts were prepared and laminin content was measured by Western blot as described in MATERIALS AND METHODS. A: Lane 1, LG; lane 2, HG; lane 3, HG + PMA (P < 0.007 vs. HG); and lane 4, HG+H-8 (P < 0.05 vs. HG). B: Lane 1, LG; lane 2, GlcN; lane 3, GlcN+PMA (P < 0.03 vs. GlcN); and lane 4, GlcN+H-8 (P < 0.01 vs. GlcN).


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Numerous studies have demonstrated that high glucose levels cause an increase in the synthesis and accumulation of ECM proteins, such as, collagen type IV, laminin, and fibronectin in cultured MES and tubule cells (3, 12, 17, 32, 34). Some of the effects of high glucose are mediated by TGF-beta acting in an autocrine/paracrine fashion. TGF-beta increases the accumulation of ECM proteins in these cells, and neutralizing antibodies against TGF-beta attenuate the stimulation of matrix proteins by high glucose (33, 41). Glucose is an important regulator of cell growth and metabolism, and it is likely that some of the adverse effects of high glucose are mediated by normal regulatory pathways. The HBP, which converts fructose-6-phosphate to glucosamine-6-phosphate with glutamine as the amino donor, has been hypothesized to be a sensor for glucose and therefore a mediator of glucose regulation in a variety of cell types (5-8, 15, 24, 25). In kidney MES cells, glucosamine was more potent than glucose in stimulating TGF-beta mRNA transcription and bioactivity (8, 20), and the inhibition of GFA activity by the glutamine analogue AZA or antisense oligonucleotide against GFA blocked the high glucose-induced expression of TGF-beta and matrix protein synthesis (20). Likewise, hexosamines regulate TGF-beta 1 transcription in rat kidney cells (mesangial and proximal tubule) and rat vascular cells via regulation of the TGF-beta 1 promoter (8, 33).

We sought to investigate whether the effects of high glucose on ECM are mediated by the HBP. Here, we show that glucosamine mimicks glucose in its ability to increase laminin synthesis. These effects of high glucose and glucosamine required at least 24 h before they were observed. The effects of hexosamines are not specific to laminin alone as both high glucose and glucosamine also increased fibronectin synthesis in MES cells. The role of the HBP in mediating the effects of glucose on the ECM is supported by the ability of two inhibitors of GFA to blunt the high glucose-induced increases in laminin and fibronectin. However, glucosamine-enhanced ECM synthesis is not affected by these GFA inhibitors as glucosamine enters the HBP distal to the rate-imiting enzyme GFA.

These observations support the notion that the HBP mediates the effects of glucose in the mesangium. Kolm-Litty et al. (20) also reported that treatment of primary cultures of porcine glomerular MES cells with 12 mM glucosamine for 48 h leads to a 2.3-fold increase in fibronectin synthesis. In the present study, the effect of glucosamine on laminin and fibronectin was observed at a much lower concentration than glucose (1.5 mM glucosamine vs. 25 mM glucose), and, in fact, in our experimental system glucosamine concentrations greater than 2.5 mM drastically reduced cell growth and viability. The reason for this discrepancy may be the differences in cell types used for the studies, but it is an important consideration given the potential, specific untoward effects of glucosamine in certain cells (14). Nevertheless, the fact that others have seen similar effects in primary cells makes it less likely that the observations reported here are unique to a transformed cell line. In additon, the importance of the HBP on mediating the assembly of the ECM is supported by the demonstration of increased fibronectin secretion in the studies mentioned above (20) and in preliminary data from our laboratory that demonstrates increased secretion of laminin in MES cells cultured in high glucose and glucosamine (not shown).

The underlying mechanism(s) by which high glucose and glucosamine enhance matrix protein synthesis are not fully understood. PKC is a candidate mediator for the induction of TGF-beta synthesis by high glucose because agents, including high glucose, that increase PKC activity also increase TGF-beta transcription and bioactivity (2, 27, 35, 38, 39, 42). The role of PKC in laminin synthesis is not entirely clear as recent reports have not supported a role for PKC in the regulation of the laminin C1 promoter (30). Similarly, an increase in PKC activity as such may not be required for TGF-beta -mediated matrix protein production (36).

Like their effects on laminin, high glucose and glucosamine increased membrane PKC activity in MES cells. This indicated a possible role for PKC in HBP-mediated ECM regulation. It was confirmed that hexosamines increase laminin via a PKC mechanism as long-term exposure of cells to PMA blocked the glucosamine-induced increase in laminin. Unlike high glucose, we did not see an effect of glucosamine on total PKC activity. However, in membrane fractions, both high glucose- and glucosamine-stimulated PKC activity are about twofold, suggesting a role for the hexosamine pathway in PKC distribution between cytosolic and membrane fractions.

