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发表于 2009-4-22 09:42 |只看该作者 |倒序浏览 |打印

作者：Satish P. Ramachandra Rao,, Richard Wassell, M. Alexander Shaw, and Kumar Sharma,作者单位：1 Dorrance H. Hamilton Research Laboratories, Department of Medicine, 3 Center for Novel Therapies for Kidney Disease, and 2 Proteomics Core Facility, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania * \/ }, E; K5 r4 N) G2 |# O: l) b+ F
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      【摘要】3 m$ E9 N! s1 ]
   Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dysregulation. In the membrane subproteome, of the 92 spots that were matched across all gels, HG induced significant downregulation of only 4 protein spots. The dysregulated spots from the membrane subproteome included binding protein (BiP), calreticulin precursor protein, a 63-kDa transmembrane protein from a ER/Golgi intermediate, and -subunit of collagen proline 4-hydroxylase. In the cytosolic subproteome, of the 122 spots that were matched across all gels, HG induced downregulation of 3 protein spots and upregulation of 2 protein spots significantly. Enolase 1, annexin VI, and 2 -actin were decreased, whereas heat shock protein-70 kDa and calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited a marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiological relevance of CaM upregulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor- and insulin in mesangial cells. In conclusion, using a combination of cell fractionation and protein expression profiling, we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells. & F/ p- |9 z, x. H6 E+ S2 N! c* X1 c1 V
      【关键词】 proteomics subcellular fraction diabetic nephropathy
   DESPITE TREMENDOUS ADVANCES in our understanding of the molecular basis of diabetes, substantial gaps remain both in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment ( 8, 30 ). Diabetic nephropathy (DNP), known to account for nearly 50% of all new cases of end-stage renal disease ( 7 ), is a major long-term complication of both types of diabetes ( 11 ). Present conventional anti-DNP therapies notwithstanding, DNP prevalence continues to be a major public health problem, emphasizing the need for developing new insights into DNP mechanism. Proteomics, a robust discovery platform ( 35 ), helps in an understanding of the disease process not only at the global level, but at a more localized and specific level as well, and hence holds great promise to enable development of novel therapeutic strategies. Since mesangial cells (MC) are pivotal in the glomerulus in both normal and disease states, and are an accepted in vitro model for events that occur in DNP development ( 12, 18, 19, 36, 55 ), we sought to study the proteome- level changes induced in MC by high glucose (HG), a diabetes-mimicking stimulus.
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To overcome the complexity if the entire human mesangial cell (HMC) proteomes were studied, we fractionated the HMC proteins into cytosolic and membrane proteins. The fractionated samples were then analyzed by two-dimensional electrophoresis (2DE) coupled with DeCyder analysis for differential regulation of proteins. We show by proteomic analyses that HG regulates protein expression in the HMC cytosol in a different manner than in HMC membrane and that calmodulin (CaM), a highly conserved, ubiquitously present calcium-binding protein, is upregulated in the HMC cytosol. We validated the proteomic data of HG-induced CaM upregulation by HMC immunostaining. We also found that kidneys of rats and mice rendered diabetic by streptozotocin (STZ) have increased CaM and, finally, that CaM is required for growth factor-induced glucose transport in MC., o" C* q' K" o% w) Y" H- d

MATERIALS AND METHODS: T$ @1 a7 a6 v7 X3 Q% q" D2 l$ V
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Cell culture. Primary HMC (Clonetics, Walkersville, MD) ( 4 ) and SV40 transformed murine mesangial cells (MMCs) ( 25 ) were cultured as previously described. HMC ( passages 4 - 6 ) modulation for 7 days was performed in 5 mM [normal glucose (NG)] or 30 mM D -glucose (HG) in MsBM medium (Clonetics) containing 5% serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. HMC culture medium was changed every other day as described by Clarkson et al. ( 4 ).

