点击显示 收起
【关键词】 cells
Department of Medicine/Nephrology, The University of Texas Health Science Center, South Texas Veterans Health Care System, San Antonio, Texas
ABSTRACT
ANG II regulates growth factor expression in the kidney. We investigated whether ANG II regulated vascular endothelial growth factor (VEGF) synthesis in proximal tubular epithelial (MCT) cells. ANG II (1 nM) increased VEGF protein expression within 5 min, the effect lasting for 30 min. There was no change in VEGF mRNA levels or mRNA stability, and transcription inhibitors did not affect ANG II-induced VEGF expression. Regulation of VEGF translation was investigated. Polyribosomal analysis revealed selective enrichment of heavy ribosomes (polysomes) with VEGF mRNA transcripts compared with light ribosomes in ANG II-treated cells, although distribution of GAPDH was unaltered. In vitro translation of total RNA from polysomal fractions showed selective increase in VEGF protein synthesis in ANG II-treated cells. Preincubation with LY-294002, a PI 3-kinase inhibitor, or expression of dominant-negative Akt prevented ANG II-stimulated increase in VEGF translation. ANG II increased phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1, critical events that regulate the initiation phase of protein translation. ANG II failed to increase VEGF mRNA translation in cells stably expressing the phosphorylation mutant of 4E-BP1. Our data illustrate that a rapid increase in VEGF protein expression by ANG II is regulated at the initiation phase of translation of VEGF mRNA in renal epithelial cells. Regulation of VEGF translation by ANG II represents a novel pathway of renal response to injury.
protein expression; polysomes; nephropathy
RENAL INJURY IN DIABETES IS the composite result of hemodynamic and cell biological processes with genetic determinants regulating predisposition to development of nephropathy (22). ANG II is an important mediator of renal injury regulating hemodynamic and cellular processes that lead to renal matrix expansion, proteinuria, and kidney failure in diabetes (9, 27). The systemic renin-ANG II axis is suppressed in diabetes (34), suggesting a role for locally produced ANG II in renal disease. Critical components of the ANG II synthetic pathway are expressed in proximal tubular epithelial cells (46). Renal tissue levels of ANG II can be severalfold higher than systemic levels and affect local physiological events (41). ANG II causes proteinuria, which may, by itself, lead to proximal tubular epithelial cell toxicity and progressive tubulointerstitial scarring (35). Remedial effects of ACE inhibitors and ANG II receptor blockers on hypertrophy and matrix expansion in diabetic nephropathy attest to the critical role of ANG II (24, 28).
In addition to its direct effects, ANG II regulates activity of several key mediators of injury. Transforming growth factor- (TGF-) is recruited by ANG II to induce hypertrophy and stimulate extracellular matrix protein synthesis in mesangial and proximal tubular epithelial cells (21, 48, 49). In this instance, ANG II stimulates the secretion of TGF-, which, in turn, binds to its cognate receptors and regulates cell function. Recent investigations have unraveled transactivation as yet another mechanism of growth factor activity regulation by ANG II. Signal transduction pathways of PDGF and epidermal growth factor are activated by ANG II without increasing the binding of respective ligands to the receptors (29, 32). It is unclear whether other growth factors are also involved in mediating ANG II effects.
Vascular endothelial growth factor (VEGF) is among growth factors of importance in diabetic microvascular injury. VEGF is of seminal importance in the ocular microvascular complication of diabetes (4). VEGF concentrations are increased in vitreous and aqueous humors in patients with retinopathy due to diabetes (2). Neutralization of VEGF in the ocular tissue with soluble VEGF receptors (3, 45) or antibodies (1) or by antisense oligonucleotides (38) limits diabetic retinopathy. The role of VEGF in diabetic renal injury has not been extensively studied. An increase in VEGF expression in renal cortex in mice with either type 1 or type 2 diabetes coincides with hypertrophy and onset of matrix accumulation (42). Administration of a neutralizing antibody against VEGF has been shown to reduce proteinuria and renal hypertrophy in animal models of type 1 (10) or type 2 diabetes (13). These observations suggest a role for VEGF in a wide spectrum of changes present in the diabetic kidney. However, the mechanism by which the diabetic state promotes VEGF expression in the kidney is not known. As inhibitors of ANG II have salutary effects on the aforementioned parameters of renal injury, it is likely that ANG II controls the activities of putative factors involved in these processes, including VEGF. In the present study, we tested the hypothesis that VEGF expression in renal cells is under the control of ANG II. As our initial observations of VEGF expression were made in renal cortex in diabetic mice (42), we studied ANG II regulation of VEGF in the most abundant cell type in renal cortex, i.e., proximal tubular epithelial cells.
