Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M706435200 on December 20, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5699-5707, February 29, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/9/5699    most recent
M706435200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bhattacharyya, S.
Right arrow Articles by Stenina, O. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bhattacharyya, S.
Right arrow Articles by Stenina, O. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cell Type-specific Post-transcriptional Regulation of Production of the Potent Antiangiogenic and Proatherogenic Protein Thrombospondin-1 by High Glucose*

Sanghamitra Bhattacharyya{ddagger}1, Tina E. Marinic{ddagger}1, Irene Krukovets{ddagger}, George Hoppe§, and Olga I. Stenina{ddagger}2

From the {ddagger}Department of Molecular Cardiology and Joseph J. Jacobs Center for Thrombosis and Vascular Biology and the §Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio 44026

Received for publication, August 3, 2007 , and in revised form, December 18, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperglycemia is an independent risk factor for development of vascular diabetic complications. Vascular dysfunction in diabetics manifests in a tissue-specific manner; macrovasculature is affected by atherosclerotic lesions, and microvascular complications are described as "aberrant angiogenesis": in the same patient angiogenesis is increased in some tissues (e.g. retinal neovascularization) and decreased in others (e.g. in skin). Molecular cell- and tissue-specific mechanisms regulating the response of vasculature to hyperglycemia remain unclear. Thrombospondin-1 (TSP-1), a potent antiangiogenic and proatherogenic protein, has been implicated in the development of several vascular diabetic complications (atherosclerosis, nephropathy, and cardiomyopathy). This study examines cell type-specific regulation of production of thrombospondin-1 by high glucose. We previously reported the increased expression of TSP-1 in the large arteries of diabetic animals. mRNA and protein levels were up-regulated in response to high glucose. Unlike in macrovascular cells, TSP-1 protein levels are dramatically decreased in response to high glucose in microvascular endothelial cells and retinal pigment epithelial cells (RPE). This down-regulation is post-transcriptional; mRNA levels are increased. In situ mRNA hybridization and immunohistochemistry revealed that the level of mRNA is up-regulated in RPE of diabetic rats, whereas the protein level is decreased. This cell type-specific posttranscriptional suppression of TSP-1 production in response to high glucose in microvascular endothelial cells and RPE is controlled by untranslated regions of TSP-1 mRNA that regulate coupling of TSP-1 mRNA to polysomes and its translation. The cell-specific regulation of TSP-1 suggests a potential mechanism for the aberrant angiogenesis in diabetics and TSP-1 involvement in development of various vascular diabetic complications.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the significant advances in the therapeutic methods to control blood glucose and insulin levels in diabetic patients, the precise regulation of these levels remains a problem. Vascular diabetic complications remain most prevalent and dangerous and account for the greatest numbers of deaths and hospitalizations in diabetic patients. The molecular basis for the vascular complications of diabetes is not well understood. Recent reports indicate that both microvascular and macrovascular complications of diabetes correlate directly with glucose levels in both patients and animal models (1–5). Some of these reports revealed the pathogenic role of impaired glucose tolerance and post-prandial hyperglycemia even in the absence of diabetes, e.g. (6). In vascular cells, glucose regulates expression of many genes that have been linked to the development of atherosclerosis or abnormal angiogenesis (reviewed in Ref. 7). One of them is thrombospondin-1 (TSP-1),3 a cell matrix protein implicated in both atherogenesis (8–12) and angiogenesis (13–19). Several lines of evidence indicate that TSP-1 may represent a link between diabetes and vascular complications. Patients with diabetes have increased TSP-1 in their plasma and kidneys (20). The expression of TSP-1 in mesangial cells was increased by high levels of glucose (21). TSP-1 promotes the events associated with diabetic nephropathy: mesangial cell proliferation and increased matrix production by mesangial cells (21–24). A peptide blocking the activation of transforming growth factor-β activation by TSP-1 prevented progression of cardiac fibrosis and improved cardiac function in a rat model, implicating TSP-1 in development of diabetic cardiomyopathy (25). Recently, we reported increased levels of TSP-1 in macrovessels of diabetic Zucker rats (26). Moreover, the major cell types present in large vessels responded to stimulation with high glucose concentrations by increasing their expression of TSP-1. TSP-1 is produced by microvascular cells and retinal cells (27–29), and, in contrast to large vessels and kidney, TSP-1 levels in the diabetic eye are significantly decreased (28). Because TSP-1 is a potent antiangiogenic agent that in the normal eye may prevent uncontrolled growth of microvessels, such down-regulation of TSP-1 in the diabetic eye suggests its involvement in diabetic retinopathy. A direct effect of TSP-1 deficiency on retinal vascularity has been demonstrated recently in TSP-1-deficient mice (30).

Elevated glucose levels are associated with the development of diabetic vascular complications, and regulation of TSP-1 production may control both macro- and microvascular changes. The aberrant angiogenesis in diabetics (increased angiogenesis in some tissues and decreased in others in the same patient), which does not have any mechanistic explanation to date, may be dependent on a cell type- and tissue-specific production of regulators of angiogenesis. TSP-1, one of the most potent antiangiogenic proteins, is a good candidate for such a regulator.

