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Am J Physiol Renal Physiol 293: F607-F615, 2007. First published June 20, 2007; doi:10.1152/ajprenal.00497.2006
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Heterogeneous nuclear ribonucleoprotein K contributes to angiotensin II stimulation of vascular endothelial growth factor mRNA translation

Denis Feliers,1 Myung-Ja Lee,1 Goutam Ghosh-Choudhury,1,2 Karol Bomsztyk,3 and B. S. Kasinath1,2

1O'Brien Kidney Research Center, Department of Medicine/Nephrology, University of Texas Health Science Center, and 2South Texas Veterans Health Care System, San Antonio, Texas; and 3University of Washington Medicine Lake Union, University of Washington, Seattle, Washington

Submitted 14 December 2006 ; accepted in final form 8 June 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ANG II rapidly increases VEGF synthesis in proximal tubular epithelial cells through mRNA translation. The role of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in ANG II regulation of VEGF mRNA translation initiation was examined. ANG II activated hnRNP K as judged by binding to poly(C)- and poly(U)-agarose. ANG II increased hnRNP K binding to VEGF mRNA at the same time as it stimulated its translation, suggesting that hnRNP K contributes to VEGF mRNA translation. Inhibition of hnRNP K expression by RNA interference significantly reduced ANG II stimulation of VEGF synthesis. ANG II increased hnRNP K phosphorylation on both tyrosine and serine residues with distinct time courses; only Ser302 phosphorylation paralleled binding to VEGF mRNA. Src inhibition using PP2 or RNA interference inhibited PKC{delta} activity and prevented hnRNP K phosphorylation on both tyrosine and serine residues and its binding to VEGF mRNA. Under these conditions, ANG II-induced VEGF synthesis was inhibited. ANG II treatment induced redistribution of both VEGF mRNA and hnRNP K protein from light to heavy polysomal fractions, suggesting increased binding of hnRNP K to VEGF mRNA that is targeted for increased translation. This study shows that hnRNP K augments efficiency of VEGF mRNA translation stimulated by ANG II.

ANG II; VEGF; signal transduction; RNA-binding protein

RENAL HYPERTROPHY IS ONE OF the cardinal manifestations of diabetic renal disease. Recent studies have linked renal enlargement seen in early stages of diabetes to such long-term complications as accumulation of renal extracellular matrix (27, 37). These data emphasize the need to understand the pathogenesis of hypertrophy of the kidney. ANG II is one of the important mediators of renal growth (2, 12, 36). In addition to its direct effects, ANG II also promotes synthesis of effectors that in turn stimulate renal hypertrophy, e.g., VEGF.

We have previously reported that VEGF expression in the renal cortex increases at the time of hypertrophy in mice with type 1 or type 2 diabetes (29). In vitro experiments showed that VEGF induces hypertrophy in MCT cells, derived from murine proximal tubular epithelium, by promoting protein synthesis in a phosphatidyl inositol (PI) 3-kinase-dependent manner (29). Administration of VEGF-neutralizing antibodies to rodents with diabetes results in amelioration of renal hypertrophy (8). Taken together, these data suggest that VEGF contributes to renal hypertrophy in diabetes. The cellular mechanism that leads to augmented VEGF synthesis in the kidney is not well understood. In view of the importance of ANG II as an important mediator of renal injury in diabetes and nondiabetic diseases, we have examined whether ANG II regulates VEGF synthesis in MCT cells. We have focused on the events governing the translation of the protein, as it is the rate-limiting step in protein synthesis. We have previously reported that the rapid phase of VEGF synthesis induced by ANG II occurs by stimulation of cap-dependent translation of its mRNA (5, 7). In that study, we had investigated the role of factors that operate at the 5'-end of mRNA and modulate its translation. In addition to events that involve 5'-end the mRNA, recently attention has been drawn to those occurring at the 3'-untranslated region (UTR), which can affect the stability and the translation efficiency of mRNA (15). Regulation of VEGF mRNA translation by factors that bind 3'-UTR has not been well studied.