HMC subcellular fractionation and buffer exchange. Post-HG treatment for 7 days, cells were scraped out and washed with buffer A containing 20 mM Tris at pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 100 µM each of -glycero-phosphate, sodium orthovanadate, and PMSF, 2.5 mM sodium pyrophosphate, 1 µg/ml leupeptin, and complete mini-protease inhibitor cocktail (Roche Diagnostics) by centrifugation at 1,000 g for 5 min. The pellet obtained was resuspended in buffer A containing digitonin (250 µg/ml) ( 49 ), mixed gently for 30 min on ice, and spun at 15,000 g for 15 min at 4°C. The supernatant (cytosolic proteins) and pellet (membrane proteins) were separated. The pellet was washed with buffer A, until no protein was detectable in the washes by absorbance at 280 nm. This pellet was solubilized using Triton X-100 (1% vol/vol) in buffer A. Membrane/hydrophobic protein solubilization was further aided by 10 up-down movement strokes in a 25-gauge needle on ice, taking care not to increase the temperature. Subsequent to centrifugation (15,000 g for 15 min) of this preparation, the pellet was discarded. The supernatant, used as the total membrane protein, was buffer exchanged by repeated dilution and reconcentration with 10 ml (500 µl x 20 times) of the sample/rehydration buffer in a Millipore ultrafiltration device. Protein estimation was carried out with Bio-Rad (RC-DC) Protein Assay kit, and 250 µg protein of the buffer-exchanged protein preparation were then loaded on immobilized pH gradient (IPG) strips (pH 3-10) in equilibration trays for 16-20 h via passive rehydration according to the manufacturer's instructions (Bio-Rad, Hercules, CA).
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Electrophoresis, image acquisition, and DeCyder analysis of spot dysregulation. Isoelectrofocusing (IEF; PROTEAN IEF cell, Bio-Rad) and SDS-PAGE (Criterion gels, Bio-Rad) were performed according to the manufacturer's instructions. After electrophoresis, gels were fixed, stained with SYPRO Ruby (Invitrogen), and scanned using a Typhoon 9400 Variable Mode Imager (GE Healthcare). Eight membrane gel images, four NG and four HG, thus obtained were individually processed in the Differential In-gel Analysis (DIA) module of DeCyder software (version 5.01, GE Healthcare). Each of the DIA files was then imported into a single biological variation analysis (BVA) file in which corresponding protein spots were matched across the eight gel images. Raw volumes of the protein spots were exported into Excel and normalized by setting the total spot volumes of each gel equal (intergel normalization). Subsequently, for each set of matched protein spots, percent volume contribution of the spots to the total gel was determined and grouped into NG and HG sets. The resulting distributions were used to calculate both the average abundance change (extent of dysregulation) and a P value for statistical confidence in that abundance change value. The upregulation of spots in HG images by more than 200% or downregulation by less than 50% with a confidence of P 9 w7 X. T5 E5 L/ U
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In-gel cysteine reduction/alkylation and tryptic digestion of proteins. Gel pieces containing the proteins of interest were manually excised with a disposable scalpel, cut further into smaller pieces of 1.0 mm 3, and transferred to low-retention siliconized 0.5-ml centrifuge tubes. Subsequent to reduction and alkylation, proteins were digested with trypsin as previously described ( 40 ), except that 50 µl of 20 mM ammonium bicarbonate containing 20 ng/µl of sequencing grade trypsin (Promega, Madison, WI) were used in two parts for the in-gel digestion of proteins. After initial addition of trypsin (30 µl), the samples were incubated on ice for 30 min, and then trypsin (20 µl) was added again to further swell the gel. The sample tubes were sealed with parafilm and incubated at 37°C for 16-20 h in a circulating water bath. After another cycle of rehydration-dehydration of the gel pieces and second extraction of peptides from the gel, the samples were dried in a vacuum centrifuge.
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Mass spectrometry and peptide mass fingerprinting. The peptide fragments from in-gel trypsin digests of the protein spots of interest were analyzed by surface-enhanced laser desorption ionization/time-of-flight (SELDI-ToF) mass spectrometry on a Ciphergen PBS II instrument operated in linear mode. Samples were prepared on an H4 hydrophobic chip using a matrix of -cyano-4-hydroxy-cinnamic acid. ANG I (1,296.5 Da) and -endorphin (3,465.0 Da) were used as internal calibrants. Protein identification of peptide fragments was performed by using primarily the "ProFound" search engine (Rockefeller University, 129.85.19.192  /profound_bin/WebProFound.exe), and MASCOT software ( http://www.matrixscience.com ) as a secondary check. The National Center for Biotechnology Information (NCBI) protein database was restricted to mammalian entries in the preliminary rounds of analysis, and to Homo sapiens in the subsequent round of analysis. Peptides were assumed to be average mass, oxidized at methionine residues, and carbamidomethylated at cysteine residues. Up to one missed trypsin cleavage and a mass tolerance error of ±0.6 Da was allowed for matching peptide mass values. Criteria for protein confirmation included a statistically significant Z score with a probability of 1.0, at least 25% sequence coverage, and correspondence of the matched peptides with the main peptide peaks of the mass spectrum.