EXPERIMENTAL METHODS
Immunohistochemistry. Localization of VEGF was assessed by immunoperoxidase histochemistry using the avidin-biotin-complex (ABC; Vector Laboratories, Burlingame, CA) technique for immunoperoxidase according to the manufacturer's instructions as previously described (17). Acetone-fixed frozen sections (6 μm) were incubated with nonimmune IgG of the same species as the second antibody to block nonspecific binding, then with rabbit anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 10 μg/ml. The primary antibody was followed by biotin-labeled donkey anti-rabbit IgG (Chemicon International, Temecula, CA). Sections were incubated with 0.6% hydrogen peroxide in methanol to block nonspecific peroxidase activity and 0.01% avidin, 0.001% biotin to block endogenous biotin activity. Primary and second antibodies were incubated for 30 min with three washes of PBS containing 0.1% BSA between steps. Controls consisted of nonimmune rabbit IgG or PBS/BSA in place of primary antibody followed by detection procedures as outlined above.
Cell culture. SV40-immortalized murine proximal tubular epithelial cells (MCT cells; kindly provided by Dr. E. Neilson, Vanderbilt University, Nashville, TN) were grown in DMEM containing 7% FBS, 5 mM glucose, and no insulin, as recently described (43, 44). Confluent monolayers of cells were serum deprived in DMEM for 18 h before treatment. MCT cells in culture express in vivo characteristics of proximal tubular epithelial cells (18).
Immunoblot analysis. Cells were washed twice with phosphate-buffered saline and lysed in 300 μl of lysis buffer (50 mM Tris?HCl, pH 7.4, 150 mM potassium chloride, 1 mM DTT, 1 mM EDTA, 50 mM glycerophosphate, pH 7.5, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM EGTA, 2 mM benzamidine, 1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml leupeptin). Cell debris was removed by centrifugation at 12,000 rpm for 5 min and concentration of protein was measured using the Bio-Rad protein reagent (Bio-Rad, Hercules, CA). Five to thirty micrograms of lysates were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with various primary antibodies at the indicated dilutions and with peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Pharmacia Biotech, Piscataway, NJ). Alternatively, fluorochrome-coupled antibodies were used and antigen-antibodies complexes were detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).
4E-BP1 phosphorylation. The phosphorylation status of 4E-BP1 was assessed by immunoblotting as recently described (7), using an antibody that detects 4E-BP1 phosphorylated on Thr 46 or Ser 65 or Thr 70 residue (New England Biolabs, Beverley, MA). The content of 4E-BP1 was assessed by immunoblotting with an antibody against 4E-BP1 (Santa Cruz Biotechnology).
Polyribosome assay. Polyribosome preparation was performed as described by (20). After treatment of serum-starved cells with or without 1 nM ANG II for the indicated duration, MCT cells were washed in PBS and pelleted by centrifugation. Pellets were lysed in 0.4 ml of resuspension buffer containing 10 mM Tris (pH 7.5), 250 mM KCl, and 2 mM MgCl2 (12). After 5 min on ice, 150 μl of a 10% Tween 80, 5% (wt/vol) deoxycholate mix was added to the lysate. Lysates were kept on ice for 15 min and centrifuged for 10 min at 14,000 rpm. The cytosolic supernatants (600 μl) were laid on top of 1540% sucrose gradients (1.5 ml, made in DEPC water) and centrifuged for 90 min at 200,000 g. After centrifugation, the gradients were separated into 4 fractions of 400 μl. RNA was extracted from each fraction using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Semiquantitative RT-PCR amplification of VEGF or GAPDH transcript was performed in polyribosomal fractions using the Superscript One-Step RT-PCR kit from Invitrogen and employing the following specific primers: VEGF sense (5'-ACATCTTCAAGCCGTCCTGTGTGC-3'), VEGF antisense (5'-AAATGGCGAATCCAGTCCCACGAG-3'), GAPDH sense (5'-CGATGCTGGCGCTGAGTAC-3'), and GAPDH antisense (5'-CGTTCAGCTCAGGGATGACC-3'). PCR amplification was performed using the following conditions: denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s for a total of 32 cycles. Fifteen-microliter samples from each 25-μl PCR product were removed and analyzed by electrophoresis on 1% agarose gel.