We now report a finding of a cell type- and cell origin-specific post-transcriptional mechanism of down-regulation of TSP-1 expression by glucose. This mechanism is not present in endothelial cells (EC) from large blood vessels, vascular smooth muscle cells (SMC), and fibroblasts, where the transcriptional increase in TSP-1 mRNA upon acute glucose stimulation of cells leads to the increased expression of TSP-1 protein (26). However, in microvascular endothelial cells (MVEC) and in retinal pigment epithelial (RPE) cells, the expression of TSP-1 protein is dramatically down-regulated despite increased TSP-1 mRNA levels. The mechanism controlling TSP-1 production is activated by high glucose, operates at the level of mRNA translation and is controlled by untranslated regions of mRNA. The finding of such a cell type- and cell origin-specific mechanism of suppression of the protein production may provide a molecular basis for aberrant angiogenesis in diabetic individuals. Thus, cell- and tissue-specific regulation of TSP-1 production may represent an important link between diabetes, hyperglycemia, and a variety of vascular complications of diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Stimulation with Glucose, TSP-1 Protein, and mRNA Detection—Primary vascular cells were purchased from Cambrex or kindly provided by the investigators listed in the Acknowledgments. The cell lines were from ATCC unless otherwise stated. CDC.EU/HMEC1 were obtained from Dr. Candal (Centers for Disease Control, Atlanta, GA). The stimulation with 30 mM glucose was performed as described (26). TSP-1 protein and mRNA were detected by Western or Northern blotting as described before (26).

Labeling of Protein with [35S]Methionine and Immunoprecipitation of Labeled TSP-1—Labeling of proteins began by the addition of 10 µCi/ml of [35S]methionine for 2 h either 1) prior to stimulation with glucose in the case of experiments on protein degradation/secretion or 2) 22 h after the start of stimulation with glucose in the case of detection of de novo synthesized TSP-1 protein. In protein degradation/secretion experiments, synthesis of labeled protein was stopped by an excess of cold methionine 1 h before the experimental time point zero. TSP-1 was precipitated from cell lysates overnight at 4 °C using anti-TSP-1 antibody (Lab Vision), followed by a 2-h incubation with anti-mouse IgG antibody (Bio-Rad) and protein A/G magnetic beads (Dynal). Precipitated proteins were resolved in 8% SDS-PAGE. The gels were dried and exposed to x-ray film (Amersham Biosciences) to detect TSP-1.

Short Pulse Labeling—200 µCi/ml of [35S]methionine was added for 5 min to RF/6A cells treated with 30 mM glucose for 0, 1, 2, 3, and 6 h. Prior to glucose stimulation, the cells were incubated overnight in low glucose, 0.2% fetal bovine serum medium without methionine. Labeling was stopped by the addition of excess of cold methionine and 10 µg/ml cyclohexamide, and the cells were immediately lysed. Immunoprecipitation of TSP-1 was performed as described above.

Reporter Construct Design—5'- and 3'-untranslated regions (UTR) of TSP-1 mRNA were amplified by PCR from clones IMAGE 6718543 (MRC Geneservice, Cambridge, UK) and IMAGE 6028666 (ATCC), respectively. We used primers 5'-TCTAGAGTCATCAAATTGTTGATTGAAAG-3' and 5'-GGCCGGCCGTTGTTCCTTGTACATAAGAA-3' for 5'-UTR and 5'-AAGCCTAGCCGCTGCGCCCGAGCTGGCC-3' and 5'-CCATGGTGGAGCTGTTGGTGCCCAGCAGG-3' for 3'-UTR. 5' was cloned into HindIII and NcoI restriction sites of the pGL3 Control vector (Promega), and 3' was cloned in FseI and XbaI sites of the same vector. Thus, the final constructs included the full 5'-UTR inserted immediately before the luciferase cDNA, the full 3'-UTR inserted immediately after the luciferase cDNA, or both full 5'-UTR and full 3'-UTR.

Transient Transfections and Luciferase Assay—Lipofectin reagent (Invitrogen) was used for transient transfections according to the manufacturer's instructions. After a 24-h recovery, full growth media were changed to a low glucose medium (5 mM or 1 mg/ml glucose, 5% fetal bovine serum) 24 h prior to a 24-h stimulation with 30 mM glucose. Luciferase activity was measured in 100 µg of total protein using reagents from Promega according to the manufacturer's instructions.

Fractionation of RNA on Sucrose Gradient—RF/6A cells transfected with TSP1–5',3'-UTR/luciferase cDNA plasmid were grown in 150-mm cell culture dishes to 100% confluency in Dulbecco's modified Eagle's medium/Ham's F-12 medium, 10% fetal bovine serum (Atlanta Biologicals), 0.1% G418 (Mediatech, Inc.). Medium was changed to a low glucose (5 mM) medium (0.2% fetal bovine serum) 24 h before the experiment. Cyclohexamide (CHX; 50 µg/ml) was added to each plate 5 min before lysing. The cells were washed with 5 ml of ice-cold phosphate-buffered saline containing CHX 50 µg/ml, and then the cells were scraped in phosphate-buffered saline/CHX and transferred to a tube, collected at 600 x g, washed twice with phosphate-buffered saline/CHX, resuspended in 500 µl of ice-cold polysome lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 300 mM KCl in DEPC (diethyl pyrocarbonate) water; 10 mM DTT, 100 units/ml ribonuclease inhibitor (Promega), 200 µg/ml CHX, protease inhibitor mixture (Roche Diagnostics), passed through a 27-gauge needle, incubated on ice for 5 min, and spun down at 1000 x g for 5 min. 1 mg of protein (0.5 ml) was layered on 4.5 ml of 30% sucrose prepared in polysome lysis buffer and spun down at 150,000 x g at 4 °C for 2 h.

After centrifugation, the supernatant (nonpolysomal fraction) was collected into a new tube and precipitated with 1/10 volume of 3 mol/liter NaOAC, 2.5 volumes of ethanol overnight at -80 °C. RNA was purified from nonpolysomal fraction using TRIzol reagent. The pellet (polysomal fraction) was resuspended in 100 µl of polysome lysis buffer, and RNA was extracted with 750 µl of TRIzol reagent.