Confocal analysis of CaM immunoreactivity in the HMC. HMC were grown on no. 1 coverslips pretreated with poly- D -lysine (0.1 mg/ml for 5 min). After a 7-day treatment with HG/NG/mannitol, HMC were treated with a mouse monoclonal anti-CaM antibody (Upstate, Lake Placid, NY; 1:100 dilution, 60 min, 37°C) and Alexa-fluor-568-conjugated goat anti-mouse antibody (1:100 dilution; Molecular Probes, Eugene, OR; 30 min, 37°C) as previously described ( 36 ). Coverslips were mounted with SlowFade (Molecular Probes) and were visualized with a Bio-Rad MRC-600 confocal laser-scanning microscope. Control cells were stained only with the secondary antibody.) `  ~4 u! {9 S% D: l2 v  d8 p
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Protein analysis. SDS-PAGE (4-15% gradient Readygel precast gels; Bio-Rad) and wet transfer to a nitrocellulose membrane (Bio-Rad) were performed as previously described ( 25 ). The membrane was blocked in 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 (TBST) and incubated overnight at 4°C with the mouse monoclonal anti-CaM antibody (Upstate; 1:1,000 dilution). After three washes in TBST, membranes were probed with a secondary goat anti-mouse (Jackson Immunolabs, West Grove, PA) conjugated to horseradish peroxidase (HRP). The HRP-catalyzed chemiluminescence reaction was developed with SuperSignal West Pico substrate (Pierce Biotechnology, Rockford, IL), allowing the detection of immunoreactive protein bands. Finally, the membrane was probed with mouse anti- -actin antibody (Sigma, St. Louis, MO) to serve as a loading control.9 K% B5 T* K7 H/ W2 M
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Analysis of CaM protein levels in kidneys of rat and mouse models of type 1 diabetes: CaM in early-stage diabetes. To study CaM regulation in short-term diabetes, Sprague-Dawley rats weighing 265-311 g were made diabetic by a single intraperitoneal injection of STZ (65 mg/kg body wt) in 10 mmol/l sodium citrate, pH 5.5, as described earlier ( 33 ). After 2 wk of diabetes, kidneys were isolated and weighed (1.74-2.02 g diabetic; 1.37-1.9 g nondiabetic) and processed for kidney protein preparation by homogenization in 10 volumes of buffer A with 1% (vol/vol) Triton X-100. Centrifugation and protein quantification were similar to those done for HMC membrane proteins. Resolution of 50 µg protein from each sample by SDS-PAGE and all further procedures were as detailed for immunoprobing of CaM from the HMC cytosol.1 @9 v: y- |' n1 Q4 |2 y
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CaM in late-stage diabetes. Analysis of CaM protein levels in kidneys of mice was carried out as follows. C57Black/J mice weighing 16-23 g were made diabetic by following the Diabetes Induction by Low-Dose Streptozotocin Induction Protocol (mouse; 50 mg·kg -1 ·day -1 x 5 consecutive days) in 10 mmol/l sodium citrate, pH 5.5, as elaborated ( 14, 34 ). After 12 mo of diabetes (blood glucose 300 mg/dl), kidneys from these mice were isolated and processed for kidney protein preparation as detailed for rat kidney protein analysis.