Northern blotting and RT-PCR. Northern blot analysis was done on total RNA isolated from cells incubated with or without ANG II for the indicated duration, as described previously (17). Equal amounts of total RNA were electrophoresed on a 1% agarose, 2.2 M formaldehyde gel with 1x 4-morpholinepropanesulfonic acid buffer, transferred to a nylon membrane using the TurboBlotter System (Schleicher and Schuell, Keene, NH), and cross-linked by UV light. Prehybridization was done in 50% formamide 5x SSC, salmon sperm DNA, sodium EDTA buffer for 4 h at 42°C. Hybridization was done in the same buffer with 7.9 to 2.26 x 106 cpm/ml of 32P-labeled partial cDNA probes for VEGF and GAPDH (VEGF probe was kindly provided by A. Karumanchi and V. Sukhatme, Beth Israel New England Deaconess Medical Center, Harvard Medical School, Boston, MA). The blot was washed two times each in 2x SSC + 0.1% SDS and 0.1x SSC + 0.1% SDS at 42°C and autoradiographed. RT-PCR was performed with primers for VEGF and GAPDH as described above.
mRNA stability assay. Quiescent MCT cells were treated with 5 μM actinomycin D for 30 min before treatment with 1 nM ANG II for 15 and 30 min. Control and ANG II-treated cells were processed for RNA isolation in TRIzol as described above. Ten nanograms of RNA were used for RT-PCR using primers for VEGF and GAPDH (6).
In vitro translation assay. An in vitro translation assay was performed employing the rabbit reticulocyte method and the Transcend nonradioactive detection system from Promega, according to the manufacturer's instructions. Four percent of total RNA extracted from cytosolic material in heavy ribosomal fraction was incubated with rabbit reticulocyte lysate, a mix of amino acids without methionine and leucine and a biotin-labeled lysine tRNA. After 1-h incubation at 30°C, 1 to 5 μl of the reaction mixture were used for electrophoresis on a 12.5% gel. After transfer onto nitrocellulose membrane, proteins were detected using an avidin-alkaline phosphatase solution for 1 h at room temperature. VEGF was detected by immunoblotting of an aliquot of the reaction mixture.
RESULTS
VEGF is expressed in the proximal tubule from mice kidneys. We performed immunoperoxidase staining of normal murine kidney sections using an antibody directed against VEGF-A. Figure 1 shows strong staining in the proximal tubules and weaker staining in the distal tubules and the glomeruli. This result is in accordance with a study by Kim et al. (23) showing the presence of VEGF in the proximal tubule of rat kidney by immunoelectron microscopy. Having established the presence of VEGF in proximal tubule epithelial cells in vivo, we used MCT cells, a proximal tubular epithelial cell line, to study the regulation of VEGF by ANG II.
ANG II increases VEGF protein but not mRNA in MCT cells. ANG II regulation of VEGF was investigated in MCT cells, employing physiologically relevant concentrations of ANG II. Quiescent MCT cells were incubated with 1 nM ANG II for up to 2 h. ANG II rapidly caused a twofold increment in VEGF protein expression in cell lysates, starting within 5 min of incubation, peaking between 5 and 15 min and being sustained for 30 min (Fig 2A; 3 experiments, P < 0.01 by ANOVA). These changes were associated with an increase in VEGF secretion into the medium that was also evident at 5 min of incubation with ANG II (data not shown).
We next investigated if ANG II induction of VEGF was due to an increase in its mRNA levels. There was no change in net VEGF mRNA content corrected to GAPDH expression in cells treated with ANG II at 15 and 30 min as assessed by Northern blot analysis (Fig. 2B) or semiquantitative RT-PCR (data not shown). Furthermore, cells were pretreated for 3 h with inhibitors of transcription, dichlorobenzimidazole-1--D-ribofuranoside (DRB), and actinomycin-D, before incubation with 1 nM ANG II. Neither transcription inhibitor affected ANG II-stimulated VEGF protein expression at 15 min (Fig. 2C). Taken together, these results suggest that ANG II stimulation of VEGF expression involves nontranscriptional mechanisms. These may include regulation at the level of mRNA stability or protein translation.
Prolongation of VEGF mRNA half-life can also lead to greater synthesis of VEGF protein. Accordingly, VEGF mRNA stability was evaluated in control and ANG II-treated cells over 30 min of incubation. Quiescent MCT cells were treated with a transcription inhibitor (5 μM actinomycin D) to prevent further transcription of the VEGF gene, so that decay of existing VEGF mRNA can be measured. ANG II did not significantly affect VEGF mRNA stability for up to 30 min of incubation (Fig. 3).
ANG II promotes translation of VEGF mRNA. Having excluded changes in mRNA level or mRNA stability as mechanisms leading to increased VEGF synthesis by ANG II, we investigated whether increased efficiency in translation of mRNA could be involved. We examined the initiation phase, as it is the rate-controlling step in protein translation. Aggregated ribosomes or polysomes associate with mRNA that is targeted for increased translation. On a sucrose gradient, ribosomes are distributed according to their density, with more aggregated ribosomes being distributed to progressively heavier fractions. Total RNA was extracted from cytosolic fractions separated on a sucrose gradient. VEGF and GAPDH mRNA species were detected by RT-PCR with the latter being employed as a control. In control cells, distribution of VEGF mRNA was minimally increased in the heavy ribosomal fractions compared with the light ribosomal fractions. At 15 min of stimulation with ANG II, a trend toward a shift in distribution of VEGF mRNA into heavier polysomal fractions was evident; at 30 min, only the heavy polysomal fraction contained VEGF mRNA (Fig. 4B). In contrast, there was no change in the ribosomal distribution of the GAPDH mRNA at any time of ANG II treatment. These results constitute evidence that ANG II facilitates selective segregation of VEGF mRNA into polysomal fractions that are involved in amplifying its translation into a peptide.
We then evaluated whether the mRNA distributed to the heavier polysomes can be employed in an in vitro translation system to test whether VEGF protein synthesis is augmented by ANG II. Four percent of RNA extracted from the heavy ribosomal fraction was incubated with rabbit reticulocyte lysate, a mix of amino acids containing biotin-labeled lysine tRNA to translate mRNA transcripts into proteins. After 1-h incubation at 30°C, an aliquot of the reaction mixture was used for electrophoresis to separate the translation products. After transfer onto nitrocellulose membrane, proteins were detected using an avidin-alkaline phosphatase solution or by immunoblotting using a VEGF antibody. There was no change in the intensities of most bands in ANG II-treated cells compared with control cells (Fig. 4C). However, a selective and time-dependent increase in the intensity of two protein bands of approximate size 23 and 30 kDa was seen in ANG II-treated cells. The 23-kDa band was confirmed to be VEGF by immunoblotting with a VEGF antibody (Fig. 4D); the identity of the 30-kDa band is unknown. These data confirm that ANG II selectively promotes translation of VEGF protein.
ANG II regulates regulatory steps in initiation phase of translation. As improved efficiency of translation initiation was the underlying mechanism of rapid increment in VEGF expression induced by ANG II, the steps in the initiation phase of translation that may be regulated by ANG II were next examined. In resting cells, eukaryotic initiation factor 4E, an mRNA cap binding protein that has important regulatory effects on mRNA translation, is normally held in an inactive complex with its binding protein belonging to the 4E-BP family. Phosphorylation of 4E-BP1 (14) leads to dissolution of the eIF4E/4E-BP1 complex and permits free eIF4E to form the eIF4F complex (16). Regulation of 4E-BP1 phosphorylation was studied by immunoblotting with a phospho-specific antibody. Within 5 min of incubation, ANG II increased the intensity of the slower migrating band of 4E-BP1, which is more heavily phosphorylated. The effect peaked at 15 min and lasted for nearly 1 h (Fig. 5A). Densitometric readings showed that ANG II caused a two- to threefold increase in 4E-BP1 phosphorylation (P < 0.05 by ANOVA, 3 experiments). Phosphorylation of 4E-BP1 occurs on several serine and threonine residues and a variety of kinases, including the PI 3-kinase-Akt axis, have been implicated in phosphorylation of specific sites. Preincubation of cells with LY-294002, an inhibitor of PI 3-kinase, prevented phosphorylation of 4E-BP1 induced by ANG II (Fig. 5B). We evaluated the role of Akt by expressing a dominant-negative form of the kinase carried by an adenovirus (Ad-DN-Akt) in which the serine and threonine phosphorylation sites are mutated to alanine, whereas the control cells were infected with an adenovirus carrying green fluorescent protein (Ad-GFP) (42). Compared with Ad-GFP-infected control cells, Ad-DN-Akt abolished ANG II-induced 4E-BP1 phosphorylation (Fig. 5B). These data demonstrate that ANG II-induced 4E-BP1 phosphorylation is dependent on activation of PI 3-kinase and its downstream target, Akt.
Phosphorylation of eIF4E on Ser209 was evaluated using a phospho-specific antibody (47). ANG II stimulated a twofold increase in phosphorylation of eIF4E that was also seen at 5 min and lasted for 30 min (Fig. 5C, P < 0.01). We previously showed that ANG II promotes dissociation between eIF4E and 4E-BP1 under these experimental conditions in MCT cells (43). These data demonstrate that ANG II regulates events of regulatory significance in the initiation phase of translation.
Signaling events in ANG II regulation of VEGF translation. We next examined whether abolition of PI 3-kinase and Akt activity would interfere with ANG II regulation of the initiation phase of VEGF translation. We previously showed that ANG II stimulates the activation of these kinases in MCT cells. We preincubated serum-starved MCT cells with LY-294002, a PI 3-kinase inhibitor, before adding 1 nM ANG II and separated polyribosomal fractions on a sucrose gradient before extracting RNA. We used RNA extracted from heavy fractions for in vitro translation assay (Fig. 6A). This assay confirmed that ANG II-induced increment in synthesis of VEGF protein (23 kDa) and the unidentified 30-kDa protein was also abrogated by the inhibitor of PI 3-kinase (Fig. 6B). Akt, a downstream target of PI 3-kinase, is prominently involved in regulation of 4E-BP1 phosphorylation (Fig. 5B). Compared with Ad-GFP-infected control cells, Ad-DN-Akt abolished stimulation of synthesis of both 30- and 23-kDa proteins (Fig. 6C); the latter was identified as VEGF by immunoblot (Fig. 6D). These data demonstrate that activation of PI 3-kinase-Akt axis is required for ANG II stimulation of VEGF translation.
Requirement of 4E-BP1 phosphorylation for ANG II regulation of VEGF translation. We next evaluated the importance of 4E-BP1 phosphorylation in ANG II stimulation of VEGF translation. To this end, we examined the role of phosphorylation of Thr37 and Thr46 residues on 4E-BP1 by employing MCT cells stably expressing a Thr37,46Ala37,46 mutant (mut. 4E-BP1) (42, 43). In vitro translation of total RNA isolated from heavy ribosomal fraction demonstrated increase in synthesis of the 23- and 30-kDa proteins in MCT overexpressing the empty vector, but not mut. 4E-BP1 (Fig. 7A). The 23-kDa protein was shown to be VEGF by immunoblot (Fig. 7B). Cells stably expressing phosphorylation mutant of 4E-BP1 also failed to show increase in VEGF protein on an immunoblot in contrast to cells stably transfected with a plasmid vector (Fig. 7C). Thus ANG II stimulation of increased VEGF translation is dependent on functional 4E-BP1.
DISCUSSION
Our results provide the first evidence that ANG II rapidly increases VEGF expression in proximal tubular epithelial cells by stimulating cap-dependent translation of its mRNA. This effect is rapid, starting at 5 min of ANG II treatment, and brief, returning to basal level within 45 min. ANG II recruits critical events in the initial phase of translation including phosphorylation of 4E-BP1 and eIF4E and distribution of VEGF mRNA to polysomes to promote VEGF synthesis. Activation of PI 3-kinase and its downstream target Akt are necessary for ANG II stimulation of VEGF translation. It is probable that these kinases act by increasing the phosphorylation of 4E-BP1 that permits dissociation of the eIF4E/4E-BP1 complex allowing eIF4E to promote cap-dependent translation of VEGF. It is unlikely that release of VEGF from intracellular storage sites accounts for the ANG II effect as total cell lysates were used in the analysis of VEGF protein levels. Furthermore, an increase in VEGF synthesis was corroborated by finding an increase in secretion of VEGF into the culture medium. The mechanism of ANG II regulation of VEGF may depend on duration of exposure to the agonist. Although our data support a translational mechanism for rapid phase effects, a longer duration of exposure to ANG II is known to be associated with transcriptional regulation of VEGF (31, 37). Thus distinct transcriptional and translational mechanisms are employed in ANG II regulation of VEGF synthesis.
In contrast to transcriptional regulation of VEGF, control of its translation has not been well studied. Previous studies showed that the presence of extensive secondary structures in 5'-UTR in an mRNA transcript inhibits the ability of the ribosome to scan for the first AUG codon and initiate protein synthesis. The VEGF 5'-UTR is known to be GC-rich and unusually long (5). On computer modeling, we confirmed the presence of extensive secondary structures in the 5'-UTR of VEGF; the energy of formation (G) of 5'-UTR was estimated at 168 kcal/mol (data not shown). Ribosomal scanning for the initiator AUG codon is inefficient in mRNAs with G greater than 50 kcal/mol (25). Translation of such transcripts is facilitated by several initiation factors, particularly eIF4E, the mRNA cap binding protein, which is normally held inactive by its binding protein, 4E-BP1 (33). After agonist stimulation, e.g., ANG II, 4E-BP1 is phosphorylated with dissolution of the dimeric complex and release of eIF4E (33). eIF4E forms an eIF4F complex in association with eIF4G and eIF4A. eIF4G and 4E-BP1 share a consensus sequence for binding to eIF4E, and the two proteins compete with each other for the latter (30). eIF4G has distinct binding sites for eIF4E and eIF4A and serves as a bridge between the mRNA and the ribosome (16). eIF4A, assisted by eIF4B, functions as a helicase, unwinding secondary structures in the 5'-UTR of the mRNA and facilitating the ribosome to successfully scan for the initiator methionine codon (16). In agreement with the above schema, ANG II stimulated important events in cap-dependent translation including phosphorylation of 4E-BP1 and eIF4E. Our studies in MCT cells have shown that phosphorylation of 4E-BP1 by ANG II is associated with dissolution of the eIF4E-4E-BP1 complex (43). The inability of ANG II to induce VEGF expression in cells expressing 4E-BP1 phosphorylation mutant further confirmed cap-dependent translation as the underlying mechanism. Although not addressed in our studies, cap-independent mechanisms of VEGF translation have also been reported, in which ribosome scans for the first AUG codon by accessing internal ribosomal entry sites (IRES) without the help of initiation factors (5).