Real Time RT-PCR—First strand cDNA was synthesized using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen) from 1 µg of RNA isolated from polysomal or nonpolysomal fractions. iQTM SYBR Green Supermix (Bio-Rad) was used for PCR. The primers used were: luciferase, 5'-gacaattgcactgatcatgaactcct and 5'-cagcgcactttgaatcttgtaatcct; TSP-1, 5'-ccaaagacgggtttcattagag and 5'-ttcaggtcagagaagaacacca; β-actin, 5'-catgtacgttgctatccaggc and ctccttaatgtcacgcacgat. Ct values for TSP-1 and luciferase were normalized to the Ct value for β-actin (dCt), dCt values for each time point of glucose treatment were normalized to the control value (ddCt), and the value for fold change represents the exponential of ddCt. β-Actin Ct remained constant in response to glucose treatment in all of the cell types in both polysomal and nonpolysomal fractions.

In Situ mRNA Hybridization and Immunohistochemistry—12-Week-old control and diabetic Zucker rats (Genetic Models, Inc.) were sacrificed by CO2 inhalation. Rat eyes were enucleated and placed into ice-cold 4% paraformaldehyde in 50 mM phosphate buffer, pH 7.4, following a penetrating incision made through the middle of the cornea. After overnight fixation at 4 °C, anterior segments and the lens were removed under preparative microscope, and the posterior eyecups were dehydrated, embedded in paraffin, and cut into 10-µm-thick sections. In situ mRNA hybridization was performed as described previously (26). Immunohistochemistry was performed as described previously (26) with minor modifications: biotinylated anti-TSP-1 antibody (Lab Vision) and Texas Red-labeled streptavidin were used.

Statistical Analysis—All of the described experiments were performed at least three times, and the data were presented as the mean values ± S.E. The p values were determined from the t test using Microsoft Excel. Differences with p values ≤ 0.05 were considered statistically significant. The analyses of images obtained using in situ mRNA hybridization and immunohistochemistry were performed using ImagePro 4.5.1 in eye sections obtained from four eyes for each experimental condition (30 microscopic fields from each eye).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased TSP-1 Production in Macrovascular EC—We recently reported that the expression of TSP-1 is increased in the aorta and carotid artery of diabetic Zucker rats at the protein and mRNA levels (26). This increase in production of the potent antiangiogenic protein TSP-1 in macrovessels was associated with the decreased number of vasa vasorum, or small blood vessels, growing through the vascular wall and feeding the inner layers of vascular cells. TSP-1 was up-regulated by glucose stimulation in all major vascular wall cell types from large vessels (EC, smooth muscle cells, and fibroblasts) in vitro (26). Both the protein and mRNA for TSP-1 were rapidly up-regulated after stimulation of cells with 10–30 mM D-glucose but not biologically inactive L-glucose or sorbitol (26, 31). In addition to our published data, similar experiments have been performed in human pulmonary artery EC, several isolates of human aortic EC, and human coronary artery EC with similar results (Fig. 1). In all the isolates of primary EC from large blood vessels, TSP-1 protein production was increased in response to high glucose.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 1.
Stimulation with high glucose results in increased production of TSP-1 in various macrovascular EC. Aortic, pulmonary artery, and coronary artery EC were stimulated with 30 mM glucose as described previously for human umbilical vein EC, aortic smooth muscle cells, and fibroblasts (26) and analyzed by Western blotting.

 
Decreased TSP-1 Production in MVEC and RPE in Response to High Glucose—In contrast to the increased TSP-1 protein production in cultured cell types from macrovessels (26), levels of TSP-1 were dramatically reduced in lysates of primary MVEC and RPE (human dermal MVEC, rat lung MVEC, and human RPE) in response to stimulation with 30 mM glucose (Fig. 2). The decreased levels of TSP-1 in primary MVEC in response to high glucose were described earlier by Sheibani et al. (28). Interestingly, cell lines derived from MVEC and RPE (MVEC, CDC.EU/HMEC1 and RF/6A; RPE, ARPE19 and D407) retained this ability to suppress TSP-1 production upon incubation with high glucose (Fig. 2), suggesting that the differential regulation of TSP-1 in MVEC and RPE versus macrovascular EC and other cells from large blood vessels is an inherited property that is preserved in cell culture and in transformed cell lines. All of the MVEC and RPE produced high levels of TSP-1 in basal conditions.

The dissimilarity in the regulation of TSP-1 protein expression by high glucose between macrovascular cell types versus MVEC and RPE is not due to a difference in transcriptional regulation or to a decreased stability of TSP-1 mRNA, because mRNA levels were increased in response to high glucose in all cell types (Fig. 3), similar to macrovascular cells (26). These disparities between mRNA levels and protein levels identified the mechanism of TSP-1 regulation in MVEC and RPE as post-transcriptional.

Decreased TSP-1 Protein Synthesis in Glucose-stimulated Cells—Decreased levels of TSP-1 protein in cell lysates may result from an increased rate of TSP-1 secretion, increased rate of protein degradation, or a post-transcriptional regulation of protein synthesis. To address these possibilities, we: 1) examined the levels of TSP-1 in cell culture supernatants; similar to the levels of TSP-1 in cell lysates, the TSP-1 levels in supernatants were decreased (Fig. 4A), suggesting that the secretion of TSP-1 is not increased in response to high glucose and cannot account for the decrease in intracellular TSP-1; and 2) monitored the change in the levels of prelabeled intracellular TSP-1 in control and high glucose-stimulated cells (Fig. 4B). In ARPE19 and RF/6A, proteins were labeled by the addition of 10 µCi of [35S]methionine for 2 h, and then an excess of cold methionine was added for 1 h to stop the synthesis of newly labeled protein, and the cells were stimulated with high glucose for 1 h. Starting at this time (time point zero), the cells were incubated for 15 min to 20 h and lysed. Labeled TSP-1 synthesized before stimulation with glucose was immunoprecipitated using two anti-TSP-1 antibodies (Labvision; Ab4 that recognizes the middle part of TSP-1 and Ab3 that recognizes the N terminus of TSP-1) and resolved in SDS-PAGE. As shown in Fig. 4B, the amount of labeled TSP-1 in cell lysates declined with time, presumably because of the secretion of the protein. However, there was no difference in labeled TSP-1 levels between samples from control cells and high glucose-stimulated cells at any given time point. This observation suggested that the decrease in TSP-1 levels was not due to the increased secretion or degradation of the protein but rather was due to decreased synthesis of the protein.


