Hexosamines and TGF- 1 use similar signaling pathways to mediate matrix protein - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:16 |只看该作者 |倒序浏览 |打印

作者：Lalit P. Singh, Kenneith Green, Michelle Alexander, Shira Bassly, and Errol D. Crook作者单位：Department of Internal Medicine, Division of Nephrology, Wayne State University School of Medicine and the John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201 . p( r6 w$ {5 s/ P9 S7 c) I9 Y





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      【摘要】
   Hyperglycemia-induced alterations in mesangial (MES) cell function and extracellular matrix (ECM) protein accumulation are seen in diabetic glomerulopathy. Transforming growth factor- 1 (TGF- 1 ) mediates high-glucose-induced matrix production in the kidney. Recent studies 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 (GlcN) 6-phosphate. We previously showed that the high-glucose-mediated fibronectin and laminin synthesis in MES cells is mediated by the HBP and that GlcN is more potent than glucose in inducing TGF- 1 promoter luciferase activity. In this study, we investigated the hypothesis that the effects of glucose on MES matrix production occur via hexosamine regulation of TGF- 1. Culturing simian virus (SV)-40-transformed rat kidney MES cells in 25 mM glucose (HG) for 48 h increases cellular fibronectin and laminin levels about twofold on Western blots compared with low glucose (5 mM). GlcN (1.5 mM) or TGF- 1 (2.5-5 ng/ml) for 24-48 h also increases ECM synthesis. However, the effects of HG or GlcN with TGF- 1 are not additive. The presence of anti-TGF- 1 antibodies (20 µg/ml) blocks both TGF- 1 - and GlcN-induced fibronectin synthesis. TGF- 1 increased ECM levels via PKA (laminin and fibronectin) and PKC (fibronectin) pathways. Similarly, TGF- 1 and hexosamines led to nonadditive increases in phosphorylation of the cAMP responsive element binding transcription factor. These results suggest that the effects of excess glucose on MES ECM synthesis occur via HBP-mediated regulation of TGF- 1.
      【关键词】 diabetic nephropathy hexosamine pathway transforming growth factor extracellular matrix protein cell signaling
   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 ( 26, 33 ). Hyperglycemia is the primary etiologic factor in the metabolic abnormalities and vascular complications of diabetes ( 26 ). For example, prolonged exposure to high glucose (HG) is an important contributor to the development of diabetic nephropathy both in types 1 and 2 diabetes ( 9 ). Although the mechanisms underlying the effects of chronic hyperglycemia on the kidney are not fully understood, transforming growth factor- 1 (TGF- 1 ) and protein kinases (PK)C and PKA have been implicated in HG-mediated ECM production seen in diabetic glomerulopathy ( 2, 7, 11, 19, 30, 35, 38, 45, 46 ).
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Recent studies demonstrated that some of the effects of HG 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-liming enzyme glutamine:fructose-6-phosphate amidotransferase (GFA) ( 3 - 6, 15, 27, 29 ). We showed that HG and glucosamine (GlcN) increase fibronectin and laminin synthesis in mesangial cells ( 40, 41 ) and that PKC and PKA signaling pathways may participate in the hexosamine-induced synthesis of these ECM components. The mechanism of hexosamine-mediated action on the ECM may involve PKC/A regulation of cAMP responsive element binding transcription factor (CREB) ( 40 ). Indeed, HG and GlcN increase CREB phosphorylation and CRE-binding activity of nuclear extracts in mesangial (MES) cells ( 21 ). In addition, we and others ( 6, 21 ) demonstrated regulation of TGF- by the HBP in kidney and vascular cells., G* ~" Q! \* H9 W2 _4 R: I+ m