Glucose uptake studies. Glucose uptake measurement was performed in six-well plates, with 2-deoxy-o-glucose (2-DOG) as the substrate as previously described ( 10 ). MMC exposed to various concentrations of TGF- (Peprotech, Rocky Hill, NJ) as indicated, with or without 100 nmol/l insulin (Sigma), or CaM inhibitors (trifluoperazine, W7 and W13, concentration as indicated) were incubated in Krebs-Ringer phosphate (KRP)-HEPES buffer 10 mM, pH 7.45, and 0.5% BSA with 2-deoxy-[1- 3 H]-o-glucose (0.4 µCi/well; Amersham Pharmacia, Arlington Heights, IL) and 0.1 mmol/l of unlabeled 2-DOG for 5 min. Incubation was terminated by rapidly aspirating the buffer and washing with cold PBS (3 x 1.0 ml). Cells were harvested with NaOH (1 N, 1 ml) and rinsed with HCI (1 N, 1 ml) for -emission counting. Glucose uptake was measured in triplicate, and cell protein was determined as above.

Statistical analysis. Comparisons between two groups were analyzed by Student's unpaired t -test.
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RESULTS
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HG treatment alters protein expression differently in HMC membrane and cytosol. The number of spots detected by the DeCyder software in the three NG membrane subproteome images and three HG membrane subproteome images ranged from 753 to 970. One NG membrane image and one HG membrane image are shown, respectively, in Fig. 1, top and bottom. The gel image with the maximum number of protein spots was designated the master image, and 350-370 protein spots were matched to the Master image ( Table 1 ); 92 of the matched spots were present in all 6 gel images. Only four spots exhibited statistically significant differences in expression level between the two conditions as indicated by a Student's t -test value of 0.05 or less; all four were decreased in HG subproteome over their NG counterpart.
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Fig. 1. Downregulation of membrane subproteome in high glucose (HG). The 2-dimensional (2-d) electrophoretic pattern of total membrane proteins of human mesangial cells (HMC) grown at normal glucose (NG; top ) or HG ( bottom ) is shown. Labeled spots 3, 4, and 5 were downregulated in HG. Spots 1 and 2 were used as internal control for the process of spot excision and protein identification and were identified to be the same protein, namely BiP (see also Table 1 ). Identification of other proteins and number labeling correspond to the spot number in Table 1. Right : molecular weight marker values. Data are representative of 3 independent experimental determinations.- w% c& @$ f/ u) z

Table 1. DeCyder analysis of HMC membrane HG subproteomes (gels 1-3) and NG subproteomes (gels 4-6)+ `& }2 ]* d$ M
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On the other hand, the number of spots detected by DeCyder software in three NG cytosolic subproteome images and three HG cytosolic subproteome images ranged from 800 to 950. One NG cytosolic image and one HG cytosolic image are shown, respectively, in Fig. 2, top and bottom. Between 370 and 400 protein spots were matched to the Master image ( Table 2 ); 122 of the matched spots were present in all 6 gel images. Statistically significant ( t -test value of 0.05 or less) differences in the level of expression between the two conditions were found for eight protein spots; three spots had lower expression levels in HG subproteomes, whereas two spots had higher expression levels in HG subproteomes. Low volumes of the other three spots precluded their further analysis. Thus the same HG stimulus induced different modes of overall regulation of protein expression in HMC membrane and cytosol.8 B9 D. G5 w" ]2 \# `) b6 {
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Fig. 2. Up- and downregulation of cytosolic subproteome in HG. The 2-d electrophoretic pattern of cytosolic proteins of HMC grown at NG ( top ) or HG ( bottom ) is shown. Right : molecular weight marker values. The arrow mark indicates the protein spot that was excised, trypsinized, and identified as calmodulin by SELDI-ToF-MS. Data are representative of 3 experimental determinations.
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Table 2. DeCyder analysis of the HMC cytosolic HG subproteomes (gels 1-3) and NG subproteomes (gels 4-6)

Four proteins are downregulated in the HG membrane subproteome. SELDI-ToF-MS analyses of tryptic digests of the four downregulated proteins against the NCBI database identified them as BiP protein, calreticulin precursor protein, collagen-proline hydroxylase -subunit, and a 63-kDa transmembrane protein from the endoplasmic reticulum (ER)/Golgi intermediate ( Table 3 ). These were, respectively, called spots 2, 3, 4, and 5. As an internal control, the spot corresponding to 2 from the HG membrane subproteome, called spot 1, was excised out of the HG gel, trypsinized, and identified by SELDI-ToF as BiP, the same protein as spot 2 from the NG membrane subproteome. Successful identification of spots 1 and 2 and their identification as the same protein served as a good internal control for both gel running as well as the process of identification. The physical details of these spots are summarized in Table 3.