Figure 2
View larger version (60K):
[in this window]
[in a new window]

  		FIGURE 2.
Stimulation of MVEC and RPE with high glucose results in decrease of TSP-1 protein levels. Cultured human dermal MVEC, rat lung MVEC as well as CDC.EU/HMEC1 and RF/6A cell lines derived from MVEC (A) and human primary RPE, D407, and ARPE19 cell lines derived from RPE (B) were preincubated in low glucose (5.5 mmol/liter) for 24 h and stimulated with 30 mM D-glucose for indicated times. Proteins in cell lysates were resolved in 8% SDS-PAGE, and TSP-1 was detected in Western blotting. Representative blots are shown. Upper panels, TSP-1; lower panels, Coomassie staining of membrane (loading control). C, densitometric scans of more than four Western blots for RF/6A and human primary retinal pigment epithelial cells (hpRPE) were quantified.

 
To confirm that the synthesis of TSP-1 protein is inhibited in response to high glucose, we have performed a short pulse labeling (200 µCi/ml of [35S]methionine) of newly synthesized proteins 1, 2, 3, and 6 h after stimulation of RF/6A with high glucose. Labeled TSP-1 was immunoprecipitated from the lysates and detected in SDS-PAGE (Fig. 4C). The difference in the amount of TSP-1 synthesized during the pulse time (5 min) was detected as early as 2 h after the glucose treatment, and the newly synthesized TSP-1 was barely detected after 6 h of treatment.

Additionally, ARPE19 and HASMC were stimulated with 30 mM glucose, and the newly synthesized proteins were labeled with [35S]methionine for 2 h prior to the cell harvesting at the 24-h time point. ARPE19 and HASMC, two cell types with different mechanisms of TSP-1 protein regulation in response to glucose, were used in these experiments. TSP-1 was immunoprecipitated from the control of each cell type, and the cells were stimulated with 30 mM glucose for 24 h. At 24 h of stimulation, up-regulation of TSP-1 reached maximal levels in HASMC (26), whereas maximum down-regulation of TSP-1 in ARPE19 is detected at the same time point. Although in HASMC the synthesis of TSP-1 was dramatically increased in cells stimulated with high glucose as compared with control cells, in ARPE the synthesis of new TSP-1 was notably decreased (Fig. 4D). No labeled protein was detected at this time point in the medium, suggesting that 2 h (labeling time) are not sufficient for the secretion of detectable amount of TSP-1, and the decrease of newly synthesized TSP-1 in lysates in cells stimulated with high glucose is not a result of increased secretion. These series of experiments confirmed that the decrease in intracellular TSP-1 is due to the decreased synthesis of the protein rather than its increased secretion or degradation.

Control of the Reporter Gene Expression by Untranslated Region of TSP-1 mRNA—UTR of mRNA frequently control protein production by inhibiting translation of a protein or sequestering mRNA (reviewed in Ref. 32, 33). We examined the expression of luciferase in cells transfected with plasmids containing the complete 5'-UTR of TSP-1 cloned in front of the luciferase cDNA, the complete 3'-UTR cloned immediately at the 3' of the luciferase cDNA, or both 5' and 3'-UTR. HASMC, bovine aortic endothelial cells (BAEC), ARPE19, and retinal microvascular endothelial cell line RF/6A were transiently transfected with these constructs and stimulated with 30 mM glucose for 24 h. In response to high glucose, the activity of 5'TSP-1/luciferase was increased up to 4-fold (Fig. 5) in all of the cell lines tested. Luciferase expression was inhibited by TSP-1 3'-UTR in all four cell types; the basal expression was inhibited by several orders of magnitude (not shown), and there was no increase in luciferase activity in response to glucose (Fig. 5). However, when the luciferase construct contained both 5'- and 3'-UTR of TSP-1, a significant difference in reporter activity was observed in MVEC and RPE versus macrovascular cell types. The activity of luciferase and the response to high glucose were restored in macrovascular cell types (HASMC and BAEC) but not in ARPE19 or RF/6A. These data suggest that differential cell type-specific regulation of TSP-1 synthesis is controlled by a cell type-specific post-transcriptional mechanism, which depends on the UTR of TSP-1 mRNA. As expected, based on the experiments that determined the increased levels of endogenous TSP-1 mRNA in all glucose-stimulated cells (Fig. 3), the levels of luciferase mRNA were not reduced by glucose treatment (Fig. 5E). These data suggest that differential cell type-specific regulation of TSP-1 synthesis is controlled by a cell type-specific post-transcriptional mechanism, which depends on the UTR of TSP-1 mRNA.


Figure 3
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 3.
Stimulation of MVEC and RPE with high glucose results in increased mRNA levels. A, cells were stimulated with high glucose as described above. TSP-1 mRNA was detected by Northern blotting in 7 µg of total RNA. HDMEC, human dermal microvascular endothelial cells; RLE, rat lung MVEC; RF/6A, retinal MVEC transformed cell line; hpRPE, human primary RPE; ARPE, RPE cell line. B, quantification of more than four densitometric scans of Northern blots.

 


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 4.
Decrease in TSP-1 protein levels is caused by a decreased de novo production of TSP-1 protein. A, TSP-1 levels in supernatants of cultured MVEC and RPE in response to 30 mmol/liter glucose. Supernatants (50 µl) were analyzed by SDS-PAGE and Western blotting. B, proteins were prelabeled with [35S]methionine in RF/6A and ARPE19 cells, and the cells were treated with glucose for 1 h (experimental point zero). The labeled TSP-1 synthesized before stimulation with glucose was immunoprecipitated (IP) at the indicated time points from the cell lysates and resolved in 8% SDS-PAGE. C, short pulse [35S]methionine-labeled TSP-1 was immunoprecipitated from the lysates of RF/6A stimulated with 30 mM glucose for indicated time and analyzed by 8% SDS-PAGE. D,[35S]methionine-labeled TSP-1 was immunoprecipitated from the lysates of ARPE19 and HASMC after stimulation with 30 mM glucose for 24 h and analyzed by 8% SDS-PAGE.