Regulation at the level of translation of mRNA offers a way for the cell to rapidly increase synthesis of target proteins within minutes. One reason for the rapidity of translational response could be that activity of proteins that regulate translation is controlled by changes in phosphorylation status. Changes in activities of kinases and phosphatases that govern phosphorylation status of these proteins can occur within seconds to minutes following agonist application. Thus signaling pathways figure prominently in regulation of translation, including that of VEGF. ANG II stimulates production of reactive oxygen species in MCT cells (Feliers D and Kasinath BS, unpublished observations), which has been shown by Zeng et al. (51) to be mediated through activation of Rac1 in the induction of p27KIP1 in rat mesangial cells.
Abolition of ANG II-induced phosphorylation of 4E-BP1 by PI 3-kinase inhibitor and dominant-negative Akt suggests important roles for these kinases. There are multiple serine and threonine phosphorylation sites on 4E-BP1, which are thought to be selectively phosphorylated by distinct kinases including Akt-mTOR, ATM and cdc2 kinases (14, 15, 19, 50). The identity and role of specific serine/threonine residues involved in dissolution of eIF4E-4E-BP1 complex are controversial (11). Also controversial is the functional role of eIF4E phosphorylation in regulation of protein synthesis. Whereas in Drosophila it is essential to promote insect cell growth and maturation (26), phosphorylation of eIF4E is thought to inhibit cap binding due to electrostatic repulsion (40, 52). In our studies, ANG II stimulation of eIF4E phosphorylation coincided with an increase in VEGF synthesis; however, whether it facilitates or inhibits VEGF synthesis is not clear.
Translation has not received much attention as an independent site of regulation in renal disease. Our observations on ANG II regulation of VEGF synthesis could have implications for renal disease, e.g., diabetes. ANG II is acknowledged as a master regulatory molecule mediating the harmful effects of high plasma glucose levels on the kidney (36); its pathogenic importance is confirmed by the ameliorative effects of ANG II receptor blockers on the course of renal disease in diabetes (8). ANG II has been shown to increase VEGF expression in retinal microvessels (39), and it would be of interest to investigate whether this regulation involves translation. Both ANG II and VEGF have been implicated in renal hypertrophy occurring in diabetes (10, 42, 49). Recent in vivo observations have shown that ANG II infusion increases VEGF expression in the renal tissue by regulating its transcription (37). Our data extend the mechanism by which ANG II regulates VEGF expression by demonstrating a novel mechanism, i.e., regulation of translation. In diabetes, tides of hyperglycemia may provoke an increase in local production of ANG II in the proximal tubule, which could rapidly stimulate VEGF synthesis by translation. VEGF may, in turn, regulate important cellular processes such as protein synthesis (11). Blocking translation of VEGF mRNA could provide an additional tool in modulating effects of ANG II on the kidney.
GRANTS
This work was supported by National Institutes of Health (NIH) grants (O'Brien Kidney Center grant, B. S. Kasinath, G. Ghosh-Choudhury), The American Diabetes Association (B. S. Kasinath), VA Research Service Merit Review Grant and the Research Enhancement Award Program (B. S. Kasinath, G. Ghosh-Choudhury), The Juvenile Diabetes Research Foundation (D. Feliers/B. S. Kasinath), and The National Kidney Foundation of South and Central Texas (D. Feliers and B. S. Kasinath). G. Ghosh-Choudhury is supported by a grant from The Juvenile Diabetes Research Foundation and NIH Grants R01-DK-55815 and R01-DK-50190.
ACKNOWLEDGMENTS
We thank Dr. E. Neilson for mouse proximal tubular epithelial cells, Dr. N. Sonenberg for 4E-BP1 mutant constructs, and Drs. A. Karumanchi and V. Sukhatme for VEGF probes.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Adamis AP, Shima DT, Tolentino MJ, Gragoudas ES, Ferrara N, Folkman J, D'Amore PA, and Miller JW. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol 114: 6671, 1996.
Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pasquale LR, Thieme H, Iwamoto MA, Park JE, Nguyen HV, Aiello LM, Ferrara N, and King GL. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 331: 14801487, 1994.
Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, and Smith LE. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92: 1045710461, 1995.
Aiello LP and Wong JS. Role of vascular endothelial growth factor in diabetic vascular complications. Kidney Int Suppl 77: S113S119, 2000.
Akiri G, Nahari D, Finkelstein Y, Le SY, Elroy-Stein O, and Levi BZ. Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription. Oncogene 17: 227236, 1998.
Bermont L, Lamielle F, Lorchel F, Fauconnet S, Esumi H, Weisz A, and Adessi GL. Insulin upregulates vascular endothelial growth factor and stabilizes its messengers in endometrial adenocarcinoma cells. J Clin Endocrinol Metab 86: 363368, 2001.
Bhandari BK, Feliers D, Duraisamy S, Stewart JL, Gingras AC, Abboud HE, Choudhury GG, Sonenberg N, and Kasinath BS. Insulin regulation of protein translation repressor 4E-BP1, an eIF4E-binding protein, in renal epithelial cells. Kidney Int 59: 866875, 2001.
Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, and Shahinfar S. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 345: 861869, 2001.
Burns KD. Angiotensin II and its receptors in the diabetic kidney. Am J Kidney Dis 36: 449467, 2000.
De Vriese AS, Tilton RG, Stephan CC, and Lameire NH. Vascular endothelial growth factor is essential for hyperglycemia-induced structural and functional alterations of the peritoneal membrane. J Am Soc Nephrol 12: 17341741, 2001.
Ferguson G, Mothe-Satney I, and Lawrence JC Jr. Ser-64 and Ser-111 in PHAS-I are dispensable for insulin-stimulated dissociation from eIF4E. J Biol Chem 278: 4745947465, 2003.
Flaim KE, Liao WS, Peavy DE, Taylor JM, and Jefferson LS. The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation, and distribution of albumin mRNA. J Biol Chem 257: 29392946, 1982.
Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, and Rasch R. Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes 51: 30903094, 2002.
Gingras AC, Gygi SP, Raught B, Polakiewicz RD, Abraham RT, Hoekstra MF, Aebersold R, and Sonenberg N. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev 13: 14221437, 1999.
Gingras AC, Kennedy SG, O'Leary MA, Sonenberg N, and Hay N. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev 12: 502513, 1998.
Gingras AC, Raught B, and Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68: 913963, 1999.
Ha TS, Barnes JL, Stewart JL, Ko CW, Miner JH, Abrahamson DR, Sanes JR, and Kasinath BS. Regulation of renal laminin in mice with type II diabetes. J Am Soc Nephrol 10: 19311939, 1999.
Haverty TP, Kelly CJ, Hines WH, Amenta PS, Watanabe M, Harper RA, Kefalides NA, and Neilson EG. Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J Cell Biol 107: 13591368, 1988.
Heesom KJ, Gampel A, Mellor H, and Denton RM. Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1). Curr Biol 11: 13741379, 2001.
Ivester CT, Tuxworth WJ, Cooper GT, and McDermott PJ. Contraction accelerates myosin heavy chain synthesis rates in adult cardiocytes by an increase in the rate of translational initiation. J Biol Chem 270: 2195021957, 1995.
Kagami S, Border WA, Miller DE, and Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 93: 24312437, 1994.
Kasinath BS. Lipoxygenases in renal injuryloading the matrix. Kidney Int 64: 19181919, 2003.
Kim BS, Chen J, Weinstein T, Noiri E, and Goligorsky MS. VEGF expression in hypoxia and hyperglycemia: reciprocal effect on branching angiogenesis in epithelial-endothelial co-cultures. J Am Soc Nephrol 13: 20272036, 2002.
Kohzuki M, Yasujima M, Kanazawa M, Yoshida K, Fu LP, Obara K, Saito T, and Abe K. Antihypertensive and renal-protective effects of losartan in streptozotocin diabetic rats. J Hypertens 13: 97103, 1995.
Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 83: 28502854, 1986.
Lachance PE, Miron M, Raught B, Sonenberg N, and Lasko P. Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth. Mol Cell Biol 22: 16561663, 2002.
Leehey DJ, Singh AK, Alavi N, and Singh R. Role of angiotensin II in diabetic nephropathy. Kidney Int Suppl 77: S93S98, 2000.
Lewis EJ, Hunsicker LG, Bain RP, and Rohde RD. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N Engl J Med 329: 14561462, 1993.