Table 3. SELDI-ToF-Bioinformatic analysis of HMC membrane protein tryptic digests from gel areas of interest that were significantly HG dysregulated3 j; Q2 k# B5 W; a3 m. w6 b
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HG induces downregulation of three proteins and upregulation of two proteins in the cytosolic subproteome. Table 4 shows details of the SELDI-ToF-MS analyses of tryptic digests of the five most prominently dysregulated proteins against the NCBI database. Whereas Enolase 1, p68 annexin VI, and 2-actin were downregulated, heat shock 70-kDa protein and CaM were upregulated in the HG cytosolic subproteome. CaM, a well-characterized protein involved in Ca 2  signaling, had the largest degree of upregulation of all the dysregulated proteins. We therefore attempted to further validate the proteomic data for CaM by other methods such as immunofluorescence confocal image analysis and Western immunoblot analysis ( Fig. 3 ). Confocal analysis revealed that CaM was primarily in the cytosol of HMC and was greatly increased by HG ( Fig. 4, A - D ). This effect was not observed with mannitol treatment, indicating that hyperosmolality was not a stimulus for CaM upregulation. By immunoblotting cytosolic protein, CaM was found to be clearly upregulated by HG conditions in MC ( Fig. 4 E ).1 x; G% J' `1 e! d4 k) o/ _

Table 4. SELDI-ToF-Bioinformatic analysis of the HMC cytosolic protein tryptic digests from gel areas of interest that were significantly HG dysregulated' b3 ]# e- D/ G& b$ D/ o0 V

Fig. 3. Calmodulin-SELDI-ToF mass spectrum. Peptide fragments from in-gel trypsin digests of the selected spot (arrow from Fig. 2, bottom ) were analyzed by SELDI-TOF mass spectrometry. Details are as described in MATERIALS AND METHODS. Protein identification was performed by using the ProFound search engine and MASCOT software. NCBI protein database was restricted to Homo sapiens entries. The mass spectrum characteristic of CaM tryptic fragments is shown.8 P, o$ \& A; r& c% b  O

Fig. 4. Upregulation of calmodulin (CaM) in HMC with HG and in diabetic kidneys. Immunostaining for CaM in HMC for CaM immunoreactivity in HMC grown in NG (5 mM; A ) and HG (30 mM; B ) and osmolar control (25 mM mannitol 5 mM glucose; C ) is shown. D : autofluorescence control. After treatment as detailed in MATERIALS AND METHODS, mouse monoclonal anti-CaM antibody at 1:100 dilution and goat anti-mouse antibody at 1:100 dilution conjugated to Alexa Fluor-568 were used to detect CaM. Bio-Rad MRC-600 confocal laser-scanning microscope mounted on a Zeiss Axiovert 100 fluorescent microscope with rhodamine filter was used. Representative regions are shown. Control cells, stained only with secondary antibody, showed minimal background fluorescence. E : CaM immunoreactivity from HMC cytosol. Equal amounts of cytosolic proteins obtained from HMC grown under NG ( lane 1 ) or HG ( lane 2 ) were resolved by SDS-PAGE and immunoblotted with antibody for CaM ( top ), and the same membrane was stripped and immunoblotted with antibody to -actin ( bottom )., e/ S, T2 Y2 s9 [3 l5 e