Analysis of the 3'-UTR of the VEGF mRNA shows cytidine-uridine (CU)-rich regions (31) which may represent potential binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) (25). hnRNP K is a factor involved in a host of processes involving nucleic acids; it is an RNA/DNA-binding protein initially identified as a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex (18, 34). Recently, hnRNP K has been found not only in the nucleus but also in the cytoplasm, where it has been implicated in the regulation of mRNA translation (4, 19, 22). hnRNP K contains multiple modules that, on the one hand, bind kinases (1, 23, 28, 35) while, on the other hand, recruit factors involved in processes such as translation (32, 38). Regulation of hnRNP K-mediated interactions appears to involve signaling reactions (9, 10, 13, 21). These observations are consistent with hnRNP K acting as a docking platform to integrate signaling cascades by facilitating cross talk between kinases and factors that mediate nucleic acid-directed processes (3). In the present study, we tested the hypothesis that a 3'-UTR-binding protein, hnRNP K, participates in ANG II regulation of VEGF mRNA translation in MCT cells.


   EXPERIMENTAL METHODS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. SV40-immortalized murine proximal tubular epithelial cells (i.e., MCT; provided by Dr. Eric Neilson, Vanderbilt University, Nashville, TN) were grown in DMEM containing 5 mM glucose and 10% FBS (17, 30). MCT cells in culture express in vivo characteristics of proximal tubular epithelial cells (11). Confluent monolayers of cells were serum deprived in DMEM for 18 h before treatment.

Transfection experiments. Transfections were carried out with Superfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions, using 200 nM small interfering RNA (siRNA)/well in six-well plates. hnRNP K siRNA was purchased from Ambion (Austin, TX): sense sequence, 5'-CCUUAUGAUCCCAACUUUU-3', and antisense sequence, 5'-AAAAGUUGGGGAUCAUAAGG-3'. Src siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunoblotting and immunoprecipitation. Immunoblotting experiments were performed as previously described (5, 6). After treatment, MCT cells were washed twice with ice-cold PBS and lysed in lysis buffer (50 mM Tris·HCl, pH 7.4, 150 mM KCl, 1 mM EDTA, 50 mM beta-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM EGTA, 0.5% Nonidet P-40, and protease inhibitor mix, Sigma, St Louis, MO). Protein concentration was measured, and indicated amounts of lysates were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with various primary antibodies, and IRDye800- or IRDye700-coupled secondary antibodies were used for detection using the Odyssey Infrared Imaging System (LiCor Biosciences, Lincoln, NE). Immunoblots were conducted within the linear range of the assay, 20–40 µg of protein (Fig. S2; all supplementary data for this article are available in the online version of the article on the journal web site).

Immunoprecipitation experiments were carried out on 500 µg of cell lysates to which 5 µg of antibody was added for overnight incubation at 4°C with rotation. Protein A/G-agarose slurry was added for 1 h at 4°C with rotation. After pelleting of the agarose beads, pellets were washed three times with lysis buffer and twice with PBS. The agarose beads were then suspended in Laemmli sample buffer and boiled for 5 min.

Pull-down assays. Twenty microliters of a polycytidylic acid- or polyuridylic acid-immobilized on cross-linked 4% beaded agarose [poly(C)-agarose or poly(U)-agarose, Sigma] were added to equal amounts (500 µg protein) of cell lysates. Samples were incubated at 4°C for 2 h with constant rotation. After three washes with lysis buffer followed by two washes with PBS, the agarose beads were suspended in Laemmli sample buffer and boiled for 5 min.

Association of hnRNP K and mRNA. 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. After 5-min incubation on ice, 60 µl of a 10% Tween 80–5% 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 supernatants were used for immunoprecipitation using anti-hnRNP K antibody as described above. RNA was extracted from pellets using TRIzol (Invitrogen, Carlsbad, CA). RT-PCR amplification of VEGF or GAPDH transcript was performed using the Superscript One-Step RT-PCR kit from Invitrogen and employing the primers and conditions described previously (5). Measurements of VEGF and GAPDH mRNAs were conducted in the linear range of the RT-PCR assay: 10–30 cycles (Fig. S1).