We hypothesized that the effects of glucose in the diabetic mesangium are mediated by its metabolism through the HBP. We hypothesize further that these effects on MES ECM are mediated via hexosamine regulation of TGF- 1. In the present study, we investigate whether HG, GlcN, and TGF- 1 use similar signaling pathways to increase synthesis of fibronectin and laminin in rat kidney MES cells. We show that HG, GlcN, and TGF- 1 have similar and nonadditive effects on ECM production and CREB phosphorylation. In addition, the hexosamine-mediated effects of glucose on PKC/A are mediated, in part, by upstream effects on TGF- 1.
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MATERIALS AND METHODS
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Materials. Goat anti-rat fibronectin antibodies, Bis-I, and H-89 were purchased from Calbiochem-Novabiochem (San Diego, CA). A subcellular proteome extraction kit (S-PEK) was purchased from EMD Biosciences (La Jolla, CA). A PKC antibody panel was obtained from Oxford Biomedical Research (Oxford, MI). TGF- 1, laminin B1/B2 chain, anti-phosphorylated CREB, and nonphosphorylated CREB antibodies were from Upstate Biotechnology (Lake Placid, NY). The ECL detection system for Western blot analysis was obtained from Amersham. An intracellular ATP assay kit was purchased from Sigma (St. Louis, MO). All other reagents and chemicals were of reagent or analytic grade.
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Cell culture. SV-40-transformed rat kidney MES cells were cultured in media containing DMEM and F-12 Nutrient Mixture (Ham's) (3:1 ratio) with 10% fetal calf serum and 0.5 mg/ml gentamicin ( 40, 41 ). Cells were routinely passaged at confluence every 4 days using 10-cm culture dishes. Approximately 50% confluent monolayers were incubated in the above medium supplemented with 2.25% fetal calf serum and the desired concentrations of glucose and GlcN for 48 h [low glucose (LG) 5 mM, HG 25 mM, GlcN 1.5 mM LG] or 24 h with TGF- 1 (2.5-5 ng/ml). For the blockade of PKC activities, Bis-I (1 µM) or PMA (2 µM) was included as described previously ( 40, 41 ). For the inhibition of PKA, H-8 or H-89 (2 µM) was added to the culture. At the end of the incubation, the dishes were rinsed twice with extraction buffer A (50 mM -glycerophosphate, pH 7.3, 1.5 mM EGTA, 1 mM dithiothreitol, 0.2 mM Na-orthovanadate, 1 mM benzamidine, 10 µg/ml aprotonin, 20 µg/ml leupeptin, 1 mM NaF, 0.5 µg/ml microcystin, and 2 µg/ml pepstatin A) and then harvested in 1 ml of the same buffer using a rubber policeman. The cells were centrifuged at 16,000 g for 5 s, resuspended in 200 µl of extraction buffer A, immediately frozen in liquid nitrogen, and stored at -80°C until use. Cells were subsequently thawed, sonicated for 20 s, and centrifuged at 4°C for 10 min. The supernatant was removed as the cytosolic fraction. To isolate membrane fractions, the pellet was washed and resuspended in buffer A containing 1% Triton X-100, sonicated for 20 s, and centrifuged at 16,000 g for 10 min. The supernatant was collected as the membrane fraction ( 41 ). For obtaining nuclear fractions, the pellet, after removal of the cytosolic fraction, was extracted in buffer A containing 1% (vol/vol) Triton X-100 and 400 mM KCl, sonicated, and centrifuged as above. The supernatant was collected as the nuclear fraction. Protein concentrations in cell extracts were determined by the method of Bradford using BSA as the standard.5 L& H3 ]8 G/ ^4 f% O
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Western blot analysis. Cell extracts (30 µg protein) were applied by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride filter membrane. The blot was blocked with 5% nonfat dry milk in 10 mM Tris·HCl, pH 7.6, containing 150 mM NaCl and 0.05% Tween 20 ( buffer B ) for 20 min. The filter was washed in buffer B and incubated with anti-laminin (1:3,000 dilution), anti-fibronectin (1:5,000 dilution), or anti-phosphorylated CREB (1:2,000 dilution) antibodies at 4°C overnight with continuous shaking in buffer B containing 5% nonfat dry milk. The membrane was then washed with buffer B (5 min x 4 times) and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution) at room temperature for 1.5 h. Immunoreactive bands were detected with the ECL system, and the intensity of the bands was measured by a Molecular Dynamics Densitometer for quantitation.5 d; s8 H6 E7 \0 u6 t

The anti-phosphorylated CREB antibodies also detected a band at 38 kDa, which is consistent with activating transcription factor (ATF)-1 bearing a peptide sequence homologous to phosphorylated CREB (see Figs. 6 and 7 ). Phosphorylated ATF-1 can also form heterodimers with phosphorylated CREB to regulate CRE promoter activity. The level of phosphorylated ATF-1 detected with the anti-CREBp antibodies used in this study differs between antibody batches and from experiment to experiment; therefore, ATF-1 cannot be accessed accurately with this antibody ( 40 ).
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Fig. 6. Phosphorylation of HG-, GlcN-, and TGF- 1 -induced CREB is not additive. MES cells were cultured for 48 h with HG and GlcN in the presence or absence of TGF- 1 for 24 h or together and harvested. CREB phosphorylation was detected with the anti-phosphorylated CREB antibody. Lane 1, LG (basal); lane 2, HG (3.56 ± 1.42, P & l% ]2 s6 O, E, ~- o" o8 {! y
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Fig. 7. Effects of PKC and PKA inhibition on TGF- 1 -induced phosphorylation of CREB. MES cells were cultured for 24 h at LG, LG TGF- 1 in the presence or absence of Bis-I (PKC inhibitor), or H-89 (PKA inhibitor) and harvested. Nuclear fractions were prepared and phosphorylation of CREB on Ser133 was examined by anti-phosphorylated CREB antibodies on Western blots as described in MATERIALS AND METHODS. Figure shows a representative blot and the densitometric data of the blots ( n = 4). * P - }' q, ]! K2 t( K$ u8 f0 z

Determination of PKC and PKA activities. PKC and PKA activities in extracts from MES cells treated with glucose, GlcN, and/or TGF- 1 were determined using specific peptide substrates for each kinase as described previously ( 41 ). Briefly, the reaction was carried out in 30 µl containing 20 mM Tris·HCl, pH 7.5, 10 mM Mg-acetate, 0.9 mM CaCl 2, 0.4 mM EGTA, 30 mM -mercaptoethanol, 25 µg/ml micellar phosphatidylserine, 0.4 µM PKA inhibitor peptide (PKI), 4 µM compound R24571  (an inhibitor of Ca 2  /calmodulin-dependent protein kinases), 100 µM PKC peptide pseudosubstrate, 5 µg protein of total or membrane extract, and 250 µM [ - 32 P]ATP (800-1,000 cpm/pmol). After 15 min at 30°C, 25 µl of the reaction mixture were spotted on P-81 phosphocellulose filters. The filters were washed four times (5 min each wash) with 0.75% (wt/vol) phosphoric acid and 32 P 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. 32 P incorporated into PKC peptide without adding cell extracts was negligible.

Phosphorylation of PKA substrate Kemptide was carried out in 30 µl containing 20 mM Tris·HCl, pH 7.5, 100 mM KCl, 1.0 mM DTT, 15 mM Mg-acetate, 4 µM PKC inhibitor peptide, 4 µM compound R24571 , 250 µM Kemptide, 5 µg of total protein extract, and 250 µM [ - 32 P]ATP. After 15 min at 30°C, 25 µl of the reaction mixture were spotted on P-81 phosphocellulose filters, and radioactivity incorporated into Kemptide was determined as described above. The addition of 0.5 µM PKI to the reaction completely blocked the phosphorylation of Kemptide, indicating a specific phosphorylation of the peptides by PKA.

Protein subcellular fractionation and PKC isoform analysis. Protein fractions from the cytosol, membrane, nucleus, and cytoskeleton were prepared using the S-PEK from EMD Biosciences and Calbiochem according to the manufacturer's instruction. MES cells were treated with glucose, GlcN, or TGF- 1, cells were harvested, and the pallet was subjected to sequential protein extraction steps that yield protein fractions from the cytosol, membrane and mitochondria, soluble nuclear proteins, and finally cytoskeletal proteins. Protease inhibitors and Benzonase, a nonspecific nuclease, were also included in appropriate fractions. Protein concentrations were determined and were subjected to Western blotting with isoform-specific anti-PKC antibodies. ECL visualized protein bands." ^) B3 R/ O, v
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Cellular ATP measurement. MES cells were cultured in DMEM containing glucose, GlcN, or TGF- 1 for 24 or 48 h. Cells were harvested by pancreatic digestion, washed twice with DMEM, and resuspended in the same medium. Cell numbers were determined microscopically using a hemocytometer. The amount of ATP released from intact cells was measured by a luciferase-luminol-based ATP assay kit from Sigma according to the manufacturer's instructions using a Monolight Luminometer. A standard curve for varying ATP concentrations was also constructed to determine the cellular ATP level.
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Statistical analysis. Results are expressed as means ± SE of the indicated number of experiments. Student's t -test and ANOVA were used to compare differences between cultures. A P value of
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RESULTS

Hexosamines and TGF- 1 stimulate fibronectin and laminin synthesis and their effects are not additive. We previously showed that the effects of HG on fibronectin and laminin in MES are mediated by glucose metabolism through the HBP ( 40, 41 ). Several studies suggest that the HG-mediated ECM protein synthesis is mediated via activation of TGF- in kidney ( 6 ). Therefore, we examined the effect of GlcN and TGF- 1 on fibronectin and laminin and whether they will produce additive effects when present together. Figure 1 A shows that HG (1.87 ± 0.26, P

Fig. 1. High-glucose (HG), glucosamine (GlcN), and transforming growth factor- 1 (TGF- 1 ) action on fibronectin and laminin are not additive. Simian virus (SV)-40-transformed rat kidney mesangial (MES) cells were cultured for 48 h in the presence of low glucose (LG; 5 mM), HG (25 mM), or GlcN (5 mM glucose 1.5 mM GlcN), and/or TGF- 1 for 24 h (5 mM glucose 2.5 ng/ml TGF- 1 ) and harvested as described in MATERIALS AND METHODS. Cell extracts (30 µg protein) were separated on SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with anti-fibronectin and laminin antibodies. Immunoreactive bands were detected by an ECL detection system. The intensity of the bands was measured by a Bio-Rad GS-700 Imaging densitometer. A : representative blot of the effect of HG, GlcN, and TGF- 1 on fibronectin and the densitometry data. The results, expressed as a percentage of control at LG, are means ± SE; P

HG- and GlcN-mediated fibronectin synthesis is blocked by anti-TGF- 1 antibodies. The results described above suggest that HG, GlcN, and TGF- 1 may use similar pathways or mechanisms to induce matrix protein synthesis in MES cells. We hypothesized that metabolism of glucose via the HBP upregulated TGF- 1 leading to increased ECM. To investigate this notion, we examined whether antibodies directed toward TGF- 1 will nullify the effects of HG and GlcN on ECM. As shown previously, HG, GlcN, and TGF- 1 stimulate fibronectin synthesis approximately twofold ( Fig. 2 ). However, the presence of pan-specific anti-TGF- 1 antibodies (20 µg/ml) in cultures blocked the HG-, GlcN-, and TGF- 1 -induced fibronectin production.

Fig. 2. Neutralizing anti-TGF- 1 antibodies blunt HG- and GlcN-induced fibronectin synthesis. MES cells were cultured in LG, HG, and GlcN for 48 h in the presence of 20 µg/ml anti-TGF- 1 antibodies, which inhibit TGF- 1 action in cells in culture. Cells were also treated with TGF- 1 plus the anti-TGF- 1 antibodies to establish the specificity of the antibodies on fibronectin production. The antibodies were added together with the agents and were present throughout the time of culture. Western blot analysis was performed for fibronectin in cell extracts similar to those described in Fig. 1. A representative Western blot for fibronectin and the densitometric analysis are shown. The values are expressed as a percentage of respective controls at LG and are means ± SE. Lane 1, LG (relative basal value); lane 2, HG; lane 3, GlcN; lane 4, TGF- 1; lanes 1 - 4, * P 4 G# ^9 `& |/ A8 B/ T$ L
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TGF- 1 stimulates PKC and PKA activities. We showed that HG and GlcN stimulate PKC and PKA activity in cultured MES cells ( 41 ). Here, we examined whether TGF- 1 also produces a similar effect on PKC and PKA activity and whether this effect might be mediated by the HBP. As shown in Fig. 3, A and B, TGF- 1 stimulates PKC and PKA activities in the membrane fraction by 44 ± 17 and 42 ± 13% ( P ' H! ?6 Y6 ^7 b  }, ]) C$ q+ m
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Fig. 3. Effect of TGF- 1 and the hexosamine biosynthesis pathway on PKC and PKA activities. MES cells were cultured for 24 h with 2.5 ng/ml TGF- 1 alone and in the presence of HG or GlcN as described in MATERIALS AND METHODS. PKC ( A ) and PKA ( B ) activities were assayed in the membrane fractions as described previously ( 41 ). Values are means ± SE of 3 different experiments performed in duplicate. The basal activity for PKC and PKA at LG was 0.3 and 0.4 pmol 32 P incorporated into peptide substrates·µg protein -1 ·min -1. P value for TGF- -mediated PKC and PKA activity was

Fig. 4. Western blot analysis of PKC and PKA protein levels. Equal amounts of extracts from LG-, HG-, GlcN-, and TGF- 1 -treated cells were analyzed for alterations in PKC and PKA levels using anti-type III PKC or anti-PKA RII regulatory subunit antibodies as described in MATERIALS AND METHODS. There were no significant differences in PKC content ( A ), but PKA content ( B and C ) was increased by HG (1.47 ± 0.1), GlcN (1.29 ± 0.2), and TGF- (1.60 ± 0.2) compared with LG. Results are means ± SE, n = 4. * P
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We further examined the effect of HG, GlcN, and TGF- 1 on PKC isoform distribution in MES cells. MES cells were treated with HG or GlcN for 48 h or with TGF- 1 for 24 h. Cells were harvested and proteins were extracted into cytosolic, membrane, and nuclear fractions. Expression of different PKC isoforms in these fractions was examined on Western blots using a PKC isoform panel kit. Figure 4 D shows that HG and GlcN increase the amount of PKC- in the membrane fraction, whereas HG, GlcN, and TGF- 1 increase PKC- 2 in the nucleus. PKC- was mostly located in the nucleus and was marginally increased by HG, GlcN, and TGF- 1. Other isoforms were not altered under these conditions. The presence of PKC- 2, -, and - in the nuclear fractions suggests that these isoforms may directly participate in nuclear signaling.' \/ d& ~0 j8 s

HG, GlcN, and TGF- 1 lead to a 1.2- to 1.6-fold increase ( P 3 X4 F5 W- W3 Z" M5 n' P
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Pharmacological inhibition of PKC and PKA activity impedes TGF- 1 -induced fibronectin and laminin synthesis. We previously showed that pharmacological inhibition of PKC and PKA blocks HG- and GlcN-mediated fibronectin and laminin synthesis ( 40, 41 ). Here, we investigated whether an inhibition of PKC or PKA will retard TGF- 1 effects on fibronectin and laminin in MES cells. Figure 5 shows that TGF- 1 stimulation of fibronectin was inhibited by both Bis-I and H-89. Inhibition of PKA also blocked TGF- 1 effects on laminin; however, the inhibition of PKC did not have a significant effect on TGF- 1 -mediated laminin synthesis.
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Fig. 5. Effect of inhibition of PKC and PKA on TGF- 1 -induced fibronectin and laminin synthesis. MES cells were cultured for 24 h with TGF- 1 in the presence of Bis-I (PKC inhibitor, 1.0 µM) or H-89 (PKA inhibitor, 2.0 µM) to block the kinase activity and were present throughout the period of incubation. Cell extracts were prepared, and the cytosolic fractions were analyzed for fibronectin by Western blot analysis. Figure shows representative blots for fibronectin and laminin, and their densitometic data are expressed as relative optical density values against LG. Fibronectin: lane 1, LG (basal control); lane 2, TGF- 1 ( P
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HG, GlcN, and TGF- 1 stimulate CREB phosphorylation. HG and GlcN result in an increase in CREB phosphorylation at Ser133 in MES cells, and these effects of HG are mediated by hexosamine metabolism ( 24, 40 ). Here, we investigated further the effects of TGF- 1 on CREB phosphorylation. As seen with ECM levels, the treatment of MES cells with HG, GlcN, and TGF- 1 increases CREB phosphorylation at Ser133 by about three- to fourfold ( P
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HG, GlcN, and TGF- 1 do not affect cellular ATP level. Hresko et al. ( 16 ) reported that GlcN treatment of 3T3-L1 adipocytes causes depletion of intracellular ATP and that GlcN-induced insulin resistance in these cells may be due to the loss of ATP. However, other investigators reported no effect of GlcN on ATP in MES cells ( 12 ). Therefore, we determined intracellular ATP levels in MES cells after treatment with HG, GlcN, or TGF- 1. We also did not observe any significant alteration in ATP levels in MES cells after exposure to 1.5 mM GlcN for 24 or 48 h ( Table 1 ).
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Table 1. Effect of HG, GlcN, and TGF- 1 on ATP levels in MES cells
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DISCUSSION3 v" |* H- S, v2 \/ I
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Numerous studies demonstrated that HG levels cause an increase in the synthesis and accumulation of ECM proteins in cultured kidney MES and tubular cells ( 1, 9, 44, 45 ). Some of the effects of HG on diabetic nephropathy are mediated via synthesis of TGF- (a profibrotic cytokine) that acts in an autocrine/paracrine fashion ( 32, 37 ). TGF- increases the synthesis and accumulation of ECM proteins in these cells, and neutralizing antibodies against TGF- attenuate the stimulation of matrix proteins by HG ( 31, 32 ). The HBP, which converts fructose-6-phosphate to GlcN-6-phosphate with glutamine as the amino donor, has been described as a cellular sensor for glucose and therefore a mediator of glucose regulation in a variety of cell types ( 3 - 6, 15, 27, 40, 41 ). In kidney MES cells, GlcN is more potent than glucose in stimulating TGF- 1 mRNA transcription and bioactivity, and the inhibition of GFA activity by the glutamine analog azaserine or antisense oligonucleotide against GFA blocks the HG-induced expression of TGF- 1 and matrix protein synthesis ( 6, 21 ).
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We hypothesized that the effects of hexosamines on MES ECM are mediated by hexosamine regulation of TGF- 1. We observed that TGF- 1 significantly enhances the production of ECMs in MES cells similar to HG and GlcN. However, the effects of TGF- are not additive to those of HG or GlcN. This result supports our hypothesis; however, the direct link among the HBP, TGF- 1, and increased ECM levels is not proven by this observation. Further support for this notion, however, is provided by the findings that the effect of GlcN on ECM is blocked by a TGF- 1 antibody ( Fig. 2 ).# |9 _* V( A1 n% A) N4 H( m/ n

The mechanisms by which hexosamines mediate the effects of excess glucose on MES ECM, while not fully understood, are becoming clearer. Previously, we showed that HG and GlcN increase both PKC and PKA activities in MES cells and agents that block the activity of these kinases blunt HG- and GlcN-induced fibronectin and laminin synthesis ( 40, 41 ). Here, we show that TGF- 1 also increases PKC/A activity in MES cells similar to HG and GlcN. Consistent with earlier results, HG-, GlcN-, and TGF- 1 -induced increases in PKA activity are associated with an increase in PKA protein levels. To the contrary, PKC protein content is not changed. In addition, the specific roles of PKC and PKA in the HBP or TGF- 1 -mediated regulation of laminin and fibronectin appear to be unique. Although both PKC and PKA are involved in hexosamine and TGF- 1 -mediated fibronectin synthesis, the effects of TGF- 1 on laminin appear to be mediated by PKA alone.
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Although HG, GlcN, and TGF- 1 enhance total PKC activity in MES cells, we do not know exactly which PKC isoforms are particularly important in ECM synthesis. Many isoforms of PKC are known to exist and they are categorized into three subclasses according to their structure and function ( 20, 22, 31, 32, 45 ). Kolm-Litty et al. ( 22 ) showed that exposure to 12 mM GlcN results in rapid and specific translocation of PKC-, -, and - to the membrane fraction, whereas PKC- is not affected. On the other hand, Goldberg et al. ( 13 ) observed no effect of GlcN on the translocation of PKC isoforms from the cytosol to the membrane in MES cells after 4 days of exposure. However, PKC- 1 and - activities were increased in the membrane fraction and were demonstrated to play an important role in hexosamine-mediated regulation of PAI-1 expression. Changes in PKC isoform-specific activity and translocation with exposure to HG and hexosamines probably involve diacylglycerol (DAG). For example, treatment of MES cells with 30 mM glucose results in an increase in DAG levels, which is associated with the translocation of PKC activity from the soluble to the particulate fraction ( 1 ). The effect of HG on DAG is evident at as early as 30 min and continues to be maintained for up to 1 wk of cell growth ( 1 ). The effects of hexosamine metabolites on DAG production in the mesangium are not clear but, similar to observations with PKC activity, they are likely to be similar to those of HG. Under our experimental conditions (48-h exposure of MES cells to HG or GlcN and 24-h exposure for TGF- 1 ), we observed that PKC- in the membrane was increased by HG, GlcN, and TGF- 1, whereas PKC-, -, as well as - were more prominent in the nucleus than in the cytosol or membrane ( Fig. 4 D ). Other isoforms tested were not altered by the above agents. These findings suggest that the role of PKC isoforms in cell signaling requires further investigation using isoform-specific inhibitors or gene knockout with RNAi and expression vectors. For example, specific inhibition of PKC- with LY-333531 has been shown to ameliorate the vascular complications of diabetes mellitus such as glomerular filtration rate, albumin secretion, and retinal circulation in diabetic rats ( 17 ). Future experiments will be undertaken to study the role of different PKC isoforms in nuclear signaling, transcription factor activation, and ECM gene regulation.
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Hyperglycemia and GlcN also increase the intracellular concentration of cAMP ( 25, 41, 45 ) and, therefore, may activate the PKA signaling pathway in MES cells. Because the fibronectin promoter contains CRE consensus sequences, phosphorylation of CREB by PKA and/or PKC may play a direct role in the transcriptional regulation of the fibronectin gene by TGF- 1 and hexosamines ( 8, 23, 34 ). With regard to TGF- 1 action, it appears that the PKA pathway is most important in CREB phosphorylation ( Fig. 7 ). The involvement of other kinase(s) in the phosphorylation of CREB in MES cells may not be ruled out because hexosamine-induced CREB phosphorylation is not completely abolished in the presence of PKC or PKA inhibitors together ( 40 )., F$ S; P# G  V' S4 S, Q8 l1 {
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Glucose regulates the expression of a number of genes including growth factors in a variety of cell types and some of these effects are mimicked by GlcN ( 5, 6, 21, 29, 36, 42 ). For example, GlcN was found to be more potent than glucose in stimulating TGF- gene expression in rat aortic smooth muscle cells, inducing up to a 12-fold increase in TGF- promoter reporter activity at a comparatively low concentration of 2 mM ( 29 ). Similarly, TGF- 1 transcription in rat vascular and renal cells is about two- to threefold higher with 3 mM GlcN vs. 20 mM glucose ( 6 ). These findings strongly advocate a role for the hexosamine pathway in mediating the hyperglycemia-induced growth factor regulation as seen in disease conditions.6 `5 j! l+ T/ x* e  c1 ?
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Nevertheless, the mechanism by which flux through the hexosamine pathway mediates hyperglycemia-induced increases in gene transcription is not entirely understood. One potential mechanism by which the HBP might regulate gene transcription is through enhanced O-glycosylation. Both glucose and GlcN increase the level of UDP-GlcNAc in MES cells, which is the downstream end product of the pathway and is a precursor for protein O-glycosylation ( 10, 13, 14, 18, 27, 43 ). Recently, McClain et al. ( 28 ) demonstrated that overexpression of an isoform of O-GlcNAc transferase in muscle and fat leads to insulin resistance and hyperleptinemia in transgenic mice. Observations that hyperglycemia increases Sp1 transcription factor glycosylation and activates plasminogen activator inhibitor-1 expression in MES cells and vascular smooth muscles indicate that covalent modification of Sp1 by O-GlcNAc may explain the link between HG and changes in gene expression ( 10, 12, 18, 36 ). TGF- 1 also has Sp1 sites in its promoter and increased hexosamine flux results in an increase in both Sp1 glycosylation and TGF- 1 expression ( 10 ), again indicating a relationship among the HBP, transcription factor activation, and gene expression.

We hypothesize that the HBP acts as a cellular sensor of glucose or other nutrients in MES 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 fibronectin and laminin levels when the rate-limiting enzyme in the HBP GFA is inhibited ( 21, 40, 41 ). Additional support is provided by the observation that overexpression of GFA in MES cells renders them more sensitive to the effects of glucose with respect to ECM accumulation and CREB phosphorylation ( 39 ). Thus downstream products of the HBP may upregulate second messenger proteins, growth factors, or transcription factors, resulting in enhanced ECM gene expression. The upregulation of these signaling systems in MES cells ultimately will lead to increased ECM synthesis and accumulation.

The data from the current study and past observations support the hypothesis that excess glucose leads to increased TGF- 1 transcription ( 6, 21 ) and activity ( 21 ) via metabolism through the HBP. These hexosamine-induced effects of TGF- 1 result in increases in the ECM components laminin and fibronectin. In the case of fibronectin, these effects of TGF- 1 are mediated by PKC and PKA, possibly through activation of the transcription factor CREB. TGF- 1 effects on the accumulation of laminin also involve PKA, and, therefore likely CREB, but appear to be independent of PKC. Identification of the specific regulatory steps in ECM synthesis that are influenced by the HBP will facilitate the development of novel therapeutic interventions for patients with diabetic nephropathy.

GRANTS
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Support for this work was from The Kidney Care Foundation and the Veterans Administration to E. D. Crook. Support from the Robert W. Schrier, MD Young Investigator Grant of the National Kidney Foundation to L. P. Singh is also acknowledged.3 q% s6 d& h$ L6 z- b& k$ S& x, P
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