CaM in early- and late-stage type 1 diabetes. We asked whether CaM upregulation was also observed at the organ level. Regulation of CaM in early stage of diabetes was studied in STZ-diabetic rat model ( 34 ). Immunoprobing for CaM in STZ-diabetic rat kidney by Western analysis showed that CaM was markedly upregulated in the diabetic rat kidney compared with its normal nondiabetic counterpart ( Fig. 5 A ). To study the effects of chronic diabetes on CaM expression, immunoblotting for CaM in kidney from mice with diabetes for 12 mo was carried out by Western analysis. The mice maintained blood glucose values between 300 and 600 mg/dl for 12 mo. CaM was markedly upregulated in the diabetic mouse kidney compared with its normal nondiabetic counterpart ( Fig. 5 B ). The TGF- levels in the diabetic mouse urine were markedly increased (113.5 ± 23.39 pg TGF- 1 /mg creatinine in diabetic mouse urine vs. 0.3466 ± 0.1361 pg TGF- 1 /mg creatinine in normal mouse urine; n = 3; P = 0.04).
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Fig. 5. A : CaM immunoreactivity from the kidneys of 3 normal rats and 3 STZ-diabetic rats. Equal amounts of cytosolic proteins obtained from the kidneys (normal: lanes 1-3 on left side; diabetic: lanes 1-3 on right side) were resolved by SDS-PAGE, immunoblotted with antibody for CaM, stripped, and immunoblotted with antibody to -actin. B : CaM immunoreactivity from the kidneys of 3 normal mice and 3 STZ-diabetic mice. Equal amounts of cytosolic proteins obtained from the kidneys (normal: lanes 1-3 on left side; diabetic: lanes 1-3 on right side) were resolved by SDS-PAGE, immunoblotted with antibody for CaM, stripped, and immunoblotted with antibody to -actin.
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TGF- - and insulin-induced enhancement of glucose uptake in MMC is blocked by functional CaM inhibitors. Glucose transport activity in MCs is well documented ( 12, 23, 24, 26, 47 ). Although CaM has been shown to regulate glucose entry in insulin-responsive cells such as fat, liver, and muscle ( 9, 37, 38, 52, 53 ), no studies have evaluated the role of CaM to regulate glucose entry into MC.

MMC in culture rendered quiescent by overnight serum deprivation were treated for 8 h with TGF- at 1, 5, and 10 ng/ml concentration, and glucose uptake was monitored. TGF- exhibited a dose-dependent increase in glucose uptake ( Fig. 6 A ), with maximal stimulation at 10 ng/ml. Inhibition of CaM function by TFP or W7 completely inhibited TGF- -induced glucose uptake. Similar findings were noted with W13 (data not shown).

Fig. 6. A : inhibition of CaM blocks growth factor-induced glucose uptake. Effect of different doses of TGF- on the radioactively labeled [ 3 H]2-de-oxy glucose (2-DOG) uptake in murine mesangial cells and the effect of TFP and W7 on TGF- -induced glucose uptake increase are shown. Glucose uptake measurement was performed in 6-well plates. Other details are as given in MATERIALS AND METHODS. After cells were harvested, radioactivity by -emission was recorded, and emission per mg protein was calculated. Glucose uptake and cellular protein estimation were performed in triplicate in all treatments. Comparisons between control and the TGF- -treated groups were analyzed by Student's unpaired t -test. Data are means ± SE ( n = 3). * P * A5 K; H9 \" t3 Y/ j+ w! W4 `
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Prior reports demonstrated that CaM is involved in regulating glucose uptake by insulin via translocating GluT4 to the plasma membrane. As MCs have been described to contain GluT4 and be responsive to insulin, we also evaluated the effects of insulin on MC glucose uptake in the presence of CaM antagonists. Glucose uptake in cells was modestly stimulated by insulin ( Fig. 6 B ). Inhibition of CaM with TFP blocked the insulin-stimulated glucose uptake.

