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Am J Physiol Cell Physiol 290: C352-C361, 2006. First published September 21, 2005; doi:10.1152/ajpcell.00050.2005
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VASCULAR BIOLOGY

Transcriptional and posttranscriptional regulation of angiopoietin-2 expression mediated by IGF and PDGF in vascular smooth muscle cells

Eric D. Phelps,1 Dawn L. Updike,1 Elizabeth C. Bullen,1 Paula Grammas,2 and Eric W. Howard1

1Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and 2Garrison Institute on Aging and Department of Neuropsychiatry, Texas Tech University Health Sciences Center, Lubbock, Texas

Submitted 7 February 2005 ; accepted in final form 14 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiopoietins play a significant role in vascular development and angiogenesis. Both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) bind the receptor tyrosine kinase Tie2. However, while Ang1 signaling results in the stabilization of vessel structure, Ang2 has been linked to vascular instability. The ratio of these two Tie2 ligands is thus critical for vascular stability and remodeling. This study identifies a mechanism of growth factor-mediated reduction in Ang2 expression in vascular smooth muscle cells (VSMCs). In response to PDGF, VSMCs downregulated Ang2 mRNA levels by 75% within 4 h, with a subsequent decrease in Ang2 protein levels. Quantitation of endogenous transcription rates revealed that PDGF stimulation did not alter Ang2 transcription rates, but instead induced a posttranscriptional mechanism of rapid Ang2 mRNA destabilization. The Ang2 mRNA half-life was reduced by at least 50% after PDGF treatment. The PDGF-induced mRNA turnover mechanism was dependent on several MAPK pathways, including ERK and JNK. In contrast, IGF-I, which did not significantly activate ERK or JNK, stimulated increased Ang2 expression through transcriptional activation. These findings demonstrate that VSMCs adjust Ang2 expression through multiple mechanisms, including changes in transcription as well as posttranscriptional mRNA destabilization.

platelet-derived growth factor; insulin-like growth factor

NORMAL AND DISEASE-RELATED tissue remodeling occurs concomitantly with changes to the local vasculature, without which tissue growth and viability would be severely limited. Vessel integrity is mediated by a balance between factors that favor localized vessel destabilization, a process that is critical for the vessel sprouting that occurs during angiogenesis, and factors that favor vessel stability. Angiopoietins regulate these processes by binding to the receptor tyrosine kinase Tie2 (34, 49). Tie2 was originally identified as a novel receptor tyrosine kinase whose expression was limited to endothelial cells (ECs) and their precursors (13); targeted disruption of Tie2 or its ligand, angiopoietin-1 (Ang1), resulted in the failure to develop a mature vasculature but did not inhibit EC differentiation (12, 47, 49). Ang1 is thought to stabilize vessel structure by stimulating EC recruitment of vascular smooth muscle cells (VSMCs) or pericytes, which synergize with ECs to produce a stable basement membrane; overexpression of Ang1 in transgenic mice resulted in stable, nonleaky vessels (50). Unlike the VEGFs, Ang1 does not induce EC proliferation, but instead promotes EC survival, chemotaxis, and tubular network stabilization (26, 42), and it can perform these functions in some cases even in the absence of mural cells (51). Angiopoietin-2 (Ang2) binds Tie2 with an affinity similar to that of Ang1 and thus competes with Ang1 for receptor binding (34). Interestingly, Ang2 inhibits Tie2 signaling in vascular ECs, and increased Ang2 expression leads to reduced microvascular stability (34). Ang2 is also thought to play a role in the regulation and remodeling of large vessels because of its expression in arteriole-associated VSMCs and has been demonstrated to activate Tie2 signaling during lymphangiogenesis (15). This finding suggests that Ang2 plays a much broader role in vascular development than originally thought.

Although the continual presence of Ang1 is necessary for EC survival and vessel stability, the highly regulated expression of Ang2 appears to drive new blood vessel formation. Increasing the Ang1-to-Ang2 ratio appears to favor vascular stability, whereas decreasing the ratio promotes angiogenesis (22, 57). Ang2 expression occurs at the leading front of neovascular sprouts, thereby contributing to the destabilized vessel structure necessary for EC migration (52). Ang2 acts synergistically with VEGF-A to promote angiogenesis (53, 57), and the simultaneous expression of Ang2 and VEGFR-2 (KDR/Flk1) occurs during tumor-mediated activation of the host vasculature (52). Ang2 induces vascular remodeling in the presence of VEGF but EC death in the absence of VEGF (32). However, overexpression of Ang2 resulted in poor vascular formation and inhibited tumor growth (58). Blocking Ang2’s association with Tie-2 in a mouse tumor model resulted in the cessation of tumor growth, as well as tumor regression in some animals (41). It was recently shown that blocking VEGF signaling in a tumor model caused a decrease in Ang2 expression and an increase in Ang1 expression, which then resulted in a transient increase in the number of patent, pericyte-coated vessels (55). In all of these cases, the ratio of Ang1 to Ang2 was found to be critical in determining the state of the tumor vasculature. An Ang2 imbalance has also been linked to nontumor vascular malformations (17) as well as to the capillary loss associated with glomerulonephritis (59). In vasculoproliferative diseases such as psoriasis, disease resolution is accompanied by a decrease in Ang2 levels (27). Thus regulating Ang2 levels is critical for the maintenance of vascular stability or the regulation of endothelial cell detachment as recently demonstrated in a three-dimensional culture model (48).

Although it is apparent that the role of Ang2 in vascular dynamics is critically important in both developmental and disease states, relatively little is known about the regulation of Ang2 expression. Ang1 expression was originally reported to be primarily constitutive (39), suggesting that changes in Ang2 expression may be an essential regulation point associated with angiogenesis and vessel stability. Factors such as hypoxia, VEGF, thrombin, TNF-{alpha}, and Sonic hedgehog increase Ang2 transcription rates in some cultured cells (21, 24, 35, 39, 45), whereas Ang1 and transforming growth factor-{beta}1 decrease Ang2 transcription. Herein we present evidence that the Ang1-Ang2 balance can be altered significantly by a posttranscriptional Ang2 mRNA decay mechanism induced by PDGF stimulation of VSMCs. We demonstrate that PDGF-stimulated VSMCs rapidly destabilize Ang2 mRNA, leading to a decreased Ang2 protein levels, and resulting in a significant change in the balance between Ang1 and Ang2. In contrast, stimulation with IGF-I resulted in a transcription-mediated increase in Ang2 mRNA. The different responses to these two mediators of VSMC function were traced to distinct signaling pathways and demonstrate the ability of cells to regulate the angiopoietin balance by a variety of mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and reagents. VSMCs derived from the heart microvasculature of Wistar-Kyoto (WKY) rats were cultured as previously described (4, 11, 16). All procedures involving animals were previously approved by the Institutional Animal Care and Use Committee. Cells were grown to confluence in DMEM (Life Technologies), 10% heat-inactivated FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B (Life Technologies). For all experimental data points, cells were washed once with serum-free medium, incubated overnight in serum-free medium supplemented with 0.2% lactalbumin hydrolysate (Life Technologies), and then stimulated in fresh serum-free medium plus 25 ng/ml recombinant rat PDGF-BB or 25 ng/ml IGF-I (R&D Systems). In some cases, cells were treated 30 min before growth factor stimulation with the following pharmacological inhibitors (in µM) 50 5,6-dichlorobenzimidazole 1-{beta}-D-ribofuranoside (DRB; Sigma), 10 U0126 (Calbiochem), 15 LY-294002 (Calbiochem), 5 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide (m-3M3FBS, a PLC activator; Calbiochem), 2 A23187 [GenBank] (Calbiochem), or 15 BAPTA-AM (Calbiochem). Soluble PDGF {beta}-receptor (1 µg/ml; R&D Systems) was also used to neutralize PDGF in the medium with PDGF-stimulated cells.

Semiquantitative and real-time RT-PCR. RNA was extracted using the guanidine thiocyanate method (7), modified as follows: chloroform-to-isoamyl alcohol ratio was reduced from 49:1 to 24:1, and an additional chloroform-to-isoamyl alcohol step was added before isopropanol precipitation. Total RNA was quantified using spectrophotometry and then reverse transcribed using the Superscript RT II RNase H– RT (Invitrogen) protocol plus a 30-min 52°C incubation before the inactivation step. cDNA was diluted 10-fold for subsequent PCR. Semiquantitative PCR was performed using 1 U of Taq polymerase (TaKaRa) and 5 µl of cDNA per 20-µl reaction. A cycle number within the linear range of amplification was chosen for semiquantitative analysis for each primer set (Integrated DNA Technologies). PCR products were separated using agarose gel electrophoresis and stained with ethidium bromide before we performed densitometric analysis (ChemiImager 4400; Alpha Innotech). For real-time analysis, 5 µl of cDNA were added per 50 µl of real-time PCR cocktail, including SYBR Green (Bio-Rad) and 1.25 U of Taq polymerase (Invitrogen). Amplification was performed in a Bio-Rad MyIQ thermal cycler, and the comparative cycle at threshold (Ct) method (20) was used to determine relative changes in mRNA levels of both Ang2 and the control gene tissue inhibitor of metalloproteinases (TIMP)-2. The relative ratios of Ang2 to TIMP-2 were plotted using Prism version 3.02 software (GraphPad). Oligonucleotide primer sequences used in semiquantitative PCR were as follows: Ang2 forward, AGAGTATTGGCTGGGCAACGAGTT; Ang2 reverse, TCCTTTGTGCTAAAATCACTTCCT; Ang1 forward, AAATTATACTCAGTGGCTGGAA; Ang1 reverse, TTCTAGGATTTTATGCTCTAATAA; VEGF-A forward, GTATATCTTCAAGCCGTCCTGTGT; VEGF-A reverse, CTTGCAACGCGAGTCTGTGTTTTT; VEGF-D reverse, CCTGAGAGAAAAGAGCCCCAATAA; VEGF-D forward, TATGAACACAAGCACCTCCTACAT; myosin heavy chain forward, GTAGAAGGTGCTGTCAAAGCCA; myosin heavy chain reverse, AAGGAACAAATGAAGCCTCGTT; TIMP-2 forward, CGCTGGACGTTGGAGGAAAGAAGG; TIMP-2 reverse, GGGTCCTCGATGTCAAGAAACTCC; c-fos forward, ATACGTCTTCCTTTGTCTTCACCT, and c-fos reverse, GGAGAAAGAGAAAAGAGACACAGA. Oligonucleotide primer sequences used in real-time PCR were as follows: Ang2 forward, CAGCCAACCAGGAAGTGATT; Ang2 reverse, AAGTTGGAAGGACCACATGC; TIMP-2 forward, AAGGAGATGGCAAGATGCAC; and TIMP-2 reverse, TGTAGCATGGGATCATAGGG.

Heteronuclear RNA quantitation. Oligonucleotides were designed within the rat Ang2 gene on the basis of sequence comparison with the human gene. The forward primer was complementary to the 3' end of exon 6 (ATGGACATGGGTGGAGGAGGGTGGAC), and the reverse primer was made complementary to the 5' end of exon 7 (AGTGCTCATACAGAGAGTGTGCCTCG). Intron 6 was amplified and sequenced, and another set of oligonucleotides (forward, GCTTCCACAGCATAAATGTCCCTAGGA; reverse, TACTCCATTGCCCTGCTCGGTACAAAT) was designed for amplification within intron 6 of the prespliced RNA as described previously (5, 60). To estimate the transcription rate of Ang2 in PDGF-stimulated, IGF-stimulated, or nonstimulated cultures, total RNA was isolated as described above and converted to cDNA using Moloney murine leukemia virus RT (Fisher) and random hexanucleotides. cDNA was amplified using a range of cycle numbers, and PCR products were resolved on 1.5% agarose gels. Gels were denatured in 1 mol/l NaCl and 0.5 mol/l NaOH twice for 15 min and then neutralized by being soaked twice for 15 min in 0.5 mol/l Tris and 1.5 mol/l NaCl, pH 7.5. The cDNA was then transferred onto MagnaGraph nylon membranes (MSI, Westborough, MA) through wicking in 10x SSC overnight. Membranes were cross linked with UV light at 120 mJ/cm2 and then hybridized with 0.5 µg of the amplified portion of intron 6 of the Ang2 gene (MiracleHyb; Stratagene), which was labeled with 50 µCi [32P]dCTP using a nick translation kit (Amersham Biosciences). To demonstrate that this methodology measured relative transcription rates accurately, we also amplified intron 1 of c-fos, an immediate early gene known to be activated rapidly and transiently by PDGF stimulation (33). The transcription rate of c-fos was evaluated in PDGF-stimulated and nonstimulated cells. Oligonucleotide primers were similarly designed at the intron-exon boundaries of intron 1: forward, CTACTACCATTCCCCAGCCGACTC; and reverse, CTCTACTTTGCCCCTTCTGCCGAT. Intron 1 was sequenced and used to design additional primers within the intron: forward, GCGCGGTCAGAGCAGCCTTAGCCT; and reverse, AGCGGAGGTGAGCGAGGAGGTTC.

Immunoprecipitation and Western blot analysis. Conditioned medium was collected from serum-starved VSMCs were treated with or without 25 ng/ml PDGF for 48 h. Recombinant mouse Tie2/Fc chimera (5 µg; R&D Systems) was added to the conditioned medium and rocked overnight at 4°C. Protein G slurry (30 µl, 50%; Upstate Biotechnology) was added to the conditioned medium and Tie2/Fc mixture and rocked at 4°C for 2 h. Protein G beads were pelleted using gentle centrifugation and washed six times in 1 ml of Nonidet P-40 (NP-40) wash buffer (150 mM NaCl, 1.0% NP-40, and 50 mM Tris·HCl, pH 8.0). SDS sample buffer plus 0.25 mM DTT were added to the pellet, which was boiled for 3 min before being separated using 10% SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes, and specific protein bands were detected using a primary antibody against Ang2 (SC-7015; Santa Cruz Biotechnology). Whole cell lysates were also collected from PDGF-, IGF-, or nontreated VSMCs after 10 min of stimulation using standard RIPA buffer plus protease and phosphatase inhibitors. Proteins were detected using primary antibodies specific to the phosphorylated and nonphosphorylated forms of Akt, ERK, JNK, PLC-{gamma}1 (Cell Signaling Technology), and {beta}-tubulin (Sigma-Aldrich). Proteins were visualized using chemiluminescence detection (EpiChem II imager; UVP) with secondary antibodies conjugated to alkaline phosphatase (Jackson ImmunoResearch).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ang2 mRNA levels are increased in IGF-I-stimulated VSMCs and decreased in PDGF-BB-stimulated VSMCs. VSMCs play a significant role in blood vessel dynamics and are known to express angiopoietins (10, 34, 35), but relatively little is known about angiopoietin regulation in these cells. We observed that VSMCs isolated from the rat heart microvasculature decreased Ang2 mRNA expression rapidly when stimulated with serum; however, under serum-free conditions, Ang2 expression was readily detectable using PCR amplification (Fig. 1A). This finding suggested the possibility that a factor in serum suppressed Ang2 expression. PDGF-BB, an important mitogen for VSMCs and a constituent of serum (18, 57), was sufficient to elicit a rapid decrease in Ang2 expression in serum-starved cells (Fig. 1B) but did not stimulate similar changes in the message levels of Ang1 or in other angiogenic mediators, such as VEGF-A or VEGF-D, within 8 h of stimulation (Fig. 1C). The expression of the VSMC marker smooth muscle-myosin heavy chain demonstrates the smooth muscle identity of our cells; in response to longer-term PDGF stimulation (>24 h), it and other SMC markers became significantly downregulated (data not shown). PDGF-induced Ang2 mRNA decreases were consistently demonstrated using both semiquantitative and real-time RT-PCR, with an estimated half-life of <3 h in the presence of PDGF compared with another secreted protein, TIMP-2, that is not regulated by PDGF (Fig. 1, B and C). Concomitant with Ang2 mRNA disappearance, Ang2 protein levels were significantly decreased in the medium conditioned by PDGF-stimulated VSMCs compared with nonstimulated cells (Fig. 1D). Together, these data demonstrate that PDGF-stimulated VSMCs can decrease Ang2 mRNA rapidly and specifically and consequently can decrease protein levels.



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  		Fig. 1. PDGF induces specific angiopoietin-2 (Ang2) downregulation at the mRNA and protein levels in vascular smooth muscle cells (VSMCs). A: serum-starved VSMCs were treated with 1% FBS and harvested at the times indicated. Semiquantitative PCR was used to compare changes in Ang2 and {beta}-actin levels after treatment. B: graph showing real-time PCR and semiquantitative PCR histograms obtained using RNA isolated from VSMCs treated with PDGF-BB (25 ng/ml) for the indicated times. Relative changes in Ang2 mRNA levels are compared with tissue inhibitor of metalloproteinases (TIMP)-2, a secreted protein that is not significantly regulated by PDGF stimulation in this model. C: semiquantitative RT-PCR was used to analyze the relative mRNA levels of several genes in the absence or presence of PDGF. D: VSMCs were treated with or without PDGF for 24 h, and Ang2 protein was detected in whole cell lysates (WCL) using Western blot analysis. Ang2 protein was immunoprecipitated (IP) from conditioned medium collected from PDGF-treated cells for 48 h using a soluble Tie2 receptor-immunoglobulin Fc chimera before Western blot analysis was performed.

 
Like PDGF-BB, IGF-I is an important mediator of VSMC phenotypic change during normal and disease processes (61). Interestingly, in response to IGF-I, our VSMC model demonstrated increased Ang2 mRNA levels (Fig. 2A). This increase was dose dependent, with increasing amounts of IGF-I stimulating increasing expression of Ang2 (Fig. 2B). Other angiogenic mediators, such as VEGF-A, VEGF-D, and Ang1, were not similarly upregulated (Fig. 2C).



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  		Fig. 2. IGF-I upregulates Ang2 levels in VSMCs. A: RNA from VSMCs treated with or without 25 ng/ml IGF-I for 12 or 24 h was evaluated by performing semiquantitative RT-PCR to detect changes in Ang2 and TIMP-2 mRNA levels. B: real-time PCR was performed to quantitate changes in Ang2 and TIMP-2 mRNA levels from VSMCs treated with increasing concentrations of IGF-I for 24 h. Triplicate experiments are represented here as a ratio of Ang2 to TIMP-2. C: semiquantitative RT-PCR was used to analyze the relative mRNA levels of several genes in the absence or presence of IGF.

 
PDGF stimulation does not alter Ang2 transcription rates, whereas IGF does. To begin to determine the mechanisms associated with growth factor-induced changes in Ang2 message levels, we assessed the endogenous transcription rate of the Ang2 gene directly by measuring the levels of nonspliced Ang2 message (heteronuclear RNA; hnRNA) in both stimulated and nonstimulated cells as previously described (5, 60). This method allowed us to measure endogenous Ang2 transcription accurately independently of transiently introduced promoter fragments and without the effects of posttranscriptional mRNA regulation. This approach consistently demonstrated that the relative rate of Ang2 transcription did not significantly change in response to PDGF stimulation as measured in four independent experiments for each time point (Fig. 3, A and B). To demonstrate that no significant contaminating genomic DNA was present in these samples, we used the same amplification conditions in samples that did not undergo reverse transcription (Fig. 3A, lanes 4, 8, and 12). Similar analysis of the c-fos gene, the transcription of which is known to increase significantly and transiently in response to growth factors (33, 57), demonstrated a rapid increase in c-fos hnRNA production followed by a decrease in expression by 60 min (Fig. 3C). This finding was consistent with the kinetics of c-fos mRNA steady-state levels (Fig. 3D). In contrast to PDGF, IGF-I-stimulated VSMCs showed increased Ang2 transcription rates by 12 h, with a return to nonstimulated levels by 24 h (Fig. 4). On the basis of these findings, we hypothesized that although IGF-I-mediated increases in Ang2 mRNA levels were likely due to increased transcription, the PDGF-dependent decrease in Ang2 mRNA levels was not due to decreased Ang2 transcription. This finding suggested a PDGF-mediated posttranscriptional level of message regulation.



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  		Fig. 3. PDGF stimulation does not alter Ang2 transcription rates. A: serum-starved VSMCs were incubated in serum-free medium alone or with 25 ng/ml PDGF-BB, and RNA was harvested at 4 or 8 h poststimulation. A portion of intron 6 within Ang2 heteronuclear RNA (hnRNA) was then amplified using PCR and detected by performing Southern blot analysis to quantitate PDGF-induced changes in the Ang2 transcription rate; samples that were not reverse transcribed (RT–) were included to control for the presence of contaminating genomic DNA. B: autoradiograms from 4 separate experiments were quantified using densitometry. C: to further validate the methodology described above, VSMCs treated with 25 ng/ml PDGF were similarly used to quantify the induction of c-fos transcription, which was previously shown to be induced rapidly and transiently by growth factor stimulation. D: steady-state c-fos mRNA levels were evaluated using semiquantitative RT-PCR.

 


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  		Fig. 4. IGF transcriptionally upregulates Ang2. A: VSMCs were treated with or without 25 ng/ml IGF-I for 12 or 24 h, and RNA was reverse transcribed using random hexamers. The same portion of intron 6 of Ang2 hnRNA shown in Fig. 2 was amplified by performing PCR and detected using Southern blot analysis to evaluate changes in Ang2 hnRNA transcription rate in response to IGF. Samples that were not reverse transcribed (RT–) were included to control for the presence of contaminating genomic DNA. B: autoradiograms obtained from 4 separate experiments were quantified using densitometry.

 
PDGF stimulation leads to decreased stability of Ang2 mRNA. On the basis of our findings that the rapid, PDGF-mediated decrease in Ang2 mRNA levels was not a result of decreased Ang2 transcription, we considered the possibility that changes in mRNA stability might play a role in this regulation. To measure the mRNA decay rate of Ang2 in the absence of new transcription contributing to the existing pool of Ang2 mRNA, we incubated VSMCs with a pharmacological transcription inhibitor, DRB, and estimated an Ang2 mRNA half-life of 6.1 h (Fig. 5). In contrast, PDGF stimulation decreased the half-life to 3.0 h, suggesting that PDGF stimulated a twofold increase in Ang2 mRNA instability, even in the presence of continuous Ang2 transcription. Incubation of cells with both PDGF and DRB unexpectedly resulted in the same Ang2 mRNA half-life as DRB treatment alone, suggesting that inhibiting transcription blocked the ability of PDGF to stimulate rapid Ang2 turnover, perhaps by preventing the expression of an essential component of the turnover mechanism or through the direct inhibition by DRB of factors involved in mRNA turnover. Similar results were obtained using another transcription inhibitor, actinomycin D (data not shown).



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  		Fig. 5. PDGF decreases the half-life of endogenous Ang2 mRNA in VSMCs. A: serum-starved VSMCs were treated with PDGF (25 ng/ml), 5,6-dichlorobenzimidazole 1-{beta}-D-ribofuranoside (DRB; 50 µM), or both. RNA was analyzed using semiquantitative RT-PCR. B: densitometric quantification of ethidium bromide-stained PCR product intensities was performed in 7 separate experiments to evaluate PDGF-induced acceleration of Ang2 mRNA decay. Slopes within the linear range of mRNA decay (between 1 and 4 h post-PDGF addition) were determined by performing linear regression analysis. Note that PDGF accelerated Ang2 mRNA turnover compared with pharmacological (DRB) inhibition of transcription alone. This effect was also observed when transcription was inhibited using actinomycin D (data not shown). t, half-life.

 
Increased transcription of Ang2 message in response to IGF-I is dependent on the PI3K signaling pathway. We next sought to determine the critical differences in signal transduction pathways that led to such divergent effects by VSMCs stimulated with PDGF-BB vs. IGF-I. In particular, PDGF-BB was able to stimulate Akt, ERK, and PLC-{gamma} effectively, whereas IGF-I only transiently activated Akt and ERK and failed to activate PLC-{gamma} (Fig. 6). On the basis of these findings, we investigated the role of phosphatidylinositol 3-kinase (PI3K)/Akt activation in Ang2 expression and found that the specific PI3K inhibitor LY-294002 was able to inhibit IGF-I-mediated Ang2 mRNA upregulation (Fig. 7A). Blocking PI3K did not affect PDGF-mediated Ang2 downregulation (Fig. 7B), consistent with the idea that signaling pathways associated with Ang2 transcriptional upregulation are distinct from growth factor-induced message destabilization. These results link Ang2 upregulation with PI3K/Akt activity and Ang2 message destabilization through pathways other than PI3K/Akt.



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  		Fig. 6. IGF and PDGF have distinct signal transduction characteristics in VSMCs. A and B: serum-starved VSMCs were treated with 25 ng/ml IGF (A) or 25 ng/ml PDGF (B), and cell lysates were harvested at the indicated times. Western blot analysis was used to detect changes in phosphorylation of Akt or ERK, and non-phospho-ERK was used to demonstrate equal loading. Note the differences in the duration of phosphorylation between IGF and PDGF. C: phospho-PLC-{gamma}1 was detected in lysates from VSMCs treated with PDGF or IGF for 10 min. {beta}-tubulin was used as a loading control.

 


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  		Fig. 7. IGF-I upregulates Ang2 via the phosphatidylinositol 3-kinase (PI3K) pathway. A and B: serum-starved VSMCs were pretreated with LY-294002 (15 µM) for 30 min before IGF-I (A) or PDGF-BB stimulation (B). RNA was harvested for RT-PCR 6 h after growth factor addition, and Ang2 or TIMP-2 mRNA levels were detected. Phosphorylated or nonphosphorylated forms of Akt were detected using Western blot analysis from whole cell lysates collected after 10 min of growth factor stimulation (bottom row).

 
PDGF-mediated MAPK and PLC-{gamma} activation results in Ang2 mRNA decay. PDGF is known to activate many signal transduction pathways through the PDGF {beta}-receptor, including Ras/ERK, PI3K, and PLC-{gamma} pathways (19), and it did so in our cell model. To identify PDGF-induced signaling pathways that are responsible for Ang2 mRNA turnover, we treated cells with pharmacological inhibitors or activators specific to individual signaling pathways. U0126, an ERK/MAPK pathway inhibitor, was able to block ERK phosphorylation completely (Fig. 8A, bottom), but alone it was unable to inhibit PDGF-induced Ang2 mRNA turnover (Fig. 8A, top). Similarly, SP-600125, an inhibitor of several pathways, particularly JNK, alone was unable to block Ang2 turnover (Fig. 8B). However, VSMCs stimulated with PDGF-BB in the presence of both inhibitors were unable to downregulate Ang2 message levels (Fig. 8C). We next stimulated cells with the PLC activator m-3M3FBS (Fig. 9A). This compound was able to induce the same rapid Ang2 mRNA turnover as PDGF alone, yielding a similar decay rate. Because PLC-{gamma}, which is activated by the PDGF receptor, is known to activate the mobilization of intracellular Ca2+ from internal stores (2), we investigated the potential role of Ca2+ signaling in the regulation of Ang2 mRNA stability. Cells treated with A23187 [GenBank] , a Ca2+ ionophore that increases the concentration of intracellular Ca2+, stimulated the rapid reduction in Ang2 mRNA levels similar to those observed using the PLC activator or PDGF (Fig. 9B). To demonstrate a direct link between PDGF, Ca2+ signaling, and Ang2 mRNA turnover, we pretreated cells with the cell-permeable Ca2+ chelator BAPTA-AM to inhibit intracellular Ca2+ signaling before the addition of PDGF or the PLC activator (Fig. 9C). In both cases, inhibition of Ca2+ signaling was sufficient to block either PLC- or PDGF-mediated Ang2 turnover. We also attempted to block PDGF-mediated PLC-{gamma} signaling with the PLC-{gamma} inhibitor U73122 [GenBank] . Interestingly, the addition of U73122 [GenBank] downregulated Ang2 message in the absence of PDGF (data not shown). It was previously shown that this PLC inhibitor can activate of Ca2+ signaling directly in some systems (3, 38), and this fact precluded its use in our cells. Together, these data suggest that the inability of IGF-I to stimulate MAPK and PLC-{gamma} pathways profoundly alters its effect on Ang2 expression.



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  		Fig. 8. PDGF-mediated Ang2 turnover is signaled through JNK and ERK. A: serum-starved VSMCs were pretreated with U0126 (10 µM) for 30 min before PDGF-BB stimulation (25 ng/ml). RNA was harvested for RT-PCR 6 h after PDGF addition; whole cell lysates were harvested after 10 min of PDGF stimulation in parallel cultures. Ang2 and TIMP-2 mRNA levels were analyzed using semiquantitative RT-PCR (top two images), and Western blot analysis was performed to detect phosphorylated or nonphosphorylated forms of ERK (bottom two images). B: VSMCs were pretreated with SP-600125 (25 µM), with or without PDGF. Treated cells were harvested after 6 h of treatment, and Ang2 and TIMP-2 mRNA levels were evaluated by performing semiquantitative RT-PCR (top two images). Western blot analysis was performed in whole cell lysates obtained after 10 min of PDGF treatment to detect phosphorylated forms of JNK and ERK (bottom two images). Note that the JNK inhibitor did not inhibit PDGF-mediated ERK phosphorylation. C: VSMCs were pretreated with U0126 and/or SP-600125 before PDGF treatment and then harvested 6 h later. Changes in Ang2 or TIMP-2 mRNA levels were assayed using semiquantitative RT-PCR.

 


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  		Fig. 9. PDGF induces Ang2 mRNA turnover through PLC-mediated changes in Ca2+ signaling. Serum-starved VSMCs were treated with the PLC-specific activator 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide (m-3M3FBS; 5 µM) (A) or the Ca2+ ionophore A23187 [GenBank] (2 µM) (B). RNA was harvested at the indicated times, and RT-PCR was used to analyze changes in steady-state Ang2 and TIMP-2 mRNA levels. C: serum-starved VSMCs were pretreated with the Ca2+ chelator BAPTA-AM 30 min before addition of the PLC activator or PDGF. RNA was harvested after 6 h of treatment, and RT-PCR was performed to determine changes in Ang2 and TIMP-2 mRNA levels.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Using a VSMC culture model, we have determined that IGF-I, through its ability to activate PI3K, could induce an increased level of Ang2 by stimulating a transient increase in Ang2 transcription rates. In contrast, PDGF-BB, although it similarly stimulated the PI3K pathway, induced Ang2 message instability because of its ability to activate several pathways not activated by IGF-I in this cell model. These results suggest that Ang2, which by necessity is likely to be highly regulated at multiple levels, is subject to both transcriptional and posttranscriptional mechanisms of regulation, depending on the intracellular signaling context.

PDGF stimulation could induce a rapid decrease in the stability of Ang2 mRNA, leading to a loss of Ang2 protein but without an apparent change in Ang2 transcription rates. Previous studies demonstrated an Ang2 mRNA half-life of ~3–4 h in bovine retinal ECs with or without VEGF stimulation (39). Hypoxia has been shown to increase this half-life to almost 5 h (44) and to increase Ang2 transcription. One important feature that distinguishes our culture models is our ability to perform these experiments in serum-free medium. Previous studies in which researchers reported relatively short Ang2 mRNA half-lives were performed in the presence of serum (39) or with relatively brief periods of serum starvation (21), which may accelerate Ang2 mRNA decay. We have found that stimulating our VSMC model with serum led to a rapid loss of Ang2 mRNA. Interestingly, Ang2 mRNA was downregulated rapidly in hemangioma-derived ECs in response to serum, but this effect was not induced by PDGF (58). We have found evidence that other mediators of VSMC function, including thrombin, also stimulate Ang2 message turnover (Phelps E and Howard E, unpublished observations). We think that VSMCs rapidly downregulate Ang2 expression in response to conditions that mimic vascular injury, including exposure to serum factors. Additional studies are needed to confirm this hypothesis.

Through the use of the transcription inhibitor DRB, we calculated the endogenous half-life of Ang2 mRNA to be ~6 h. PDGF addition, in the absence of the transcription inhibitor, accelerated this rate of decay to yield a half-life of only 3 h. To our surprise, the addition of both PDGF and DRB resulted in the same half-life determined using DRB alone. These observations have several possible explanations. First, Ang2 message destabilization may require the presence of one or more newly transcribed factors that PDGF stimulation induces; hence the ability of transcription inhibitors to block the process. Second, transcription inhibitors have been demonstrated to interfere selectively with the degradation of one type of AU-rich element-containing mRNA vs. another (43). In addition, different groups have reported different degradation characteristics of the same mRNA in different cell types (6, 28). Because transcription inhibition actually blocked PDGF-mediated Ang2 mRNA turnover, we were unable to quantitate the increased rate of Ang2 turnover accurately in PDGF stimulated cells. Our estimate of an Ang2 half-life in the presence of PDGF is likely to be too high, given that Ang2 transcription remained constant under these conditions (Fig. 2). Despite the complications inherent in using DRB or actinomycin D, we conclude on the basis of the present study that PDGF induces a reduction in Ang2 mRNA stability compared with nonstimulated cells, resulting in dynamic changes in Ang2 expression.

The results presented herein suggest that VSMCs have the ability to regulate Ang2 levels precisely through transcriptional and posttranscriptional mechanisms that are highly responsive to extracellular factors. In the case of transcriptional upregulation, we have identified the PI3K pathway as an important component in growth factor-mediated increases in Ang2 expression. We are currently investigating the mechanisms involved in increased Ang2 transcription; however, we can conclude that this process is completely distinct from growth factor-induced Ang2 message destabilization. This destabilization appears to involve multiple pathways, at least in the response of VSMCs to PDGF-BB. Our results suggest the involvement of multiple MAPK pathways. Similarly, we have linked Ca2+-mediated signaling pathways to the destabilization of Ang2 mRNA. We base this finding on the ability of the Ca2+ ionophore A23187 [GenBank] to induce Ang2 mRNA turnover rapidly and on the ability of the intracellular Ca2+ chelator BAPTA-AM to block PDGF-mediated turnover. Our findings are consistent with the involvement of PLC-{gamma} on the basis of the ability of a synthetic PLC activator to induce turnover, as well as on the ability of PDGF, but not IGF-I, to induce turnover. A23187 [GenBank] is known to induce the stabilization of AU-rich mRNA through a mechanism independent of transcription or translation (25); in our VSMCs, we also observed A23187 [GenBank] -mediated message stabilization of two different classes of AU-rich mRNA (data not shown). Proteins required for the dynamic regulation of AU-rich mRNA also have been demonstrated to be upregulated in cells treated with A23187 [GenBank] (46). Ca2+-induced stabilization of AU-rich mRNA has been linked to multiple signaling pathways (56), but it is not yet clear how Ca2+ signaling leads to the instability of Ang2 mRNA in our model system. It is possible that growth factor-mediated Ca2+ release leads to the activation of MAPK pathways that then initiate the Ang2 message destabilization mechanism; in fact, Ca2+/CaM signaling has been linked to both JNK and ERK activation and may provide an explanation of the ability of Ca2+ influx into cells to stimulate Ang2 message destabilization (9, 14).

Several instances in which Ang2 expression significantly decreased over time in both in vitro and in vivo models have been documented. In several cases of experimentally induced ischemia, Ang2 levels were transiently upregulated and then decreased significantly over time (1, 36, 37). During normal excisional wound healing, Ang2 was shown similarly to spike and then decline (23, 54). In cultured cells, previous studies demonstrated an increase in Ang2 mRNA in response to TNF-{alpha} within 6 h, with a rapid decrease to baseline within the next 4 h (24). Similarly, serum caused decreased Ang2 mRNA levels in hemangioma-derived ECs within 8 h (58). We postulate that regulated message stability plays a role in this regulation, and we have found that multiple extracellular signals lead to what we call the Ang2 instability phenotype. The observation that PDGF activation of VSMCs leads to decreases in Ang2 levels may, in fact, have relevance to vascular homeostasis. The importance of PDGF in vascular development has been well documented (19, 29, 31, 40). The PDGF {beta}-receptor is of critical importance for initiating VSMC migration during the stabilization of primitive EC tubes. Lack of a functional PDGF {beta}-receptor in the developing retina resulted in a vasculature that was completely deficient in pericytes and VSMCs, thereby resulting in microvascular leakage (51). Interestingly, addition of recombinant, modified Ang1 that was not subject to Ang2 inhibition increased EC integrity in the absence of pericytes or VSMCs and prevented vascular hemorrhage (51). This finding suggests that the leakiness of the retinal microvasculature may be an indirect result of PDGF {beta}-receptor inhibition and that PDGF-mediated changes in angiopoietins are critical for normal development. Similarly, PDGF-deficient mice have been shown to die as a result of vessel leakiness and a lack of pericytes, particularly in the kidney (29, 31). Our results suggest that without PDGF {beta}-receptor function, VSMCs may not normally turn over Ang2 message. The resultant increased expression of Ang2 mRNA shifts the Ang1-to-Ang2 ratio and favor Tie2 inhibition. Without Ang1-dependent Tie2 signaling, VSMCs or pericytes are not recruited to stabilize the primary vasculature, thereby resulting in the microvascular leakage and immature vascular structure reported previously (51).

A number of important genes associated with vascular remodeling and contractility are regulated to a significant degree by altered message stability. For example, VEGF-A message is stabilized in response to hypoxia by factors that bind to a sequence in its 3'-UTR (30). Similarly, the mRNA of several VSMC contractile proteins are controlled in part by mRNA turnover; for example, smooth muscle {alpha}-actin mRNA levels sharply decrease upon PDGF-induced activation through mechanisms that may include decreased message stability (8). Because Ang2 levels play a critical role in vessel stability and angiogenesis, it is not surprising that its regulation is tightly controlled. A constitutive but low level of Ang2 transcription, coupled with the ability to control mRNA stability and thus protein synthesis, would yield a rapid and flexible mechanism to control Ang2 expression precisely. We predict that this mechanism is a primary tool used by vascular associated cells to adjust the levels of Ang2 rapidly.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Oklahoma Center for the Advancement of Science and Technology Grant HN5-030, National Institutes of Health Grants HL-62268, GM-63638, and AG-020569, and American Heart Association Predoctoral Fellowship 0215187Z.


   ACKNOWLEDGMENTS
 
We thank Brian Ceresa and George Risinger for critical evaluation and helpful suggestions during the preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. W. Howard, Dept. of Cell Biology, BMSB 566, Univ. of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104 (e-mail: eric-howard{at}ouhsc.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.


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