There are at least 11 PKC isoforms and they are categorized into 3 subclasses according to their structure and function. PKC isoforms, alpha , beta 1, beta 2, and gamma  are classified in the conventional group which are regulated by diacylglycerol, phosphatidylserine, and Ca2+; novel PKCs (delta , varepsilon , eta  and phi ), which are also regulated by diacylglycerol and phosphatidylserine; and atypical (zeta , iota , and lambda ), whose regulation has not been clearly established, although their activity can be stimulated by phosphatidylserine (12, 28). We did not find any change in protein content of conventional PKCs on Western blots with anti-type III PKC antibodies which recognize PKCalpha , beta 1, beta 2, and gamma . Different PKC isoforms, however, have different enzymatic properties and their distribution changes after cell activation. Some isoforms are translocated from the cytosolic compartment to cellular membranes while others are translocated into the nucleus where they play a major role in signaling (12, 19). It is not known which of the isoforms are activated or translocated by high glucose or glucosamine. Answering this question and how these PKC isoforms participate in ECM regulation will be the focus of future experiments.

Hyperglycemia also increases the intracellular concentration of cAMP (22, 44) and, therefore, may activate the PKA pathway in MES cells. We observed that high glucose increases cAMP content in MES cells and glucosamine mimics the effect of high glucose on cAMP levels. As observed with PKC, hexosamines appear to be involved in PKA translocation, however, unlike PKC, hexosamines appear to regulate PKA at the protein level (Fig. 6C). The ability of high glucose and glucosamine to increase PKA activity and the ability of the PKA inhibitor, H-8, to decrease their effects on laminin synthesis, suggest that a PKA-mediated pathway may be important in ECM regulation. This is supported by the work of other investigators that has demonstrated regulation of PKA by TGF-beta in the mesangium (37).

We hypothesize that the HBP acts as a cellular glucose sensor in target cells. Abnormalities in flux through or regulation of this pathway may lead to altered cellular responses to glucose. This is supported by the loss of glucose-induced increases in laminin levels when GFA, the rate limiting enzyme in the HBP is inhibited by glutamine analogs like DON or AZA. Further support of this hypothesis is the observed downregulation of GFA activity (the rate limiting enzyme in this pathway) by high glucose in MES cells (4). Thus when flux through the HBP is altered, downstream products of this pathway may upregulate PKC, PKA, and TGF-beta bioactivity leading to increased ECM gene expression (8, 20) and protein synthesis. The upregulation of these signaling systems in MES cells ultimately leads to increased ECM levels. The effects of PKC and PKA inhibition on glucose- or glucosamine-induced laminin synthesis (Fig. 8) strongly support the involvement of these intracellular signaling pathways in glucose-mediated ECM synthesis.

How the HBP mediates the ECM and how PKC and PKA are involved in this process are not completely understood. The time course for the effects of hexosamines on PKC, PKA, and ECM components (>24 h) is consistent with transcriptional regulation of some regulatory gene(s). An interesting hypothesis for how the HBP has its effects is through O-linked protein glycosylation (14, 24). The substrate for this posttranslational modification of proteins is UDP-GlcNAC (Fig. 1) a downstream metabolite of the HBP (24). Among the proteins that are O-glycosylated are transcription factors and this process is mediated by hexosamine metabolism (13). UDP-GlcNAC is increased in our cells when cultured in high glucose or GlcN (not shown). We propose that increased flux through the HBP (as seen with hyperglycemia) leads to increased UDP-GlcNAC levels and altered regulation of genes that mediate synthesis of the ECM. Included among these are genes encoding proteins that regulate PKC and PKA. This hypothesis is supported by the fact that PKA protein levels did change with glucose and glucosamine. This ultimately may lead to elevated ECM levels and diabetic gluomerulosclerosis. Ongoing work is focused on uncovering the mechanism by which glucose metabolism through the HBP regulates ECM.

In conclusion, the present study demonstrates that high-glucose-induced laminin synthesis in MES cells is mediated by the HBP. Glucosamine, at a much lower concentration, mimics the effects of high glucose. DON, an inhibitor of GFA, blocks the high glucose-, but not glucosamine-induced increases in laminin synthesis. The data also suggest that both PKC and PKA signaling pathways may participate in high-glucose and glucosamine regulation of matrix protein production and that these proteins are regulated by the HBP. Elucidation of the various signaling pathways mediated by hyperglycemia and excess hexosamines may allow the development of novel means for therapeutic intervention and treatment of patients with diabetic nephropathy.


    ACKNOWLEDGEMENTS

Support for this work was from The Robert Wood Johnson Foundation (E. D. Crook) and Kidney Care Foundation. E. D. Crook is also supported by a Career Development Award from the Veterans Administration. Portions of this work were presented at the 1997 American Society of Nephrology Meeting in San Antonio, TX.


    FOOTNOTES

Address for reprint requests and other correspondence: E. D. Crook, Dept. of Medicine, Div. of Nephrology, 2500 N. State St., Jackson, MS 39216-4505 (E-mail: ecrook{at}medicine.umsmed.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 8 November 1999; accepted in final form 5 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 279(4):F646-F654