DISCUSSION
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We herein report our findings on HMC subproteome profiling subsequent to ambient HG exposure. We resorted to HMC subcellular fractionation and separate analyses of membrane and cytosolic subproteomes for two reasons: 1 ) to reduce the complexity of the analysis that would ensue if entire HMC proteomes were studied and 2 ) to amplify the inherent difference in properties of proteins from the membrane (generally hydrophobic) and those from cytosol (hydrophilic) by the virtue of which different handling is required. Indeed, HG-induced dysregulation was different in the membrane and cytosolic subproteomes. Thus, by combining subcellular fractionation with 2-DE, we show that in addition to enhancing the number of proteins that can be rendered detectable, subcellular fractionation further delineates the dissimilar nature of protein expression regulation in different cellular compartments.$ ~- n* m( t. ^6 a3 M# S
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Of the nine dysregulated proteins in both compartments, CaM was the most significantly upregulated. We sought further validation of CaM upregulation by nonproteomic means. CaM upregulation was corroborated by Western blotting and immunostaining of MC. Furthermore, CaM upregulation was also demonstrated in whole kidney protein preparation of STZ-diabetic rats, reflecting the potential physiological relevance of the proteomic data. Consistent with these results, CaM upregulation in the kidney of the diabetic OVE26 mice, also using a proteomic approach, was recently reported ( 43 ). CaM has been shown to play a fundamental role in regulated exocytosis, transcytosis, and endocytotic membrane trafficking ( 29, 41, 48 ), and inhibitors of CaM function have been shown to affect glucose uptake and metabolism in several insulin-responsive cell types ( 38, 51 ). In view of our findings that CaM is distinctly upregulated in HG, we investigated whether glucose uptake in the MC may be affected by CaM inhibitors. As enhanced action of the prosclerotic cytokine TGF- 1 may be mediated, in part, by enhanced glucose uptake ( 15 ), we examined the role of CaM in TGF- - and insulin-induced mesangial cell glucose uptake. The TGF- -induced increase in glucose uptake was completely inhibited by CaM antagonists. Similarly, insulin-stimulated glucose uptake was completely inhibited by CaM antagonists. Thus glucose uptake studies in the presence of CaM-specific antagonists further demonstrated that CaM plays an important role in mesangial cell glucose uptake in response to growth factors. Although the presence of CaM in HMC was not surprising due to its ubiquitous nature, this is the first report to show upregulation of CaM with HG in HMC and to show that CaM plays a critical role in glucose uptake in MC.

TGF- is a multifunctional cytokine involved in regulation of proliferation and metabolism of extracellular matrix proteins in many cell types including MC. In animal models, TGF- is shown to promote renal cell hypertrophy as well as stimulate extracellular matrix accumulation, the two hallmarks of diabetic renal disease ( 2, 54 ). In this communication, we show that the TGF- -induced increase in the mesangial cell glucose uptake is reversed by CaM antagonists. In view of this finding, it would indeed be interesting to study whether and how the increased glucose uptake contributes to the hypertrophic and fibrogenic effects of this cytokine, and the molecular dileniation of how it gets altered by CaM inhibitors.

Apart from CaM, other proteins that were dysregulated by HG in the cytosolic subproteome included enolase 1, annexin VI, 2-actin, and heat shock 70-kDa protein 8. Enolase 1, also called phosphorpyruvate hydratase, catalyzes the penultimate step of the glycolytic pathway: i.e., 2-phosphoglycerate (2PG) conversion to phosphoenolpyruvate (PEP). It is also a multifunctional enzyme that plays a role in various processes such as growth control, hypoxia tolerance, and allergic responses. Mammalian enolase is located in the cytoplasm and can translocate to the plasma membrane in either the homodimeric ( 2) or heterodimeric ( - ) form ( 3, 6 ). Its decrease with HG to almost half the control levels, as observed in our experiments (ratio of enolase levels in HG/NG is 0.542, n = 3, P = 0.0304), may be due, in part, to the cellular response to downregulate the glucose metabolizing enzymes in the presence of HG. It would be intriguing to study whether the well-established glucocytotoxic mechanism of reduced cytosolic glyceraldehyde-3-phosphate dehydrogenase activity translocating to nucleus due to HG is the cause for the reduction in enolase levels despite elevated glucose concentration ( 1, 31, 32 ).- [! b; V) ~/ H

