A Novel Class of Vascular Endothelial Growth Factor-responsive Genes That Require Forkhead Activity for Expression*

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-κB inhibitor, constitutively active Ad-IκB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-B inhibitor, constitutively active Ad-IB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.
The mammalian members of the winged helix, or forkhead, transcription factors include FKHR (Foxo1), FKHRL1 (Foxo3a), and AFX (Foxo4). Forkhead transcriptional activity is coupled to three principal functions: growth inhibition, apoptosis, and regulation of metabolism. In non-endothelial cells, forkhead-mediated effects on cell growth and survival have been shown to be governed by cell type-specific induction of cell-cycle arrest and/or pro-apoptotic genes, including kinase inhibitor protein p27 (p27 kip1 ), growth arrest and DNA damage 45A (GADD45A), B-cell translocation gene 1 (BTG1), Fas ligand, and pro-apoptotic Bcl-2 interacting member (20 -23). Forkhead effects on metabolism are mediated by a distinct set of target genes involved in energy metabolism and stress resistance, including glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, insulin-like growth factor-1-binding protein-1, manganese superoxide dismutase (Mn-SOD), and catalase (24 -29). Studies in non-ECs suggest that agonists involved in promoting cell growth, survival, or metabolism, including growth factors, cytokines, and hormones, result in PI3K-Aktdependent phosphorylation, nuclear exclusion, and inactivation of forkhead (30 -36). According to this model, growth factor-mediated inactivation of forkhead and secondary inhibition of the forkhead-responsive genes is a prerequisite for cell proliferation and metabolism.
Consistent with this paradigm, we have recently demonstrated that VEGF signaling in ECs results in PI3K-Aktdependent phosphorylation and nuclear exclusion of forkhead transcription factors, with subsequent down-regulation of the forkhead target gene, p27 kip1 , increased cell proliferation, and decreased apoptosis (19).
In contrast to non-ECs, where insulin suppresses Mn-SOD levels via a PI3K-Akt-forkhead-dependent signaling pathway (27), VEGF was shown to induce expression of Mn-SOD in ECs (37). A positive role for forkhead in this pathway was evidenced by the observation that VEGF-mediated induction of Mn-SOD was enhanced by inhibition of PI3K or Akt or by overexpression of TM-FKHRL1 (38).
According to the classic view of forkhead as a pro-apoptotic and cell-cycle arrest gene, its deletion in mice might predict for increased cell survival and growth. However, mice that are null for FKHR demonstrate impaired vascular development and embryonic lethality (39,40). Moreover, ECs derived from FKHR Ϫ/Ϫ embryonic stem cells demonstrated an abnormal morphological response to VEGF (40). Finally, siRNA-mediated inhibition of FKHR in serum-treated human umbilical vein endothelial cells resulted in altered expression of angiogenesis-related genes (41). Together with our findings that forkhead proteins are involved in mediating VEGF induction of the antioxidant Mn-SOD (38), these data suggest that the VEGF-forkhead signaling axis plays an important role in vascular biology, over and above its ability to inhibit pro-apoptotic/ cell-cycle arrest genes. Here, we provide evidence for the existence of a subset of VEGF-responsive genes whose expression is both dependent on and modulated by forkhead activity in ECs.
Adenoviruses-HCAECs were transduced with replicationdeficient adenoviruses encoding the cDNAs of ␤-galactosidase (Adv), wild-type (WT)-FKHR, and triple mutant (TM)-FKHR as previously described (42). The triple mutant version of FKHR contains T24A, S256A, and S319A and is resistant to agonistinduced phosphorylation. Adenovirus expressing constitu-tively active IB was a generous gift from Chritiane Ferran of Beth Israel Deaconess Medical Center.
siRNA-mediated Inhibition of Endogenous FKHR-HCAECs were grown to 70 -80% confluency in 6-cm plates and transfected with siRNA against one of the following FKHR target sequences: siFKHR1, ATG GAG GTA TGA GTC AGT ATA, and siFKHR2, CAG CGC CGA CTT CAT GAG CAA (Qiagen, Valencia, CA) in Opti-MEM containing Lipofectin (10 g/ml) for 4 h. The cells were then incubated in EGM-2 medium for 24 h and serum-starved in 0.5% serum for 12-16 h before VEGF treatment for the times indicated.