Linseman DA, Benjamin CW, and Jones DA. Convergence of angiotensin II and platelet-derived growth factor receptor signaling cascades in vascular smooth muscle cells. J Biol Chem 270: 1256312568, 1995.
Mader S, Lee H, Pause A, and Sonenberg N. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 and the translational repressors 4E-binding proteins. Mol Cell Biol 15: 49904997, 1995.
Moravski CJ, Skinner SL, Stubbs AJ, Sarlos S, Kelly DJ, Cooper ME, Gilbert RE, and Wilkinson-Berka JL. The renin-angiotensin system influences ocular endothelial cell proliferation in diabetes: transgenic and interventional studies. Am J Pathol 162: 151160, 2003.
Moriguchi Y, Matsubara H, Mori Y, Murasawa S, Masaki H, Maruyama K, Tsutsumi Y, Shibasaki Y, Tanaka Y, Nakajima T, Oda K, and Iwasaka T. Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor- synthesis via transcriptional and posttranscriptional mechanisms. Circ Res 84: 10731084, 1999.
Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC Jr, and Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 371: 762767, 1994.
Price DA, Porter LE, Gordon M, Fisher ND, De'Oliveira JM, Laffel LM, Passan DR, Williams GH, and Hollenberg NK. The paradox of the low-renin state in diabetic nephropathy. J Am Soc Nephrol 10: 23822391, 1999.
Remuzzi G and Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 339: 14481456, 1998.
Rincon-Choles H, Kasinath BS, Gorin Y, and Abboud HE. Angiotensin II and growth factors in the pathogenesis of diabetic nephropathy. Kidney Int Suppl 82: 811, 2002.
Rizkalla B, Forbes JM, Cooper ME, and Cao Z. Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors. J Am Soc Nephrol 14: 30613071, 2003.
Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, and Smith LE. Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA 93: 48514856, 1996.
Sarlos S, Rizkalla B, Moravski CJ, Cao Z, Cooper ME, and Wilkinson-Berka JL. Retinal angiogenesis is mediated by an interaction between the angiotensin type 2 receptor, VEGF, and angiopoietin. Am J Pathol 163: 879887, 2003.
Scheper GC and Proud CG. Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation Eur J Biochem 269: 53505359, 2002.
Seikaly MG, Arant BS Jr, and Seney FD Jr. Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat. J Clin Invest 86: 13521357, 1990.
Senthil D, Choudhury GG, McLaurin C, and Kasinath BS. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells: a potential role in diabetic nephropathy. Kidney Int 64: 468479, 2003.
Senthil D, Faulkner JL, Choudhury GG, Abboud HE, and Kasinath BS. Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells. Biochem J 360: 8795, 2001.
Senthil D, Ghosh Choudhury G, Bhandari BK, and Kasinath BS. The type 2 vascular endothelial growth factor receptor recruits insulin receptor substrate-1 in its signalling pathway. Biochem J 368: 4956, 2002.
Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, and D'Amore PA. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35: 101111, 1994.
Tang SS, Jung F, Diamant D, Brown D, Bachinsky D, Hellman P, and Ingelfinger JR. Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system. Am J Physiol Renal Fluid Electrolyte Physiol 268: F435F446, 1995.
Tschopp C, Knauf U, Brauchle M, Zurini M, Ramage P, Glueck D, New L, Han J, and Gram H. Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies. Mol Cell Biol Res Commun 3: 205211, 2000.
Wolf G, Mueller E, Stahl RA, and Ziyadeh FN. Angiotensin II-induced hypertrophy of cultured murine proximal tubular cells is mediated by endogenous transforming growth factor-. J Clin Invest 92: 13661372, 1993.
Wolf G and Neilson EG. Angiotensin II induces cellular hypertrophy in cultured murine proximal tubular cells. Am J Physiol Renal Fluid Electrolyte Physiol 259: F768F777, 1990.
Yang DQ and Kastan MB. Participation of ATM in insulin signalling through phosphorylation of eIF-4E-binding protein 1. Nat Cell Biol 2: 893898, 2000.
Zeng L, Xu H, Chew TL, Chisholm R, Sadeghi MM, Kanwar YS, and Danesh FR. Simvastatin modulates angiotensin II signaling pathway by preventing Rac1-mediated upregulation of p27. J Am Soc Nephrol 15: 17111720, 2004.
Zuberek J, Wyslouch-Cieszynska A, Niedzwiecka A, Dadlez M, Stepinski J, Augustyniak W, Gingras AC, Zhang Z, Burley SK, Sonenberg N, Stolarski R, and Darzynkiewicz E. Phosphorylation of eIF4E attenuates its interaction with mRNA 5'-cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein. RNA 9: 5261, 2003.