PKC{delta} in vitro kinase assay. PKC{delta} was immunoprecipitated from 500 µg of MCT cell lysates as previously described. Pellets were resuspended in 20 µl of kinase buffer containing 25 mM Tris·HCl (pH 7.4), 10 mM MgCl2, 25 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 50 µM ATP, and 5 µg myelin basic protein (MBP). The reaction was carried out in the presence of 1 µCi of {gamma}-[32P]ATP for 20 min at 30°C, and stopped by the addition of Laemmli buffer and boiling for 5 min. An aliquot of reaction mixture was separated on a 15% SDS-PAGE. Radioactivity incorporated in MBP was detected by autoradiography.

Polysome assay. A polysome assay was performed as described elsewhere (5). MCT cells were lysed in polysome resuspension buffer containing 10 mM Tris (pH 7.5), 250 mM KCl, and 2 mM MgCl2. After 5 min on ice, 150 µl of a 10% Tween 80, 5% (wt/vol) deoxycholate mix was added to the lysates, which were kept on ice for 15 min, and centrifuged for 10 min at 14,000 rpm. The cytosolic supernatants (~500 µl) were laid on top of a 15–40% sucrose gradient (total volume: 1.5 ml, in DEPC water), and centrifuged for 90 min at 200,000 g. After centrifugation, the gradients were separated into 10 fractions of 200 µl. RNA was extracted from each fraction using TRIzol (Invitrogen). Semiquantitative RT-PCR amplification 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', and VEGF antisense, 5'AAATGGCGAATCCAGTCCCACGAG-3', and GAPDH sense, 5'CGATGCTGGCGCTGAGTAC-3', and GAPDH antisense, 5'-CGTTCAGCTCAGGGATGACC-3'. Measurement of VEGF and GAPDH mRNAs was conducted in the linear range of the RT-PCR assay: 10–30 cycles (Fig. S1). PCR products were removed and analyzed by electrophoresis on a 1% agarose gel.

Statistics. Data from a minimum of three experiments were expressed as means ± SE and analyzed by ANOVA for comparison among multiple groups using Bonferroni posttest analysis (GraphPad Prizm) and a t-test for comparison between two groups. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II activates hnRNP K during stimulation of VEGF mRNA translation. ANG II stimulates VEGF synthesis rapidly in MCT cells by increasing the efficiency of cap-dependent translation (5). hnRNP K activation, assessed by measuring its binding to poly(C)- or poly(U)-agarose, which serve as surrogates for CU-rich regions of mRNA, started at 5 min of ANG II treatment and lasted for up to 60 min (Fig. 1A).


Figure 1
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  		Fig. 1. ANG II activates heterogeneous nuclear ribonucleoprotein K (hnRNP K) and stimulates its binding to VEGF mRNA. Quiescent MCT cells were incubated with 1 nM ANG II for the indicated time. A: hnRNP K activation was assessed by poly(C)- and poly(U)-agarose pull-down experiments using 500 µg of lysates. IB, immunoblot. Bottom: densitometric analysis of hnRNP K activation from 3 experiments, expressed as percentage of untreated cells. *P < 0.05, **P < 0.01 vs. untreated cells by ANOVA. B: hnRNP K activation by ANG II is mediated through ANG II type 1 receptor (AT1R). Quiescent MCT cells were incubated with 10 µM ZD7155, an AT1R antagonist, before treatment with 1 nM ANG II for 30 min. hnRNP K activation was assessed by poly(C)-agarose pull-down assay described earlier. Bottom: results of densitometric analysis of 3 independent experiments, expressed as percentage of untreated cells. **P < 0.01 vs. control by ANOVA. ++P < 0.05 vs. ANG II by ANOVA. C: RT-PCR was performed on RNA extracted from hnRNP K immunoprecipitates using primers specific for murine VEGF and GAPDH mRNAs. Densitometric analysis of 3 experiments is expressed as percent of untreated cells. *P < 0.05, **P < 0.01 vs. control by ANOVA. D: MCT cells were transfected with control or hnRNP K-specific siRNA 48 h before treatment with 1 nM ANG II for 30 min. Immunoblots were performed on 20 µg protein from whole-cell lysates, quantified by densitometric analysis, and normalized against actin. Bottom: composite data from 3 experiments are expressed as percentage of untreated MCT cells transfected with the control, small interfering RNA (siRNA). *P < 0.05, **P < 0.01 by ANOVA.