 
Change in Osmolarity Regulates TSP-1 Production—The transcriptional increase of TSP-1 mRNA that we observed in all cell types is dependent on the intracellular glucose metabolism; cell-impermeable sorbitol and L-glucose used as osmolarity control failed to up-regulate TSP-1 mRNA (26, 31). In contrast, the regulation of protein production by UTR of TSP-1 mRNA in response to high glucose is regulated by a change in osmolarity; biologically inactive cell-impermeable L-glucose had a similar effect on luciferase production regulated by TSP-1 UTR and on the endogenous TSP-1 levels (Fig. 5, A–D and F, respectively).

High Glucose Regulates Coupling of TSP-1 and TSP-1–5',3'-UTR/ Luciferase mRNA with Polysomes—Actively translated mRNA can be found in association with polysomes. Uncoupling from polysomes is a common mechanism of inhibition of mRNA translation. To detect the effect of high glucose on coupling of TSP-1 mRNA with polysomes, we have performed fractionation of RNA from glucose-stimulated RF/6A, HUVEC, and HASMC followed by detection of TSP-1 mRNA in polysomal and nonpolysomal fractions by real time RT-PCR. The cells were preincubated in low glucose medium for 24 h and stimulated with 30 mM glucose for 1, 2, 3, 6, and 24 h. Polysomal fraction was separated on 30% sucrose cushion, total RNA was isolated from the polysomal, and nonpolysomal fractions and 1 µg of RNA were used for RT-PCR (Fig. 6A). In HUVEC and HASMC, the amount of TSP-1 mRNA in nonpolysomal fraction was rapidly and significantly decreased, and all of the newly transcribed TSP-1 mRNA was accumulated in polysomal fraction (4.5 ± 1.5-fold increase over nonstimulated cells in HUVEC and 2.7 ± 0.14-fold increase in HASMC at 24 h, p = 0.04 and 0.0001, respectively). These data indicated that the newly transcribed mRNA is actively translated, and this observation is consistent with increased production of the protein in both HUVEC and HASMC in response to high glucose. However, in RF/6A, the TSP-1 mRNA amount was rapidly decreased in actively translated polysomal fraction, and all the newly transcribed TSP-1 mRNA was accumulated in nonpolysomal fraction (4.1 ± 0.41-fold increase at 24 h, p = 4.9E-08). These data clearly indicate that the uncoupling of TSP-1 mRNA from polysomes is the cause of decreased production of the protein in response to high glucose.


Figure 5
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 5.
UTR of TSP-1 mRNA regulates a reporter gene expression in response to high glucose. A–D, macrovascular cells (HASMC and BAEC), MVEC, and RPE were transiently transfected with reporter plasmids containing TSP-1 5'-UTR, 3'-UTR, or both 5'- and 3'-UTR and stimulated with 30 mM D-glucose or L-glucose for 24 h. Luciferase activity was measured in lysates of transfected cells, n ≥ 4. E, total RNA from glucose-stimulated RF/6A stably expressing 5',3'-UTR-TSP-1/luciferase was analyzed by Northern Blotting using luciferase probe. F, TSP-1 protein levels were analyzed by Western blotting in RF/6A stimulated with 30 mM L-or D-glucose.

 
To confirm that UTR of TSP-1 mRNA is responsible for the translocation to the untranslated fraction of RNA and decreased production of protein, we used RF/6A stably transfected with a fusion constructs expressing heterologous coding region of luciferase surrounded with 5'-UTR, 3'-UTR, or 5',3'-UTR of TSP-1. The cells were preincubated in low glucose medium for 24 h and stimulated with 30 mM glucose for 1, 2, 3, 6, and 24 h. As shown in Fig. 6B, there was no detectable shifting of 5'TSP/luciferase mRNA between polysomal and nonpolysomal fractions. However, both 3'TSP/luciferase and 5',3'TSP/luciferase translocated to the nonpolysomal fraction, suggesting that UTR of TSP-1 mRNA controls the translocation of the heterologous luciferase mRNA.

Decreased Level of TSP-1 in RPE and Retina of Diabetic Zucker Rats—To find out whether the described mechanism is operative in vivo, we examined RPE and retina of diabetic Zucker rats. TSP-1 mRNA was detected by in situ mRNA hybridization. Comparison of control nondiabetic RPE to RPE of diabetic animals revealed that mRNA levels are increased in diabetic rats (Fig. 7). In contrast, TSP-1 protein detected in the extracellular matrix of choroid layers adjacent to RPE and in the extracellular matrix of retina was visibly down-regulated in diabetic animals (Fig. 8). The changes on the amounts of mRNA and protein were estimated by measuring the intensity of fluorescence sections from four diabetic and control rat eyes (Figs. 7B and 8B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TSP-1 expression by MVEC regulates cell survival, and the correlation between TSP-1 and TSP-2 expression and abnormal angiogenesis has been well documented in animal models and in human pathologies (34, 35). High expression of TSP-1 in RPE in normoglycemic conditions is consistent with the anatomical position of RPE in the eye, where TSP-1, a potent antiangiogenic protein, may participate in the maintenance of the normal density of microvessels in the retina and choroid. The expression of TSP-1 in the normal eye and its decreased expression in the diabetic eye were reported, as well as increased vascularity in the retina of TSP-1-deficient animals (28, 30).