Quantitative Real-time PCR-Total RNA was prepared using the RNeasy RNA extraction kit with DNaseI treatment following the manufacturer's protocol (Qiagen). To generate cDNA, total RNA (100 ng) from each of triplicate samples was mixed and converted into cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). All cDNA samples were aliquoted and stored at Ϫ80°C. Primers were designed using the Primer Express oligonucleotide design software (Applied Biosystems, Foster City, CA) and synthesized by Integrated DNA Technologies (Coralville, IA). All primer sets were subjected to rigorous data base searches to identify potential conflicting transcript matches to pseudogenes or homologous domains within related genes. Amplicons generated from the primer set were analyzed for melting point temperatures using the first derivative primer melting curve software supplied by Applied Biosystems. The sequences of the real-time PCR primers used in this study are listed in Table 1. The SYBR Green I assay and the ABI Prism 7700 Sequence Detection System were used for detecting real-time PCR products from the reverse-transcribed cDNA samples, as previously described (43). 18 S rRNA, which exhibits a constant expression level across all the HCAECs samples, was used as the normalizer. PCR reactions for each sample were performed in duplicate and copy numbers were measured as described previously (43). The level of target gene expression was normalized against the 18 S rRNA expression in each sample, and the data presented as mRNA copies per 10 6 18 S rRNA copies.
Western and Northern Blot Analyses-ECs were harvested for total protein, and Western blots were carried out as previously described (19). The phosphospecific antibodies against Ser-256 FKHR and Ser-473 Akt were purchased from Cell Signaling (Beverly, MA), and anti-␤-actin was from Sigma. Anti-Akt and anti-FKHR antibodies were obtained from Cell Signaling. Anti-HA antibody was from BD Biosciences. RNA extraction and Northern blot assays were performed as described previously (37).
Immunolocalization Studies-These studies were carried out in HCAECs grown onto four-well chamber slides (Lab-Tek, Christchurch, New Zealand) and treated with or without VEGF for the times indicated. Subcellular localization of FKHR was determined using a primary anti-FKHR antibody and a Cy3conjugated secondary antibody as described previously (19). Quantitative analyses were carried out by counting 200 cells per time point.
Complement-mediated Cell Lysis Assay-HCAECs were plated in 24-well plates and incubated at 37°C for 3 h prior to adenoviral transduction or 24 h prior to siRNA transfection.
The cells were serum-starved overnight and incubated with or without VEGF for 24 h. Calcein acetoxymethyl ester (7 L, Molecular Probes, Leiden, The Netherlands) were added to HCAECs monolayer for 30 min. Following washing with serum-starvation medium containing 1% BSA, 250 l of monoclonal anti-endoglin (CD105) antibody (IgG2a) was added to opsonize HCAECs for 30 min. HCAECs were washed with Hanks' balanced salt solution containing 1% BSA and 250 l of 5% to 20% baby rabbit complement (Serotec, Oxford, UK) was added. HCAECs were then incubated at 37°C for 30 min. The supernatant from each well was transferred to a 96-well microtiter plate. The remaining HCAECs in the 24-well plate were washed with Hanks' balanced salt solution plus 1% BSA, and the calcein remaining in the cells was released by incubation with 250 l of Hanks' balanced salt solution containing 1% BSA and 0.1% Triton X-100 (Sigma). The lysate was then transferred to another 96-well plate, the calcein was released by complement, and detergent was estimated using a fluorescence plate reader (model 680, Bio-Rad). Percent specific lysis in triplicate wells was calculated as [(complement-mediated release Ϫ spontaneous release)/(maximal release Ϫ spontaneous release)] ϫ 100%, where maximal release equals complement-mediated release plus detergent-mediated release.
Monocyte Adhesion Assay-HCAECs transduced with adenovirus (m.o.i. ϭ 5) or transfected with 75 nM siRNAs were seeded on 24-well tissue culture plate. After grown to confluence, HCAECs were serum-starved in EBM-2 containing 0.5% fetal bovine serum for 18 h, and treated with 50 ng/ml VEGF for 6 h. U937 cells were labeled with red fluorescent dye using PKH-26 staining kit (Sigma), which were added to the HCAECs-seeded 24-well plate with 3 ϫ 10 5 cells/well. 90 min later, cells were washed twice with Hank's buffered saline (Invitrogen), and examined by fluorescence microscopy. The adhesion intensities were measured with MetaMorph (Molecular Devices Corp., Sunnyvale, CA) and cell image analyzer (Kurabo, Tokyo, Japan).