 
Most of the effects of ANG II are mediated through binding to the ANG II type 1 receptor (AT1R). To check the involvement of AT1R in ANG II activation of hnRNP K, MCT cells were incubated with 10 µM ZD7155, a specific AT1R antagonist (14), before stimulation with 1 nM ANG II for 30 min. hnRNP K activation was measured by a poly(C)-agarose pull-down assay. Figure 1B shows that hnRNP K activation by ANG II (187.3 ± 7.8% of control, P < 0.01 by ANOVA) was prevented by ZD7155 (76.3 ± 9.7% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA). This shows that ANG II activation of hnRNP K is mediated by the AT1R.

Next, we directly explored the association of hnRNP K with VEGF mRNA by extracting RNA from hnRNP K immunoprecipitates and performing semiquantitative RT-PCR using primers for murine VEGF and GAPDH. ANG II stimulated association of hnRNP K with VEGF mRNA (Fig. 1C), starting at 15 min and reaching a maximum of 282.0% of control (P < 0.01 by ANOVA) at 60 min, which corresponds to the maximum redistribution of VEGF mRNA to heavy polysomal fractions (5). Binding of hnRNP K to GAPDH mRNA, employed as a loading control, did not change over this time frame.

The importance of hnRNP K in ANG II stimulation of VEGF synthesis was assessed by inhibition of hnRNP K by RNA interference. Transfection of hnRNP K siRNA in MCT cells decreased hnRNP K expression by >90% after 48 h (Fig. 1D, bottom, lanes 3 and 4). Under these conditions, ANG II stimulation of VEGF expression (153.5 ± 8.5% of control, P < 0.01 by ANOVA) was decreased by 50% (123.5 ± 1.5% of control, P < 0.05 by ANOVA) but was still significantly higher than control (Fig. 1D, P < 0.05 by ANOVA). Transfection of MCT cells with control siRNA did not inhibit hnRNP K (Fig. 1D, bottom, lanes 1 and 2) and did not affect ANG II stimulation of VEGF synthesis (Fig. 1D, top, lanes 1 and 2). These data show that ANG II promotes binding of hnRNP K to VEGF mRNA and that this binding contributes to increased VEGF synthesis.

Regulation of hnRNP K phosphorylation by ANG II. hnRNP K activity and/or localization is regulated by tyrosine and serine phosphorylation events (10, 20, 23). In quiescent MCT cells, ANG II stimulated phosphorylation of hnRNP K on both tyrosine and serine residues, with a distinct temporal profile (Fig. 2A). Tyrosine phosphorylation occurred early, peaking at 5 min and decreasing thereafter (Fig. 2A, top), whereas phosphorylation of hnRNP K on Ser302 occurred later (15 min) and peaked at 30–60 min (Fig. 2A, bottom). The peak of tyrosine phosphorylation correlated with minimal binding of hnRNP K to VEGF mRNA, whereas Ser302 phosphorylation correlated with maximum binding of hnRNP K to VEGF mRNA (Fig. 2B).


Figure 2
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  		Fig. 2. ANG II regulates hnRNP K phosphorylation. A: tyrosine phosphorylation was assessed by immunoblotting for phosphotyrosine (pY) in hnRNP K immunoprecipitates (IP). Phosphorylation of hnRNP K on Ser302 was examined by immunoblotting using a specific antibody. B: densitometric data of hnRNP K phosphorylation ({blacksquare}, tyrosine phosphorylation; {square}, Ser302 phosphorylation) from 3 experiments were superimposed on hnRNP K binding to VEGF mRNA, expressed as percentage of untreated cells (shown as bars). *P < 0.05, **P < 0.01 vs. control by ANOVA.

 
Role of src in ANG II-induced phosphorylation of hnRNP K. Tyrosine phosphorylation of hnRNP K is under the control of the src family of tyrosine kinases (3). To investigate the role of src in ANG II regulation of hnRNP K, we used PP2, a specific inhibitor of src (17). Figure 3A shows that ANG II stimulated tyrosine phosphorylation of hnRNP K at 5 min (197.2 ± 7.6% of control, P < 0.01 by ANOVA), which was markedly inhibited by PP2 (116.6 ± 14.3% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA). Because src activation can lead to stimulation of PKC{delta}, the kinase that phosphorylates Ser302 on hnRNP K (28), we assessed the requirement of src for this phosphorylation event. Figure 3B shows that ANG II resulted in increased phosphorylation of hnRNP K on Ser302 (187.5 ± 7.8% of control, P < 0.01 by ANOVA ), which was prevented by PP2 (102.3 ± 7.2% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA). Thus phosphorylation of hnRNP K on both tyrosine and Ser302 was dependent on src in ANG II-treated MCT cells.


Figure 3
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  		Fig. 3. Src is required for ANG II-stimulated hnRNP K phosphorylation and PKC{delta} activation. A: tyrosine phosphorylation of hnRNP K was examined in cells treated with ANG II for 5 min, with or without preincubation with 5 µM PP2 for 30 min, by hnRNP K immunoprecipitation followed by immunoblotting for phosphotyrosine. Densitometric analysis of 3 independent experiments is expressed as percentage of untreated cells. **P < 0.01 by ANOVA. B: hnRNP K phosphorylation on Ser302 was examined in MCT cells treated with ANG II for 30 min, with or without preincubation with PP2. Bottom: densitometric analysis of 3 independent experiments, expressed as percentage of untreated cells. **P < 0.01 by ANOVA. C: PKC{delta} activity was measured by an in vitro kinase assay, using MBP as a substrate in MCT cells treated with ANG II for up to 60 min. Total PKC{delta} in the corresponding lysates was measured by immunoblot. Incorporation of [32P]ATP into myelin basic protein (MBP) was measured by autoradiography and quantitated by densitometry. Composite data from 3 experiments are expressed as percentage of untreated MCT cells. *P < 0.05, **P < 0.01 vs. control by ANOVA. D: PKC{delta} activity was measured by an in vitro kinase assay in MCT cells treated with ANG II for 15 min, with or without preincubation with PP2. Quantitation of PKC{delta} activity was performed as described in C. **P < 0.01 by ANOVA.

 
These data suggest that src is upstream of PKC{delta} in the ANG II signal transduction cascade leading to serine phosphorylation of hnRNP K. To verify this hypothesis, we measured PKC{delta} activity by an in vitro kinase assay, using MBP as a substrate. In quiescent MCT cells, ANG II increased PKC{delta} activity that peaked at 15 min (221.6 ± 9.5% of control, P < 0.01 by ANOVA) and decreased over 60 min (Fig. 3C). Figure 3D shows that PP2 inhibited ANG II stimulation of PKC{delta} activity at 15 min (126.0 ± 6.6% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA). Together, these data show that src activity is required for ANG II stimulation of PKC{delta} activity and hnRNP K phosphorylation on Ser302.

Role of src in ANG II induction of hnRNP K activation and VEGF synthesis. Because hnRNP K phosphorylation on Ser302 correlated with its activation (Fig. 2B), the effect of src inhibition on hnRNP K activation was explored. Figure 4A shows that ANG II stimulation of hnRNP K binding to poly(C)-agarose at 30 min (187.5 ± 14.5% of control, P < 0.01 by ANOVA) was abrogated by PP2 (71.3 ± 16.1% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA). Similarly, ANG II stimulation of hnRNP K binding to VEGF mRNA (246.2 ± 7.5% of control, P < 0.01 by ANOVA) was prevented by PP2 (102.7 ± 6.4% of control, P < 0.01 vs. ANG II, not significant vs. control by ANOVA) (Fig. 4B). Together, these data show that src activity is required for ANG II activation of hnRNP K and its binding to VEGF mRNA. Next, the role of src in VEGF synthesis was examined using RNA interference. Transfection of siRNA specific for src decreased src expression by ~80% (Fig. 4C, bottom, lanes 3 and 4) and prevented the stimulation of VEGF expression by ANG II (Fig. 4C, top, lanes 3 and 4), whereas transfection with control siRNA had no effect either on src expression or on the ANG II-induced increase in VEGF expression (Fig. 4C, lanes 1 and 2). These data show that suppression of src results in a greater degree of inhibition of ANG II stimulation of VEGF expression than inhibition of hnRNP K (Fig. 1C). These observations thus suggest that src may participate in ANG II stimulation of VEGF expression by other mechanisms in addition to its contribution to hnRNP K activation.


Figure 4
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  		Fig. 4. Src is required for ANG II-stimulated hnRNP K activation and VEGF expression. A: poly(C)-agarose pull-down assay was performed as previously described. hnRNP K expression in corresponding lysates was measured by immunoblotting to assess equal loading. Bottom: densitometric analysis of hnRNP K activation from 3 experiments, expressed as percentage of untreated cells. **P < 0.01 by ANOVA. B: binding of hnRNP K to VEGF mRNA was measured as previously described in MCT cells treated with 1 nM ANG II for 30 min, with or without preincubation with PP2. Densitometric analysis of 3 independent experiments is expressed as percentage of untreated cells. **P < 0.01 by ANOVA. C: MCT cells were transfected with siRNA specific for murine src or control siRNA 48 h before ANG II treatment for 30 min. VEGF expression was detected in MCT cell lysates by immunoblotting. Src expression was measured to assess siRNA efficiency, and actin expression was measured to assess loading. Composite data from 3 experiments are expressed as percentage of untreated cells transfected with the control siRNA. ***P < 0.001 by ANOVA.

 
Association of hnRNP K and VEGF mRNA in polysomes. An mRNA targeted for augmented translation is bound by several ribosomes and forms a polysome (26). mRNAs present in polysomes can be separated from nonpolysomal mRNAs, i.e., those not targeted for augmented translation, by ultracentrifugation on a sucrose gradient. mRNA transcripts that are not targeted for increase in translation are associated with several proteins that facilitate their export from the nucleus and protect them from premature degradation (24), but unlike ribosome-bound mRNAs, they do not distribute to heavier fractions in a sucrose gradient. It is the association of mRNAs with multiple 80S ribosomal units that causes increased density that results in their distribution to heavier fractions (26). This property is exploited to isolate mRNAs targeted for augmented translation from other mRNAs that are not so regulated. Because hnRNP K binding to VEGF mRNA is maximal when its translation is stimulated by ANG II, we hypothesized that the polysomal distribution of hnRNP K would follow that of VEGF mRNA. MCT cells were incubated with ANG II for 30 min, and polysomes were separated as described elsewhere (5). Figure 5A shows a polysomal profile of ANG II-treated cells. To assess the polysomal distribution of VEGF mRNA, sucrose gradients were separated in 10 fractions, RNA was extracted from each fraction, and VEGF and GAPDH mRNAs were detected by RT-PCR (Fig. 5B). In untreated cells, VEGF mRNA was present in all fractions (10% of total VEGF mRNA in each fraction). The presence of VEGF mRNA in heavier fractions that contain polysomes in untreated cells is consistent with a low level of constitutive synthesis. However, in ANG II-treated cells, its distribution was shifted to the heaviest polysomal fractions (fractions 5–10, which contain ~17% of total VEGF mRNA). GAPDH mRNA was present in all polysomal fractions in untreated cells, and its distribution was not affected by treatment with ANG II. The polysomal distribution of hnRNP K was assessed by immunoblotting on an aliquot of each fraction. Figure 5C shows that in unstimulated cells, hnRNP K was present in the light ribosomal fractions (fractions 1–5) and in ANG II-stimulated cells it was recovered from the heaviest polysomal fractions (fractions 8–10). These data show that following ANG II stimulation, VEGF mRNA is distributed to polysomes along with hnRNP K that was bound to it. Together, these data suggest that hnRNP K binds to VEGF mRNA that is targeted for augmented translation.


Figure 5
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  		Fig. 5. hnRNP K and VEGF mRNA are associated with heavy polysomes in ANG II-treated MCT cells. Preparation of polysomes was carried out as described in EXPERIMENTAL METHODS. A: sedimentation analysis of ANG II-treated MCT cells was obtained by measuring optical density at 254 nm. B: after ultracentrifugation, sucrose gradients were separated into 10 fractions. VEGF and GAPDH mRNAs were detected in each fraction by RT-PCR on RNA extracted from each fraction. Bottom: results of densitometric analysis of 3 independent experiments. Ratio of VEGF to GAPDH mRNA present in each fraction was calculated and expressed as percentage of total VEGF/GAPDH ratio. C: hnRNP K expression was detected by immunoblot performed on 10 µl of each fraction. Bottom: results of densitometric analysis of 3 independent experiments. hnRNP K expression in each fraction is expressed as percentage of total hnRNP K.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data demonstrate the following. 1) Increased binding of hnRNP K to VEGF mRNA contributes to ANG II stimulation of VEGF mRNA translation in MCT cells. 2) Ser302 phosphorylation of hnRNP K correlates with its binding to VEGF mRNA. 3) src is required for ANG II induction of VEGF mRNA translation by stimulating phosphorylation of hnRNP K on both serine and tyrosine residues. 4) In ANG II-stimulated cells, hnRNP K cosegregates with VEGF mRNA in polysomal fractions. Our data demonstrate a role for hnRNP K in VEGF mRNA translation in renal proximal tubular epithelial cells.

ANG II has been shown to orchestrate the synthesis of several growth factors such as VEGF (5), which, in turn, promote renal cell growth (29). ANG II induces rapid synthesis of VEGF in MCT cells, which is due to stimulation of its mRNA translation (5). Regulatory events in mRNA translation involve the 5'-end or the 3'-end of mRNA. In our previous study, we had characterized the former (5); events occurring at the 3'-end of mRNA have not been well understood. In this study, under conditions of ANG II induction of VEGF mRNA translation, we report that hnRNP K, a protein that binds the 3'-end of the mRNA, increases VEGF mRNA translation efficiency, as a reduction in its amount resulted in significant inhibition of ANG II stimulation of VEGF synthesis. This is in contrast to the role of hnRNP K in 15-lipoxygenase (LOX) mRNA translation: hnRNP K inhibits translation of the LOX mRNA through binding to the differentiation control element, a CU-rich repetitive stretch found in its 3'-UTR (19). Inhibition of hnRNP K binding through tyrosine phosphorylation relieved this inhibition (19). These data show that direction of regulation of translation by hnRNP K is mRNA specific. Our previous studies had shown that ANG II induction of rapid-phase synthesis of VEGF could be largely inhibited in MCT cells expressing phosphorylation mutants of 4E-BP1 (5). In the current study, reduction in hnRNP K expression by siRNA resulted in a partial inhibition of ANG II stimulation of VEGF. Taken together, these data suggest that, at least in the current experimental context, events involving the 5'-end of VEGF mRNA have greater regulatory impact on VEGF synthesis than those involving the 3'-part of the mRNA. In the cells expressing the phosphorylation mutants of 4E-BP1, the cells still synthesized a basal amount of VEGF. It is possible that hnRNP K may contribute to basal VEGF expression in these cells. The amplitude of changes in VEGF mRNA translation in ANG II-treated cells that are treated with siRNA for hnRNP K suggests that this protein may be involved in fine tuning of VEGF mRNA translation.

Including hnRNPs and the mRNA cap-binding complex, several proteins are bound to mRNAs as they emerge from the nucleus following transcription (24). Even in the presence of these proteins, mRNAs that are not targeted for translation are recovered from the light fractions on the sucrose gradient. Studies on polysomal distribution of mRNAs show that heavy sucrose fractions contain mRNA bound to multiple ribosomes. Given that the density of ribosomes (~80S each) is much higher than that of mRNAs (6-25S), the density of polysome-bound mRNAs (150-400S) is due to the multiple ribosomal units rather than to mRNAs (26) and their associated proteins. Thus RNA-binding proteins, which would be bound to mRNA strands in cytoplasm, seem to be less important than much heavier 80S ribosomes in dictating whether an mRNA is isolated in the lighter or heavier fraction on a sucrose gradient.

As hnRNP K is a phosphoprotein, we examined signaling reactions that may regulate its activity. ANG II promoted phosphorylation of hnRNP K on both serine and tyrosine residues; however, only phosphorylation of Ser302 correlated with binding of hnRNP K to VEGF mRNA. Previous studies have shown that phosphorylation of hnRNP K on Ser302 is mediated by PKC{delta} (28). Src inhibition prevented ANG II induction of hnRNP K phosphorylation on Ser302 and activation of PKC{delta}. These data place src upstream of PKC{delta} in the signaling pathway leading to hnRNP K phosphorylation and activation. PKC{delta} differs from classic isoforms of PKC in that is activated by diacylglycerol (DAG) in the absence of calcium (33). PKC{delta} is unique among PKC isoforms in that it can be activated by tyrosine phosphorylation in the absence of DAG production (16). Phosphorylation of Tyr311 located in the hinge region of PKC{delta} is mediated by src (16) and induces a conformational change in the enzyme similar to the one induced by DAG binding and yields a lipid-independent kinase activity. Therefore, it is conceivable that hnRNP K serves as a scaffold that brings together src and PKC{delta}, as proposed by Schullery et al. (28). We propose the following model: ANG II promotes src-dependent tyrosine phosphorylation and activation of PKC{delta}, which then phosphorylates hnRNP K on Ser302, increasing its association with VEGF mRNA (Fig. 6). Studies are currently underway to verify this hypothesis.


Figure 6
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  		Fig. 6. Proposed model of ANG II regulation of hnRNP K and VEGF mRNA translation. ANG II activates src which, in turn, activates PKC{delta} by phosphorylation of Tyr311. Activated PKC{delta} phosphorylates hnRNP K on Ser302 and promotes its association with the 3'-untranslated region (UTR) of VEGF mRNA. This association increases the efficiency of VEGF mRNA translation induced by ANG II.

 
Other kinases have been shown to phosphorylate hnRNP K. For example, hnRNP K is phosphorylated by interleukin-1-responsive K-protein kinase, but the significance of these phosphorylation events is not known (3). hnRNP K is also phosphorylated by ERK, which leads to its redistribution from the nucleus to the cytoplasm (10). Whether PKC{delta}-mediated phosphorylation induces cytoplasmic accumulation of hnRNP K is not known.

We have previously shown that ANG II stimulated cap-dependent translation of VEGF mRNA, involving factors binding to the 5'-end of the VEGF mRNA, and that inhibition of cap-dependent translation markedly inhibited VEGF mRNA translation (5). These events were dependent on the generation of reactive oxygen species (7). Here we show that preventing association of hnRNP K with VEGF mRNA reduces VEGF protein expression by 50%. These data show that the primary regulation of VEGF mRNA translation occurs at the 5'-UTR and that events occurring at the 3'-UTR, e.g., binding of hnRNP K, may have a modulatory role in VEGF mRNA translation and hence VEGF synthesis.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the American Heart Association (D. Feliers), a National Institutes of Health (NIH) O'Brien Kidney Center Grant (B. S. Kasinath, D. Feliers), NIH Grants RO1 DK-55815 and DK-50190 (G. Ghosh-Choudhury), RO1-KD-45978 and GM-45134 (K. B), the American Diabetes Association (B. S. Kasinath), a Veterans Affairs (VA) Research Service Merit Review Grant (B. S. Kasinath and G. Ghosh-Choudhury), and the Juvenile Diabetes Research Foundation (D. Feliers/B. S. Kasinath). G. Ghosh-Choudhury is the recipient of a VA Research Scientist Award.


   ACKNOWLEDGMENTS
 
We thank Dr. E. Neilson for mouse proximal tubular epithelial cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Feliers, Dept. of Medicine/Nephrology, Mail Code 7882, UT Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX (e-mail: feliers{at}uthscsa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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