At the mRNA level, both macrovascular cell types and MVEC and RPE respond similarly to high glucose stimulation by dramatically increasing TSP-1 mRNA levels. Surprisingly, in response to glucose there is a striking difference between TSP-1 protein regulation in macrovascular cell types and MVEC and RPE; although the protein level is dramatically increased in macrovascular cell types (26), these levels are significantly decreased in MVEC and RPE. This observation suggests a cell type-specific post-transcriptional mechanism of TSP-1 regulation (e.g. this may be a specific mechanism suppressing the production of TSP-1 in MVEC and RPE or a specific mechanism relieving the block of protein production in macrovascular cells).


Figure 6
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 6.
Translocation of TSP-1 and reporter mRNA from polysomal fraction to nonpolysomal fraction of mRNA upon stimulation of microvascular EC with high glucose. A, TSP-1. RF/6A, HUVEC, and HASMC were stimulated with high glucose for indicated time, and RNA was fractionated as described under "Materials and Methods." TSP-1 mRNA levels were measured by real time RT-PCR in both polysomal and nonpolysomal RNA fractions. The mean values ± S.E. are presented in the graphs, An asterisk indicates the statistically significant difference (p < 0.05) from control (0 h) TSP-1 mRNA levels in polysomal fractions; # indicates the statistically significant difference (p < 0.05) from control (0 h) TSP-1 mRNA levels in nonpolysomal fractions. B, luciferase (please note logarithmic scale). RF/6A stably expressing 5'-UTR-TSP-1/luciferase, 3'-UTR-TSP-1/luciferase, or 5',3'-UTR-TSP-1/luciferase were stimulated with 30 mM D-glucose for indicated time. Luciferase mRNA levels were measured by real time RT-PCR in both polysomal and nonpolysomal RNA fractions. The mean values ± S.E. are presented in the graphs; an asterisk indicates the statistically significant difference (p < 0.05) from control (0 h) TSP-1 or luciferase mRNA levels in polysomal fractions; # indicates the statistically significant difference (p < 0.05) from control (0 h) TSP-1 or luciferase mRNA levels in nonpolysomal fractions.

 


Figure 7
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 7.
Increased TSP-1 mRNA levels in RPE of diabetic Zucker rats. A, eyes of 12-week-old control (top panels) and diabetic (bottom panels) Zucker rats were collected as described under "Materials and Methods," and TSP-1 mRNA was visualized by in situ mRNA hybridization using biotinylated antisense TSP-1 mRNA probe and Texas Red-labeled streptavidin. The blood vessels are marked by asterisks. B, fluorescence intensity was analyzed in sections as described under "Materials and Methods."

 
Decreased levels of TSP-1 protein in cell culture supernatants of glucose-stimulated cells and the identical time course of loss of labeled intracellular TSP-1 from control and glucose-stimulated cells suggest that increased secretion of TSP-1 cannot account for the difference. Indeed, TSP-1 levels decrease dramatically within the time insufficient for complete protein processing and secretion; the decrease in protein levels can be detected as early as 1 h, whereas 2 h from the start of labeling were insufficient for the detection of labeled TSP-1 in cell culture supernatants. Immunoprecipitation of labeled newly synthesized TSP-1 from lysates of microvascular EC RF/6A and ARPE 19 confirmed the decrease in TSP-1 protein synthesis in response to high glucose.

The regulation of TSP-1 production in RPE and MVEC in response to glucose occurs at the level of protein synthesis; transcriptional inhibition is excluded because of the increased levels of TSP-1 mRNA, and protein degradation and secretion are not affected, whereas the synthesis of TSP-1 protein is decreased upon glucose stimulation. UTR of mRNA frequently control the protein expression at the post-transcriptional levels. Such regulation may involve a direct inhibition of mRNA translation or sequestering of mRNA. To find out whether TSP-1 UTR controls the expression of TSP-1 protein in MVEC and RPE in response to high glucose, we decided to investigate the effect of 5'- and 3'-UTR of TSP-1 mRNA on the expression of a heterologous reporter gene in macrovascular cells versus MVEC and RPE. These experiments confirmed that TSP-1 UTR regulates the production of TSP-1 protein. When both 5'- and 3'-UTR were introduced into the luciferase-expressing plasmid, the chimeric mRNA was translated into the protein in macrovasular cell types (HASMC and BAEC), but not in RF/6A or ARPE19. According to the circular model of mRNA, 5' and 3' UTR of mRNA interact through the formation of complexes between 5' and 3' binding proteins (36). Such interaction is necessary for the initiation and for the efficient control of mRNA translation. The requirement of both 5'- and 3'-UTR for the cell type-specific regulation of TSP-1 production suggests a cell type-specific interaction between 5'- and 3'-UTR of TSP-1 upon glucose stimulation. Indeed, in response to high glucose, both endogenous TSP-1 mRNA and chimeric 5',3'-UTR-TSP-1/luciferase mRNA were translocated from actively translated polysome-associated pool of mRNA to nonpolysomal fraction in microvascular EC, but not in macrovascular EC or HASMC. It appears that 3'-UTR alone is sufficient for the translocation to the nontranslated RNA fraction and that both 5'- and 3'-UTR are required for the cell-specific regulation. Further analysis of the requirements for the inhibition of the protein production in MVEC and RPE will be needed to attempt the identification of the RNA-binding protein(s) responsible for this effect. The use of deletion/mutant UTR and identification of protein(s) binding to the UTR fragments that control the cell-specific effect will be necessary to describe the precise mechanism of the observed cell type-specific regulation.


Figure 8
View larger version (63K):
[in this window]
[in a new window]

  		FIGURE 8.
Decreased TSP-1 protein levels in RPE and retina of diabetic Zucker rats. TSP-1 in control or diabetic rats was visualized using biotinylated anti-TSP-1 antibody followed by detection using Texas Red-streptavidin (choroid, A) or horseradish peroxidase-streptavidin (retina, C). The blood vessels are marked by asterisks. B, analysis of fluorescence intensity.