Modulation of Intracellular Levels of FKHR in Primary ECs
by Adenovirus and siRNA-To determine the role of forkhead in mediating VEGF signaling in ECs, HCAECs were transduced with adenoviruses (Ad) overexpressing ␤-galactosidase (Adv), wild-type (WT)-FKHR, or constitutively active, phosphorylation-resistant triple mutant (T24A,S256A,S315A) TM-FKHR. Alternatively, HCAECs were transfected with siRNA against FKHR to reduce endogenous FKHR levels. As shown in Fig.  1A, WT-FKHR but not TM-FKHR resulted in a significant increase in phospho-FKHR in untreated HCAECs. Both TM-FKHR and WT-FKHR (each of which carries an HA tag) resulted in detectable HA protein levels after 24 and 48 h of adenoviral transduction. On the contrary, FKHR siRNA resulted in significant reduction of FKHR protein expression in HCAECs, with siFKHR2 demonstrating the greatest effect (Ͼ85% reduction) (Fig. 1B). Thus, Ad and siRNA are effective in increasing and decreasing intracellular FKHR levels in ECs, respectively.
Forkhead Differentially Modulates VEGF-responsive Genes in Primary ECs-We initially employed DNA microarrays to assay for the effect of PI3K inhibition and/or TM-FKHRL1 on VEGF-mediated gene expression in HCAECs. We used these data as a guide to identify two classes of genes: 1) classic VEGFrepressible, forkhead-responsive transcripts, and 2) novel VEGF-inducible, forkhead-responsive genes. Recognizing the primary importance of FKHR in ECs (39,40), and assuming some degree of redundancy between FKHRL1 and FKHR isoforms, we chose to confirm the existence of these distinct classes downstream of the VEGF-FKHR signaling axis. HCAECs were transduced with Adv-or TM-FKHR, or transfected with FKHR siRNA, treated in the absence or presence of VEGF and then assayed for candidate genes by reverse transcription-PCR. Table 1 shows the list of the primers used for the genes that were tested by reverse transcription-PCR.
In keeping with the results of our DNA microarray studies, two distinct patterns of response were noted ( Table 2). One group of genes, including BTG-1, GADD45A, and p27 kip1 , demonstrated the usual forkhead response pattern observed in non-ECs, namely agonist (VEGF)-mediated inhibition of expression, TM-FKHR-mediated induction of mRNA, and FKHR siRNA-mediated repression of basal mRNA levels.
A second group of genes was induced both by VEGF and TM-FKHR and super-induced by VEGF plus TM-FKHR (Table  2). VEGF-mediated induction of this novel group was blocked by FKHR siRNA, suggesting that VEGF requires FKHR for their optimal expression. These genes include bone morphogenic protein 2 (BMP2), cbp/p300-interacting transactivator 2 (CITED2), decay accelerating factor (DAF), vascular cell adhesion molecule (VCAM-1), manganese superoxide dismutase (Mn-SOD), endothelial specific molecule-1 (ESM-1, also known as endocan), RING1 and YY1 binding protein, matrix metalloproteinase-10 (MMP-10), and MGC5618. For purposes of discussion, we will refer to the first (classic) group as Class I, and the second (novel) group as Class II.
VEGF Inhibits Expression of Class I Genes in Primary ECs via an FKHR-dependent Mechanism-To further characterize the Class I genes, we focused on the expression patterns of GADD45A and BTG1 in ECs. HCAECs were pretreated in the absence or presence of the PI3K inhibitor, LY294002, or transduced with adenovirus overexpressing ␤-galactosidase (Adv), constitutively active phosphorylation-resistant triple mutant FKHR (TM-FKHR) or wild-type FKHR, and then treated with VEGF or vehicle for varying time points. Cells were harvested for RNA and assayed for gene expression using Northern blot analyses. VEGF resulted in a decrease in the expression of GADD45A and BTG1 (Fig. 2, A and B). We have previously shown that the PI3K inhibitor, LY294002, inhibits VEGF-mediated phosphorylation, nuclear exclusion, and inactivation of forkhead proteins in ECs (19). Preincubation with LY294002 resulted in increased basal levels of GADD45A and BTG1 and blocked the inhibitory effect of VEGF on these genes (Fig. 2, A  and B). TM-FKHR increased GADD45A and BTG1 mRNA levels (Fig. 2, C and D), suggesting that VEGF-mediated downregulation of Class I genes occurs through phosphorylation and nuclear exclusion of FKHR. These data are consistent with those previously reported for p27 kip1 (19) and suggest that one role for the VEGF-FKHR signaling axis is to reduce the expression of pro-apoptotic and/or cell-cycle inhibitory genes.

TABLE 2 Induction (-fold) or reduction of the genes in endothelial cells by RT-PCR analyses
The basal levels of expression for each gene in unstimulated, serum-starved HCAEC were arbitrarily considered as 1 (-fold) per 10 6 18 S mRNA copies. Numbers are expressed as -fold induction (or reduction if less than 1) over the basal levels.

VEGF-mediated Induction of Class II Genes in Primary ECs
Depends on FKHR-To further characterize the Class II genes, we focused on the expression patterns of Mn-SOD, VCAM-1, ESM-1, MMP-10, BMP2, and CITED2 in HCAECs. In Northern blot analyses, VEGF increased the mRNA levels for each of these genes (Fig. 3). TM-FKHR alone increased expression of Mn-SOD, ESM-1, MMP10, and CITED2; and together with VEGF resulted in super-induction of Mn-SOD, ESM-1, MMP10, BMP2, VCAM-1, and CITED2 (Fig. 3, A and B). VEGF-mediated induction of all the above genes, Mn-SOD, VCAM-1, ESM-1, MMP10, BMP2, and CITED2, was significantly attenuated by FKHR siRNA (Fig. 3, C and D). These findings support a role for FKHR as a positively acting transcription factor required for transduction of the VEGF signal.
VEGF-mediated Protection against Complement-mediated Lysis of ECs Is Dependent on FKHR-Expression of DAF, a membrane-bound complement regulatory protein, is induced by VEGF in ECs and provides protection from complementmediated injury/lysis of ECs (44). As shown in Table 2, VEGFmediated increase in DAF expression is dependent on FKHR ( Table 2). We next wished to determine the functional relevance of these findings. In control or VEGF-treated ECs, overexpression of TM-FKHR resulted in decreased complementmediated cell lysis (Fig. 4). In contrast, siRNA against FKHR blocked the protective effect of VEGF against cell lysis (Fig. 4). Together, these findings support an important role for FKHR in VEGF-mediated, DAF-dependent protection of ECs against complement.
VEGF-mediated Monocyte Adhesion to ECs Is Dependent on FKHR-VCAM-1, a member of the immunoglobulin superfamily of genes, promotes the adhesion of monocyte to ECs. As shown in Fig. 5, VEGF resulted in more than 3-fold induction of U937 adhesion on endothelial monolayer in vitro. As shown for VCAM-1 expression (Table 2 and Fig. 3), VEGF-induced monocyte adhesion to HCAECs was further enhanced by overexpression of a constitutively active TM-FKHR (Fig. 5A). In contrast, a reduction in the level of FKHR by siFKHR in HCAECs resulted in a decrease in the VEGF-induced monocyte adhesion (Fig. 5B), suggesting an important role for FKHR in this process.

VEGF-mediated Induction of Class II Genes Also Depends on NF-AT and/or NF-B Activity-Our findings indicate that
Class II genes are forkhead-responsive. For example, strategies that increase nuclear localization of FKHR, e.g. inhibition of PI3K-Akt or overexpression of TM-FKHR, result in increased expression of Class II genes. Moreover, VEGF-mediated induction of Class II genes requires the presence of FKHR. However, the fact that VEGF signaling promotes the nuclear exclusion, hence transcriptional inactivation, of FKHR and the observation that VEGF super-induces Class II gene expression in the presence of TM-FKHR, strongly suggest that additional positive acting transcription factors are involved.
Several transcription factors have been implicated in VEGF signaling, including NF-B and NF-AT (38,(45)(46)(47). To deter-    NOVEMBER 17, 2006 • VOLUME 281 • NUMBER 46 mine whether NF-B plays a common role in mediating VEGF induction of Class II genes, HCAECs were transduced with Adv or the inhibitor of NF-B, constitutively active Ad-IB, treated in the absence or presence of VEGF, and then assayed for candidate genes by reverse transcription-PCR. Ad-IB attenuated VEGF stimulation of every Class II gene (Table 3 and Fig. 6), suggesting an important role for NF-B in the expression of these genes. To determine whether constitutively active FKHR was sufficient for the expression of these genes when NF-B activity was inhibited, HCAECs were transduced with adenoviruses expressing Ad-IB and TM-FKHR and treated with or without VEGF for 4 h. Fig. 7 shows that overexpression of TM-FKHR could override the inhibitory effects of DN-NF-B on the VEGF-mediated expression of Mn-SOD, VCAM-1, and ESM-1. To assay for NF-AT response, HCAECs were preincubated with cyclosporin, treated in the absence or presence of VEGF, and assayed for Class II genes. As shown in Fig. 8, cyclosporin inhibited VEGF induction of Mn-SOD, VCAM-1, and DAF, but not ESM-1, suggesting that NF-AT plays a role in the transcription of some but not all of the Class II genes. In contrast, siRNA-mediated down-regulation of the Ets transcription factor, GABP-␣, had no significant effect on VEGF induction of Mn-SOD, VCAM-1, and ESM-1 (supplemental Fig. S1).  1 and 10 m.o.i.). The cells were serum-starved overnight, treated without or with VEGF (50 ng/ml) for 4 h, and then harvested for total RNA. Northern blot analyses of the genes were carried out using the same membrane. The lower panel shows ethidium bromide-stained 28 S RNA as a loading control. The blots shown are representative of at least three independent experiments. Two transcripts for Mn-SOD are indicated.

TABLE 3 Induction (-fold) or reduction of the Class II genes in endothelial cells by RT-PCR analyses
The basal levels of expression for each gene in unstimulated, serum-starved HCAEC, were arbitrarily considered as 1 (-fold) per 10 6  VEGF-mediated Subcellular Localization (Nuclear Export) of FKHR in Primary EC Is Transient in Nature-VEGF inhibition of Class I genes occurred early (at 1 h), whereas VEGF induction of Class II genes occurred only after 1 h (data not shown). We were interested in determining whether these differences correlated with the temporal pattern of FKHR nuclear exclusion in VEGF-treated ECs. VEGF-mediated phosphorylation of FKHR in HCAECs reached maximal levels between 15 and 30 min and returned to basal levels after 60 min of VEGF addition (Fig. 9A). In immunofluorescent studies of HCAECs, VEGF-mediated exclusion of FKHR from the nucleus was similarly transient in nature (Fig. 9B). In untreated serum-starved cells, FKHR was localized primarily in the nucleus (65 Ϯ 5.6% in the nucleus versus 35 Ϯ 3.1% in the cytoplasm). 30 min after addition of VEGF, this ratio was reversed, with 32 Ϯ 4.3% nuclear and 68 Ϯ 4.4% cytoplasmic. The nuclear to cytoplasmic ratio of FKHR returned to basal levels after 60 min (Fig.  9B). These data demonstrate that VEGF-mediated phosphorylation and nuclear export of FKHR in HCAECs are transient in nature.

DISCUSSION
Previous studies in non-endothelial cells have demonstrated that growth factors exert their proliferative, pro-survival, and metabolic effects in part by PI3K-Akt-dependent phosphorylation and exclusion of forkhead proteins from the nucleus, with secondary reduction in target gene expression (19,20,38,46,48,49). Among these target genes are p27 kip1 and BTG1, both inhibitors of G 1 to S cell-cycle progression (21,23), and GADD45A, a DNA-damage repair protein that regulates the G 2 /M cell-cycle checkpoint (22,50).
More recently, we have demonstrated that VEGF promotes PI3K-Akt-dependent phosphorylation and nuclear exclusion of FKHR in ECs (19). As a consequence, VEGF signaling resulted in decreased expression of p27 kip1 (19). Here, we have extended these findings by demonstrating a similar mechanism for BTG1 and GADD45A. Taken together, these data suggest that VEGF-mediated repression of p27 kip1 , BTG1, and GADD45 is a perquisite for entry into cell cycle.
Importantly, the present study provides evidence for the existence of a previously unrecognized group of FKHR-dependent genes (Class II genes) whose expression is normally FIGURE 8. VEGF-mediated induction of Mn-SOD, VCAM-1, and DAF, but not ESM-1, is dependent on NFAT signaling. HCAECs were preincubated with (Cyclo) or without (C) cyclosporine (10 L) for 30 min and then treated in the absence or presence of VEGF (50 ng/ml) for 4 h as indicated. Total RNA was extracted from HCAECs, and Northern blot analyses were performed using the radiolabeled probes as indicated. The lower panel shows ethidium bromide-stained 28 S RNA as a loading control. The data shown are representative of three independent experiments. The bar graph is a quantitative analysis of the expression of the respective mRNA from three independent Northern blots. *, p Ͻ 0.05 control versus VEGF-treated; †, p Ͻ 0.05 VEGFtreated versus cyclosporine plus VEGF-treated.

FIGURE 9. VEGF phosphorylates and modulates subcellular localization of endogenous FKHR in HCAECs.
A, serum-starved HCAECs were incubated with VEGF (50 ng/ml) for the times indicated. HCAECs were harvested for total protein, and Western blots were carried out as described under "Experimental Procedures." The membranes were probed for Ser-473-Akt (p-Akt), Ser-256-FKHR (p-FKHR), Akt, and ␤-actin. B, VEGF-mediated subcellular localization of FKHR was detected by immunofluorescence as described under "Experimental Procedures." The photomicrographs shown are superimposition of FKHR (red) and nucleus (blue) using an anti-FKHR antibody and 4Ј,6-diamidino-2-phenylindole, respectively. The bar graph in the bottom panel was generated by counting nuclear and cytoplasmic localization of FKHR in 200 HCAECs cells per time point. N, nuclear; C, cytoplasmic. *, p Ͻ 0.05. The data presented are from three independent experiments. induced by VEGF. These genes, which include BMP2, MMP-10, CITED2, VCAM-1, Mn-SOD, DAF, ESM-1, and RING1 and YY1 binding protein, encode for proteins known to be involved in metabolism, cell signaling, cell adhesion, stress response, matrix reorganization, differentiation, and other cellular functions. The functional relevance of these findings is evidenced by our studies of monocyte adhesion and complement-mediated EC lysis.
Recently, FKHR was shown to be essential for embryonic vascular development in mice (39,40). In two independent studies, deletion of FKHR resulted in embryonic lethality (at embryonic day 11) and abnormalities in angiogenesis. Furuyama et al. (40) extended their findings by showing that the ECs differentiated in vitro from isolated embryonic stem cells of FKHR-null mice had an abnormal morphological response to exogenous VEGF. These data raised an interesting question. If FKHR is a death-promoting gene that requires exclusion from the nucleus for cell viability and growth, then why did these mice not demonstrate uncontrolled cell proliferation and overgrowth? Based on the data presented in this study, we propose that FKHR is not simply a death-promoting, cell-cycle-arrest factor, but also a positive-acting transcriptional protein necessary for VEGF-mediated EC homeostasis. In other words, FKHR is an essential transacting factor that is involved in the coordinated regulation of a distinct set of genes in ECs involved in cell maintenance/health.
There are three possible explanations for the finding that VEGF induces the expression of FKHR-responsive genes (Class II), while promoting transient nuclear export of this transcription factor. One is that FKHR exerts an unusual dose response whereby too much nuclear FKHR is inhibitory and too little is insufficient to transduce the VEGF signal.
However, the observation that LY294002 or TM-FKHR result in super-induction of Class II gene expression argues against this hypothesis.
A more likely explanation is that VEGF induces the activity of other positive acting factors which, together with residual FKHR in the nucleus, transactivates downstream target genes. In support of this model is the finding that VEGF-mediated induction of Class II genes is dependent on NF-B and in some cases NF-AT. Further studies are required to determine the relative contribution of these and other non-FKHR transcription factors in mediating the effect of VEGF on Class II gene expression.
A final possibility relates to the time course of FKHR subcellular localization. Our data suggest that VEGF-mediated phosphorylation and nuclear exclusion of FKHR is transient, returning to baseline by 1 h. The initial transient nuclear exclusion of FKHR from the nucleus may explain the down-regulation of expression of Class I genes within the first 1 h of VEGF treatment. These early events may be important for promoting EC cell cycle and/or cell survival. It is possible that re-entry of FKHR into the nucleus at 1 h is necessary for mediating the positive effect of VEGF on Class II genes. Consistent with this notion is the observation that VEGF-mediated induction of this group of genes occurs only after 1 h, peaking between 2 and 4 h (data not shown). Although expression of Class II genes may depend on the reentry of FKHR into the nucleus, it seems unlikely that redistribution of FKHR to its baseline pattern is alone sufficient for inducing target gene expression. We propose that FKHR and non-FKHR transcription factors (including NF-B and NF-AT) interact in a spatially and temporally regulated manner to mediate expression of these genes (Fig. 10).
In conclusion, Class II genes represent a novel category of forkhead-responsive genes that have not been previously reported in any cell type. For this class of genes, FKHR is an important signal transducer, necessary for agonist-mediated induction of expression. Further studies to elucidate the mechanism(s) of agonist-induced, forkhead transcription factor-dependent gene expression should increase our understanding of spatial and temporal regulation of EC phenotypes.