Annexin VI and 2-actin have been shown to either directly or indirectly be involved with the cytoskeletal organization of HMC, thus reflecting the muscle-like contractile properties of HMC. Annexin VI isoform 1 or 2 differs by a 6-amino acid insert and has been implicated in mediating endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. It is thought to be located inside stress fibers that localize to a submembranous region of the cytosolic periphery. It may associate with CD21 and may also regulate the release of Ca 2  from intracellular stores ( 13, 17, 20 ). 2-Actin is a group 3 actin, found in the cytosols of nonmuscle cells. Actins are highly conserved and involved in various types of cell motility, with their primary function being the maintenance of the cytoskeleton ( 16, 28, 42, 44 ). Both annexin VI and 2-actin are decreased with HG in HMC, indicating that at least part of HMC response to increased ambient glucose would be mediated by Ca 2  -dependent cytoskeletal rearrangements.; S  d! ]& O1 f  F/ ], Q) P% [
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Heat shock-70 kDa protein 8, isoform 1 or 2 was found to be increased in the HG cytosolic subproteome. HSP70 ( 45, 50 ) is a well-known chaperone protein. It translocates rapidly from the cytoplasm to the nuclei, upon heat shock. Isoform 2 may function as an endogenous inhibitory regulator of HSC70.
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Many of the HG-dysregulated proteins in the membrane subproteome localized to ER. Prominent ones that localized to ER included BiP, also called GRP78 ( 39 ), can be stimulated by a variety of environmental and physiological stress conditions that perturb ER function ( 21, 22 ). Calreticulin, a chaperone protein, has been shown to be an ER lumen protein that assists in appropriate protein folding. The -subunit of proline 4-hydroxylase catalyzes the posttranslational formation of 4-hydroxyproline in -xaa-pro-gly- sequences mainly in collagens. It is essential to the proper three-dimensional folding of the newly synthesized procollagen chains. Due to their ER localization, the HG-induced downregulation of all these proteins suggests a role for ER stress in mediating glucotoxicity in the HMC. In the islet -cells, chronic exposure to HG and free fatty acids is shown to have dual effects, initially triggering glucose hypersensitization, and later apoptosis is linked to ER stress ( 5, 46 ). In the light of our findings of the HG-induced ER-resident protein dysregulation, whether similar mechanisms operate in the HMC merits further studies. To our knowledge, proteins such as calreticulin, BiP, and the ER/Golgi trans -membrane protein have not been shown to be involved in diabetes vascular complications. Further understanding of the roles that these proteins play in mediating glucotoxicity will likely be of relevance to diabetes complication.
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It is likely that the majority of spots that showed no significant variation with HG belong to structural and housekeeping proteins that show no significant variation due to HG. Ideally, all detected protein spots in both NG and HG subproteomes should be identified; however, it is more meaningful from an economy and disease point of view to identify only those protein spots that are significantly dysregulated. In such a case, it is appropriate that different subproteomes be studied, so that differences in the regulation mechanisms of various protein populations are understood. Using a digitonin-permeabilization protocol, we successfully isolated the total complement of cytosolic proteins from membrane proteins, quickly and efficiently. This is borne out by the identification of the ER luminal and ER/Golgi trans -membrane proteins in the membrane subfraction and not in the cytosolic subfraction, which indicates that these membrane-bound organelles were intact even after digitonin-induced plasma membrane solubilization. This further increases our confidence in digitonin treatment for successfully isolating the total complement of cytosolic proteins without resorting to elaborate protocols. In studies involving cytosolic proteins that either have a rapid turnover or suffer pH-, temperature-, or solvent-sensitive inactivation due to handling, the activity may not be detectable. This problem is compounded due to lengthy and labor-intensive protocols for purifying the protein of interest. Due to its inherent simplicity and speed, digitonin permeabilization of cells allows for developing methods that easily surmount this problem. The above protocol can be easily applied to other cell types.
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In summary, we used protein expression profiling as a tool to identify important proteins that have not previously been recognized to play important roles in diabetic vascular complications. Regulation of overall expression profiles is different in distinct cellular compartments. We demonstrated the advantage of using cell fractionation to highlight differences in the regulation patterns of proteins in different subproteomes. Using a proteomics-based approach coupled with functional studies, we identified a critical and as yet unrecognized role for CaM in glucose transport in MC.- A4 D, A+ Z/ |7 C3 l+ S# F

ACKNOWLEDGMENTS1 t- `' W- Z+ A8 n% y4 I
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We thank Dr. M. Muniswamy for very helpful discussions on digitonin permeabilization.9 T, V* a, {' l$ A- t( @0 c7 \
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