 
The difference in distribution of the endogenous TSP-1 mRNA in microvasclar EC and macrovascular cells was striking; as the levels of TSP-1 mRNA in all the cell types increased, TSP-1 mRNA accumulated in nonpolysomal fraction in RF/6A, but not in HUVEC or HASMC, where it was found with actively translated polysome-associated mRNA. The cell-specific regulation of TSP-1 mRNA translocation to the nonpolysomal fraction and inhibition of translation is clearly not a general effect of high glucose on protein synthesis; β-actin mRNA did not translocate to the nonpolysomal fraction (not shown), and there was no decrease in production of β-actin protein.

The regulation of TSP-1 protein production by UTR does not depend on the uptake and intracellular metabolism of glucose; biologically inactive and cell-impermeable L-glucose had an effect similar to the effect of D-glucose, unlike in case of the up-regulation of levels of TSP-1 mRNA that was dependent on the glucose uptake/intracellular metabolism and was not reproduced with sorbitol or L-glucose.

We have visualized the TSP-1 mRNA and protein expression in RPE of diabetic and control Zucker rats, and the results of the in situ mRNA hybridization and protein detection by immunohistochemistry revealed that TSP-1 expression is controlled in a tissue-specific manner at the post-transcriptional level; although the TSP-1 mRNA level was increased in RPE of diabetic Zucker rats, the protein level was decreased, suggesting that the cell type-specific mechanism of regulation found in cell culture is operative in vivo.

Our data suggest that TSP-1 expression is regulated by high glucose in a cell type- and tissue-specific manner by a post-transcriptional mechanism present in selected cell types. Such cell type-specific regulation would increase TSP-1 expression in response to high glucose in some tissues (wall of large vessels, skin fibroblasts, etc.) while decreasing the production of TSP-1 expression in others (retina). Cell type-specific regulation of expression of this potent antiangiogenic protein may explain the aberrant angiogenesis in diabetic patients, when at the same time the increased vascularization can be observed in selected tissues, whereas the decreased angiogenesis can cause diabetic complications in other tissues. Thus, tissue-specific regulation of TSP-1 expression in response to hyperglycemia may contribute to a variety of vascular diabetic complications.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants K01 DK62128, P50 HL077107, and R01 DK067532, American Heart Association Grant 0565284B, funds from the Lerner Research Institute (Cleveland Clinic Foundation) (to O. I. S.), and by American Heart Association Grant 0555235B (to G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. NB50, 9500 Euclid Ave., Cleveland, OH 44195. Fax: 216-445-8204; E-mail: stenino{at}ccf.org.

3 The abbreviations used are: TSP-1, thrombospondin-1; EC, endothelial cell(s); MVEC, microvascular endothelial cell(s); BAEC, bovine aortic EC; RPE, retinal pigment epithelial cell(s); SMC, smooth muscle cell(s); HASMC, human aortic SMC; UTR, untranslated region(s); CHX, cyclohexamide; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Janice M. Burke (Medical College of Wisconsin, Milwaukee, WI) for primary human RPE (supported by Core Grant for Vision Research P30 EY01931 awarded to the Medical College of Wisconsin), Dr. DiCorleto and Lori Mavrakis (Cleveland Clinic) for HUVEC, and Dr. S. Y. Desai (Cleveland Clinic) for rat lung MVEC. D407 cells were generously provided by Richard Hunt (Department of Immunology and Pathology, University of South Carolina Medical School, Columbia, SC).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Martin, A., Komada, M. R., and Sane, D. C. (2003) Med. Res. Rev. 23, 117-145[CrossRef][Medline] [Order article via Infotrieve]
  2. The Diabetes Control and Complication Trial Research Group (1993) N. Engl. J. Med. 329, 977-986[Abstract/Free Full Text]
  3. Nathan, D. M., Lachin, J., Cleary, P., Orchard, T., Brillon, D. J., Backlund, J. Y., O'Leary, D. H., and Genuth, S. (2003) N. Engl. J. Med. 348, 2294-2303[Abstract/Free Full Text]
  4. UK Prospective Diabetes Study (UKPDS) Group (1998) Lancet 352, 837-853[CrossRef][Medline] [Order article via Infotrieve]
  5. Nathan, D. M., Cleary, P. A., Backlund, J. Y., Genuth, S. M., Lachin, J. M., Orchard, T. J., Raskin, P., and Zinman, B. (2005) N. Engl. J. Med. 353, 2643-2653[Abstract/Free Full Text]
  6. Khaw, K. T., Wareham, N., Luben, R., Bingham, S., Oakes, S., Welch, A., and Day, N. (2001) Bmj. 322, 15-18[Abstract/Free Full Text]
  7. Stenina, O. I. (2005) Curr. Pharm. Des. 11, 2367-2381[CrossRef][Medline] [Order article via Infotrieve]
  8. Topol, E. J., McCarthy, J., Gabriel, S., Moliterno, D. J., Rogers, W. J., Newby, L. K., Freedman, M., Metivier, J., Cannata, R., O'Donnell, C. J., Kottke-Marchant, K., Murugesan, G., Plow, E. F., Stenina, O., and Daley, G. Q. (2001) Circulation 104, 2641-2644[Abstract/Free Full Text]
  9. Peyvandi, F., Palla, R., Ardissino, D., Foco, L., Bernardinelli, L., Bauer, K., and Mannucci, P. M. (2003) Blood 102, 314 (abstr.)
  10. Riessen, R., Kearney, M., Lawler, J., and Isner, J. M. (1998) Am. Heart J. 135, 357-364[CrossRef][Medline] [Order article via Infotrieve]
  11. Roth, J. J., Gahtan, V., Brown, J. L., Gerhard, C., Swami, V. K., Rothman, V. L., Tulenko, T. N., and Tuszynski, G. P. (1998) J. Surg. Res. 74, 11-16[CrossRef][Medline] [Order article via Infotrieve]
  12. Zhao, X. M., Hu, Y., Miller, G. G., Mitchell, R. N., and Libby, P. (2001) Circulation 103, 525-531[Abstract/Free Full Text]
  13. DiPietro, L. A., Nebgen, D. R., and Polverini, P. J. (1994) J. Vasc. Res. 31, 178-185[Medline] [Order article via Infotrieve]
  14. Canfield, A. E., and Schor, A. M. (1995) J. Cell Sci. 108, 797-809[Abstract]
  15. Sheibani, N., and Frazier, W. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6788-6792[Abstract/Free Full Text]
  16. Hugo, C. P., Pichler, R. P., Schulze-Lohoff, E., Prols, F., Adler, S., Krutsch, H. C., Murphy-Ullrich, J. E., Couser, W. G., Roberts, D. D., and Johnson, R. J. (1999) Kidney Int. 55, 2236-2249[CrossRef][Medline] [Order article via Infotrieve]
  17. Carpizo, D., and Iruela-Arispe, M. L. (2000) Cancer Metastasis Rev. 19, 159-165[CrossRef][Medline] [Order article via Infotrieve]
  18. Doll, J. A., Reiher, F. K., Crawford, S. E., Pins, M. R., Campbell, S. C., and Bouck, N. P. (2001) Prostate 49, 293-305[CrossRef][Medline] [Order article via Infotrieve]
  19. Hawighorst, T., Oura, H., Streit, M., Janes, L., Nguyen, L., Brown, L. F., Oliver, G., Jackson, D. G., and Detmar, M. (2002) Oncogene 21, 7945-7956[CrossRef][Medline] [Order article via Infotrieve]
  20. Bayraktar, M., Dundar, S., Kirazli, S., and Teletar, F. (1994) J. Int. Med. Res. 22, 90-94[Medline] [Order article via Infotrieve]
  21. Murphy, M., Godson, C., Cannon, S., Kato, S., Mackenzie, H. S., Martin, F., and Brady, H. R. (1999) J. Biol. Chem. 274, 5830-5834[Abstract/Free Full Text]
  22. Tada, H., Kawai, H., Ishii, H., Nomura, K., Urayama, T., and Isogai, S. (1995) Tohoku J. Exp. Med. 177, 293-302[Medline] [Order article via Infotrieve]
  23. Poczatek, M. H., Hugo, C., Darley-Usmar, V., and Murphy-Ullrich, J. E. (2000) Am. J. Pathol. 157, 1353-1363[Abstract/Free Full Text]
  24. Yevdokimova, N., Wahab, N. A., and Mason, R. M. (2001) J. Am. Soc. Nephrol. 12, 703-712[Abstract/Free Full Text]
  25. Belmadani, S., Bernal, J., Wei, C. C., Pallero, M. A., Dell'italia, L., Murphy-Ullrich, J. E., and Berecek, K. H. (2007) Am. J. Pathol
  26. Stenina, O. I., Krukovets, I., Wang, K., Zhou, Z., Forudi, F., Penn, M. S., Topol, E. J., and Plow, E. F. (2003) Circulation 107, 3209-3215[Abstract/Free Full Text]
  27. Hiscott, P., Larkin, G., Robey, H. L., Orr, G., and Grierson, I. (1992) Eye 6, 566-569[Medline] [Order article via Infotrieve]
  28. Sheibani, N., Sorenson, C. M., Cornelius, L. A., and Frazier, W. A. (2000) Biochem. Biophys. Res. Commun. 267, 257-261[CrossRef][Medline] [Order article via Infotrieve]
  29. Carron, J. A., Hiscott, P., Hagan, S., Sheridan, C. M., Magee, R., and Gallagher, J. A. (2000) Int. J. Biochem. Cell Biol. 32, 1137-1142[CrossRef][Medline] [Order article via Infotrieve]
  30. Wang, S., Wu, Z., Sorenson, C. M., Lawler, J., and Sheibani, N. (2003) Dev. Dyn. 228, 630-642[CrossRef][Medline] [Order article via Infotrieve]
  31. Raman, P., Krukovets, I., Marinic, T. E., Bornstein, P., and Stenina, O. I. (2007) J. Biol. Chem. 282, 5704-5714[Abstract/Free Full Text]
  32. Mazumder, B., Seshadri, V., and Fox, P. L. (2003) Trends Biochem. Sci 28, 91-98[CrossRef][Medline] [Order article via Infotrieve]
  33. Moore, M. J. (2005) Science 309, 1514-1518[Abstract/Free Full Text]
  34. Lawler, J., and Detmar, M. (2004) Int. J. Biochem. Cell Biol. 36, 1038-1045[CrossRef][Medline] [Order article via Infotrieve]
  35. Armstrong, L. C., Bjorkblom, B., Hankenson, K. D., Siadak, A. W., Stiles, C. E., and Bornstein, P. (2002) Mol. Biol. Cell 13, 1893-1905[Abstract/Free Full Text]
  36. Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A. B. (1998) Mol. Cell 2, 135-140[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
B. K. Tan, R. Adya, J. Chen, S. Farhatullah, D. Heutling, D. Mitchell, H. Lehnert, and H. S. Randeva
Metformin decreases angiogenesis via NF-{kappa}B and Erk1/2/Erk5 pathways by increasing the antiangiogenic thrombospondin-1
Cardiovasc Res, August 1, 2009; 83(3): 566 - 574.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
G. Orasanu and J. Plutzky
The pathologic continuum of diabetic vascular disease.
J. Am. Coll. Cardiol., February 3, 2009; 53(5 Suppl): S35 - S42.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Q. Huang and N. Sheibani
High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs
Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1647 - C1657.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/9/5699    most recent
M706435200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bhattacharyya, S.
Right arrow Articles by Stenina, O. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bhattacharyya, S.
Right arrow Articles by Stenina, O. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement