Published online before print March 29, 2007, doi: 10.1161/01.RES.0000265844.56493.ac
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© 2007 American Heart Association, Inc.
Molecular Medicine |
The BTB-Kelch Protein KLEIP Controls Endothelial Migration and Sprouting Angiogenesis
From the Department of Vascular Biology and Angiogenesis Research (T.G.N., A.A., K.L., C.F., H.W., H.G.A., J.K.), Tumor Biology Center, Freiburg, Germany; National Cancer Institute (T.M.), Bethesda, Md; Cardiovascular Research Institute Maastricht (F.S.C., J.W.), Maastricht University, The Netherlands; Institute of Pharmacology and Toxicology (T.W.), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; and Joint Research Division Vascular Biology of the Medical Faculty Mannheim, University of Heidelberg, and the German Cancer Research Center Heidelberg (A.A., H.G.A., J.K.), Germany.
Correspondence to Jens Kroll, PhD, Research Division Vascular Biology, Medical Faculty Mannheim, University of Heidelberg, Building 28/Level 0, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail jens_kroll{at}web.de
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Abstract |
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Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1
. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF- and basic fibroblast growth factor-induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF- or basic fibroblast growth factor-mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases.
Key Words: BTB-kelch protein KLEIP • angiogenesis • migration • hypoxia • G proteins
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Introduction |
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Angiogenesis, the formation of blood vessels from preexisting ones, is a crucial process during embryonic development and in several pathological conditions. Vascular endothelial growth factor (VEGF) and its receptors are major regulators of endothelial cell (EC) function and angiogenesis.1 VEGF is upregulated under hypoxic conditions and induces several angiogenesis-related functions in ECs, such as proliferation, migration, and NO release, which are mediated by cascades of intracellular signaling pathways.2–4 Genetic experiments suggest that VEGF receptor Flk-1/KDR signaling is required for proper positioning of hemangioblastic cells from the posterior primitive streak in the yolk sac.5 Furthermore, cells lacking Flk1/KDR are unable to coalesce to form blood islands, suggesting that Flk1/KDR signaling controls the migration of ECs.5 The signaling pathways regulating EC migration involve the phosphorylation of p38 mitogen-activated protein kinase and focal adhesion kinase,6 activation of phosphatidylinositol 3-kinase,7 as well as the phosphorylation of protein kinase Akt/protein kinase B,8,9 with subsequent formation of NO by endothelial NO synthase (eNOS).10 Yet, mechanistically, VEGF-driven EC migration is poorly understood. Notably, cytoskeletal rearrangements regulated by monomeric GTPases, such as RhoA, Rac1, and CDC42, are involved in this process.11,12
BTB-kelch proteins are regulators of the cytoskeleton acting as modulators of cellular architecture, cellular organization, and cell migration.13 The kelch motif was identified several years ago in the ORF 1 protein in Drosophila. ORF 1 was shown to bind actin and to be required for the stabilization of the ring canals within the egg chamber.14 In addition to the kelch motif, most kelch proteins contain a BTB/POZ domain (Broad complex, Tramtrack, Bric-a-brac/Poxvirus and Zinc-finger domain), which serves as a protein-protein interaction domain,15 regulates protein degradation pathways,16 and controls Golgi complex localization.17 Furthermore, several BTB-kelch proteins contain a BACK domain (BTB And C-terminal Kelch).15 So far, of the 71 known and putative kelch family molecules within the human genome, 4 have been deleted in mice.18–21 Mutations of each of these BTB-kelch molecules lead to severe and distinct developmental defects,18–21 suggesting that the hitherto not widely recognized kelch molecule family may harbor numerous other unknown molecules of major biological and biomedical relevance.
The BTB-kelch protein KLEIP (Kelch-Like ECT2 Interacting Protein), also known as Kelch-like protein X or KLHL20, has been identified in a yeast 2-hybrid screen using ECT2, a Rho guanine nucleotide exchange factor (RhoGEF), as bait.22 KLEIP contains 1 BTB/POZ domain, 1 BACK domain, and 6 kelch repeats.15 KLEIP forms dimers, colocalizes with F-actin and concentrates transiently at sites of cell adhesion on cell-cell contact.22 We have identified KLEIP as a differentially expressed endothelial cell molecule whose expression is strongly regulated by hypoxia. Consequently, the aim of this study was the functional analysis of KLEIP-mediated endothelial cell functions. These experiments have identified KLEIP as an essential mediator of VEGF- and basic fibroblast growth factor (bFGF)-induced endothelial migration and invasive sprouting angiogenesis. KLEIP associates in a VEGF-dependent manner with the nucleotide exchange factor ECT2 and is required for VEGF-mediated RhoA activation. Collectively, the data establish the BTB-kelch protein KLEIP as an essential intracellular regulator of activated endothelial cell functions.
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Materials and Methods |
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Antibodies, Plasmids, Growth Factors, Drugs, Cells, and Media
The following antibodies and plasmids were used for this study: rabbit anti-KLEIP-N antibody and plasmid encoding fusion protein green fluorescence protein (GFP)-KLEIP,22 mouse anti-KDR 3G2 antibody (Tumor Biology Center, Freiburg, Germany), mouse anti-phosphotyrosine antibody PY99 (Santa Cruz Biotechnology, Santa Cruz, Calif), rabbit anti-extracellular signal-regulated kinase (ERK)1/2 antibody K-23 (Santa Cruz Biotechnology), mouse anti-pERK antibody E-4 (Santa Cruz Biotechnology), mouse anti-CD31 antibody clone JC70A (DAKO), mouse anti-RhoA antibody 26C4 (Santa Cruz Biotechnology), horseradish peroxidase-conjugated antibodies (DAKO), rabbit anti-ECT2 C-20 (Santa Cruz Biotechnology), mouse anti-hypoxia inducible factor (HIF)-1
antibody (BD Biosciences, Heidelberg, Germany), goat anti-actin antibody I-19 (Santa Cruz Biotechnology), mouse anti-GFP antibody (Chemicon), and Alexa Fluor 568 conjugated to phalloidin (Invitrogen). Recombinant VEGF165 and bFGF were purchased from R&D Systems and CellSystems, respectively. CoCl2, desferrioxamine (DFX), dipyridyl, and ciclopirox (CPX) were purchased from Sigma and dissolved in water, DMSO (for CPX), or ethanol (dipyridyl), respectively. Hypoxic conditions were generated by using the BBL GasPak Pouch System (BD Biosciences), as recently described, which reduces oxygen to less than 2%.23,24 Human umbilical vein ECs (HUVECs) were freshly isolated from human umbilical veins of newborns by collagenase digestion.25 HUVECs and bovine aortic endothelial cells (BAECs) were cultured in ECGM (PromoCell) containing 10% heat-inactivated FCS and antibiotics.
Transfection of HUVECs and BAECs With Small-Interfering RNA Molecules
HUVECs (1.2x105 per 6-well plate) were transfected with small-interfering RNA (siRNA) (final concentration: 200 nmol/L) and 6 µL of Oligofectamine (Invitrogen) according to the protocol of the manufacturer. The transfection medium was replaced after 4 hours by ECGM containing 10% FCS and the cells were incubated for another 48 hours. BAECs (3.5x105 cells per 6-well) were transfected with 6 µg of GFP-KLEIP plasmid, together with different siRNA molecules using 6 µL of lipofectamine (Invitrogen). BAECs were analyzed 24 hours later for GFP expression by fluorescence-activated cell sorting flow cytometers and CellQuest software. The following siRNA molecules were used: KLEIP siRNA13 sense sequence: GGGCUAUGGAAUUACUGAUtt (Ambion; siRNA ID:21286); KLEIP siRNA14 sense sequence: GGGCAAUGUUCAGACUCUUtt (Ambion, siRNA ID:21381), validated nontargeting negative control no. 1 siRNA (Ambion); validated HIF-1 siRNA sense sequence: GGGUAAAGAACAAAACACAtt (Ambion, siRNA ID:42840), validated ECT2 (h) siRNAs (Santa Cruz Biotechnology) and validated GFP-22 siRNA (Qiagen).
Microarray Analysis
HUVECs were starved overnight in 5% FCS, incubated for 6 hours with CPX (10 µmol/L) or dipyridyl (100 µmol/L), followed by the isolation of RNA and microarray analysis using the U133A chip (Affymetrix).
In Vitro Angiogenesis Assay
siRNA-transfected HUVECs were used to generate spheroids of defined cell number and used for in-gel sprouting angiogenesis experiments as previously described.26
Migration Assay
siRNA-transfected HUVECs were starved overnight in 5% (or 2.5%) FCS and stimulated with 25 ng/mL VEGF. Lateral migration assay was performed as recently described.27 Chemotaxis was assessed using a modified 48-well Boyden chamber and a collagen I-coated polycarbonate membrane (8-µm pore diameters). HUVECs (3x105 cells/mL) were allowed to migrate for 4 hours.
Proliferation Assay: 5-Bromodeoxyuridine
siRNA-transfected HUVECs were seeded in 96-well plates (2000 cells per well), starved overnight in 2.5% FCS, and stimulated with 25 ng/mL VEGF or 25 ng/mL bFGF. Twenty-four hours later, new media with growth factors and 5-bromodeoxyuridine were added. 5-Bromodeoxyuridine incorporation was determined 1 day later, according to the protocol of the manufacturer (Cell Proliferation ELISA, 5-bromodeoxyuridine; Roche Applied Science).
Western Blots
siRNA-transfected HUVECs were starved overnight in 2.5% FCS, incubated for 5 minutes with 100 µmol/L Na3VO4, and stimulated with 25 ng/mL VEGF at 37°C. Cells were washed with PBS/Na3VO4, and lysed in buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4, 1% NP40 
, 10 mmol/L EDTA, 10% glycerol, 100 µmol/L Na3VO4, and protease inhibitors) followed by KDR immunoprecipitation. Immunoprecipitates were bound to Protein-G-Agarose (Roche Applied Science), washed, boiled, and loaded onto a gel. Alternatively, equal amounts of cell lysates were directly loaded onto a gel. Membranes were incubated with the indicated antibodies, followed by incubation with Western blot detection reagent (Perbio Science). To study the effect of VEGF and bFGF on the association of KLEIP with ECT2, BAECs were transfected with the GFP-KLEIP plasmid (or mock), together with lipofectamine for 24 hours. BAECs were starved overnight in 2.5% FCS and stimulated with 25 ng/mL VEGF or 25 ng/mL bFGF, followed by ECT2 immunoprecipitation and an anti-GFP Western blot.
Northern Blots and RT-PCR
HUVECs were incubated with 125 µmol/L CoCl2, 75 µmol/L DFX, or 10 µmol/L CPX or cultured under hypoxic conditions in ECGM containing 10% heat-inactivated FCS, respectively. For downregulation of HIF-1 expression, HUVECs were transfected (24 hours) with a validated HIF-1 siRNA followed by the stimulation with DFX and hypoxia, respectively. Total RNA from cells was isolated using the RNeasy Kit (Qiagen). The probe for Northern blots (427-bp length) was generated by PCR using the following primers: sense, 5'-GTG ATG GCC TGG GTC AAA TAC-3'; and antisense, 5'-GAG GAT CCA TCA TGG CCT CCT AC-3'.
Immunohistochemistry
Sections of human umbilical cord, human heart, matched pairs of human tumors (BioCat GmbH), and ECs were washed in PBS, fixed in 4% paraformaldehyde/PBS and analyzed according standard histological procedures. Samples were analyzed with an Olympus IX50 fluorescence microscope or by confocal microscopy (LSM510 Axiovert 200M, Zeiss, Oberkochen, Germany).
RhoA-GTP Precipitation Assay
siRNA-transfected HUVECs were starved overnight in 2.5% FCS followed by VEGF stimulation (25 ng/mL) for 1 minute. Cells were lysed in GST-fish buffer (10% glycerol, 50 mmol/L Tris, pH 7.4, 100 mmol/L NaCl, 1% NP-40, 2 mmol/L MgCl2), followed by incubation with Rhotekin-RBD-GST beads (tebu-bio) for 1 hour on ice. Protein complexes were washed 2 times with GST-fish buffer, boiled, and loaded onto a gel. Membranes were incubated with a RhoA-specific antibody.
Statistical Analysis and Quantification
Data were analyzed by Student t test (2-sided, unpaired). A probability value of less than 0.05 was considered statistically significant. The data are presented as means±SD. Northern blots and Western blots were quantified using TotalLab version 2.01 (Nonlinear Dynamics).
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KLEIP Is Expressed in Endothelial Cells and Is Upregulated Under Hypoxic Conditions
Genetic experiments have revealed critical and rate-limiting roles of the vascular endothelium in maintaining tissue homeostasis in response to changes in oxygen levels.28,29 We have analyzed EC transcriptomic changes in response to reduced oxygen to unravel the hypoxia response program of ECs. Chemically induced hypoxia (ciclopirox and dipyridyl30) led to the upregulation of a distinct set of genes that has previously been characterized as hypoxia-inducible genes (among others: adrenomedullin, angiopoietin-like 4, hexokinase-2, insulin-like growth factor-binding protein-3, lysyl oxidase, phosphoglucomutase-1, stanniocalcin-2, VEGF-A, VEGF-C [for details, see Table I in the online data supplement, available at http://circres.ahajournals.org]). Expression-profiling experiments of several of the unknown hypoxia-induced EC genes identified a preferential endothelial expression of the BTB-kelch protein KLEIP in ECs. Abundant KLEIP expression was detected by anti-KLEIP and anti-CD31 double immunohistochemistry in ECs of the human umbilical vein (Figure 1A through 1C), of the human heart (Figure 1D through 1F), and in ECs of other human tissues (data not shown). Furthermore, KLEIP expression was increased in ECs of a human breast tumor (Figure 1H), as compared with corresponding healthy breast tissue (Figure 1G). Likewise, GFP-KLEIP-transfected BAECs (Figure 1J), as well as cultured HUVECs (Figure 1L), express KLEIP uniformly in their cytoplasm, and GFP-KLEIP colocalizes with actin (Figure 1K), as shown by confocal microscopy analysis.
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To further study the upregulation of KLEIP under hypoxic conditions, HUVECs were treated for 8 hours with the hypoxia mimicry drugs DFX, CoCl2,31,32 CPX30 (Figure 2A), and VEGF (supplemental Figure I). In a separate experiment, HUVECs were kept under reduced oxygen tension for 8 and 24 hours, respectively (Figure 2B). Hypoxia mimicry drugs and reduced oxygen tension (Figure 2A and 2B), but not VEGF (supplemental Figure I), led to a strong induction of endothelial KLEIP mRNA levels. Increased mRNA expression of KLEIP under hypoxia is rapidly downregulated on reoxygenation (Figure 2B), indicating a rapid turnover of KLEIP. Transfection of HUVECs with a validated HIF-1-specific siRNA (Figure 2C) and subsequent exposure to hypoxia or treatment with DFX inhibited the upregulation of KLEIP mRNA levels (Figure 2C). Taken together, the data establish the hypoxia-mediated transcriptional regulation of KLEIP expression in ECs.
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KLEIP Regulates VEGF-Induced Endothelial Cell Migration and In-Gel Sprouting Angiogenesis but Not Proliferation
Two different KLEIP-specific siRNAs were validated for the silencing of KLEIP expression in ECs to pursue loss-of-function experiments. HUVECs were transfected with KLEIP-specific siRNA13 and siRNA14 for 48 hours, and expression of KLEIP was examined by RT-PCR. KLEIP-specific siRNA13 and siRNA14 robustly downregulated endothelial KLEIP mRNA levels compared with control siRNA-transfected cells (supplemental Figure IIA). Correspondingly, hypoxia or treatment with the hypoxia mimicry reagents CoCl2 and DFX failed to induce KLEIP mRNA levels in KLEIP siRNA-silenced HUVECs (supplemental Figure IIA). To verify the downregulation of KLEIP protein in siRNA-transfected cells, BAECs were transfected with a GFP-KLEIP fusion protein and GFP-KLEIP expression was analyzed by fluorescence-activated cell sorting after transfection with KLEIP-specific siRNAs. In contrast to control siRNA, both KLEIP-specific siRNAs inhibited GFP-KLEIP expression to a similar degree as a validated GFP-specific positive control siRNA (supplemental Figure IIB). Collectively, these experiments reliably validate KLEIP-specific siRNA13 and siRNA14 as effective molecular tools for the specific and selective downregulation of KLEIP mRNA and subsequently KLEIP protein.
EC migration (Figure 3A and supplemental Figure III), in-gel sprouting angiogenesis (Figure 3B), and proliferation experiments (Figure 3C) were performed to study the functional consequences of KLEIP silencing in ECs. KLEIP-silenced HUVECs did not migrate in response to VEGF (Figure 3A and supplemental Figure III). Conversely, GFP-KLEIP-transfected BAECs showed an enhanced migratory response on VEGF stimulation (data not shown). For in-gel sprouting angiogenesis experiments, KLEIP-silenced HUVECs and control cells were embedded in a collagen matrix and allowed to grow capillary sprouts in response to VEGF or bFGF. VEGF and bFGF induced a robust sprouting angiogenesis phenotype in control siRNA-transfected cells. Yet, KLEIP-silenced HUVECs failed to respond to VEGF- or bFGF-induced sprouting angiogenesis (Figure 3B). In contrast to the inhibition of VEGF-induced endothelial migration and sprouting angiogenesis, KLEIP silencing did not affect VEGF- and bFGF-induced HUVEC proliferative responses (Figure 3C). Collectively, these loss-of-function experiments establish a specific role of KLEIP in controlling the angiogenic growth factor-induced migratory and invasive response program of ECs.
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To study the relationship between hypoxia induction of EC KLEIP expression and the role of KLEIP in shaping the migratory and invasive EC phenotype, we performed in-gel sprouting angiogenesis assays under hypoxia mimicry conditions. KLEIP-silenced and control siRNA-transfected HUVECs were treated for 24 hours with the hypoxia mimicry agent DFX and analyzed for their ability to respond to VEGF in spheroid-based in-gel sprouting angiogenesis experiments (Figure 4). Treatment of endothelial spheroids with DFX alone induced a strong sprouting angiogenesis response that was quantitatively comparable to the maximum effect of VEGF stimulation under normoxic conditions. Thus, the basal DFX-induced sprouting angiogenesis is apparently independent of KLEIP, as evidenced by the observation that KLEIP silencing did not inhibit DFX-induced sprouting angiogenesis (Figure 4). Costimulation with DFX and VEGF induced a synergistic effect on sprouting angiogenesis. Most importantly, however, KLEIP-silenced HUVECs no longer exhibited a VEGF-induced sprouting response (Figure 4). Together, these data demonstrate that the DFX response program is maintained in KLEIP-silenced cells. Yet, VEGF-induced sprouting angiogenesis depends on the presence of KLEIP in the absence or presence of DFX.
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KLEIP Regulates VEGF-Induced Activation of RhoA and Interacts Physically With ECT2 on VEGF Stimulation
Several intracellular signaling cascades are activated by VEGF on receptor binding.7 VEGF-driven endothelial proliferation is primarily mediated by activation of ERK1/2.2 VEGF-induced endothelial migration involves the activation of RhoA,12 p38 mitogen-activated protein kinase,6 Akt,8 and phosphatidylinositol 3-kinase.7 Phosphorylation of the VEGF receptor KDR was not altered in KLEIP siRNA-silenced HUVECs compared with control cells on VEGF stimulation (Figure 5A). Likewise, VEGF-induced activation of ERK1/2 phosphorylation was similarly not inhibited in KLEIP-silenced HUVECs; yet, basal ERK activation in KLEIP-silenced HUVECs was weakly increased (Figure 5B). These data biochemically support the previous observation that EC proliferation is not altered in KLEIP-depleted HUVECs. Previous reports demonstrated that KLEIP directly binds to the RhoGEF ECT2, which promotes the exchange from GDP with GTP on RhoA.22 We therefore hypothesized that ECT2-mediated RhoA activation is essential to VEGF-driven endothelial sprouting angiogenesis and that interaction of KLEIP with ECT2 may be indispensable for VEGF-induced activation of RhoA. Such an interaction would also mechanistically explain the profound migratory and invasive loss-of-function phenotype of KLEIP-depleted ECs. To test this hypothesis, we first analyzed the effect of siRNA-mediated ECT2 depletion in HUVECs on VEGF-induced endothelial sprouting angiogenesis (Figure 6A). Silencing of ECT2 significantly inhibited VEGF-induced in-gel sprouting angiogenesis (Figure 6A), demonstrating that the KLEIP-binding nucleotide exchange factor ECT2 regulates endothelial VEGF responses. To address whether ECT2 and KLEIP are indispensable for VEGF-induced activation of RhoA, ECT2- or KLEIP-silenced and control siRNA-transfected HUVECs were stimulated with VEGF and analyzed for RhoA activation. VEGF stimulation of control siRNA-transfected HUVECs induced a strong activation of RhoA (Figure 6B and 6C). In contrast, VEGF failed to induce RhoA activation in HUVECs transfected with ECT2-specific siRNAs or KLEIP-specific siRNAs (Figure 6B and 6C). Based on this finding, we next tested whether VEGF induces a physical interaction of KLEIP and ECT2 and whether KLEIP/ECT2 complexes translocate from the cytosol to the plasma membrane, which would further support its role during EC migration. VEGF stimulation of GFP-KLEIP-expressing BAECs induced a translocation of KLEIP/ECT2 complexes from the cytosol to the plasma membrane, and KLEIP/ECT2 complexes were highly concentrated at the plasma membrane (Figure 7A). In addition, VEGF as well as bFGF induced the physical association between KLEIP and ECT2, as determined by KLEIP/ECT2 coimmunoprecipitation experiments (Figure 7B and supplemental Figure IV).
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Discussion |
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Based on the following findings, we establish the BTB-kelch protein KLEIP as a novel and essential intracellular component in the EC response program to angiogenic activation: (1) KLEIP is preferentially expressed in ECs, (2) endothelial KLEIP expression is controlled by hypoxia, (3) KLEIP mediates VEGF-induced endothelial migration and in-gel sprouting angiogenesis, and (4) KLEIP regulates VEGF-mediated activation of RhoA by complex formation with the RhoGEF ECT2 in ECs.
Many BTB-kelch proteins are intracellular proteins that regulate cytoskeletal organization and modulate cellular architecture.13 As such, BTB-kelch molecules control fundamental cellular processes that are involved in response programs to different microenvironmental stimuli. The BTB-kelch protein KLEIP was identified as a nucleotide exchange factor ECT2-binding and actin-binding protein.22 Although no distinct cellular functions of KLEIP have been identified thus far, these findings suggested a role of KLEIP in cellular processes involving cytoskeletal changes. Likewise, KLEIP expression has been described in model cells such as MDCK and HeLa,22 but the physiological cellular functions of KLEIP in vivo have not yet been identified. We have identified KLEIP as a hypoxia-regulated gene that is preferentially expressed by ECs. Kelch molecules are expressed by many cell types. Yet, they also display rather specific expression patterns, suggesting cell-type-specific rather than pleiotropic functions. For example, the BTB-kelch protein KLHL6 is expressed in embryonic ECs and in hematopoietic cells only. Correspondingly, KLHL6-deficient mice have a severe phenotype in B lymphocyte function.19 Similarly, preferential endothelial expression of KLEIP and its regulation by hypoxia implied specific KLEIP functions in vascular remodeling processes including angiogenesis. VEGF-driven EC migration and sprouting is a complex process that requires the reorganization of the cytoskeleton, which involves activation of monomeric GTPases of the Rho subfamily including RhoA.11,12 The results of the present investigation show that the BTB-kelch protein KLEIP is an essential regulator of EC sprouting and migration during VEGF-induced angiogenesis. Using several biochemical avenues, we could dissect the pathways that are regulated by KLEIP. VEGF-mediated phosphorylation of VEGF receptor KDR and ERK activation2 were not affected in KLEIP-deficient HUVECs. Instead, KLEIP regulates the activation of RhoA in ECs. A previous report has shown that KLEIP interacts with the protooncogene ECT2.22 However, factors triggering the interaction between KLEIP and ECT2 have not been described thus far. ECT2 is a RhoGEF that triggers activation of G proteins and binds specifically to RhoA.33 ECT2 is involved in different cell activities, such as regulation of cell polarity,34 in cytokinesis,35 and during cell migration.36 We have found that the expression of KLEIP and ECT2 is required for VEGF-driven nucleotide exchange on RhoA in ECs. Furthermore, VEGF induces the physical interaction between KLEIP and ECT2, and ECT2, by itself, also regulates VEGF-induced sprouting of ECs. These findings suggest that the VEGF-driven association between KLEIP and ECT2 is essential for VEGF-mediated RhoA activation and subsequently for VEGF-driven sprouting angiogenesis. Apparently, bFGF and its receptor are activating a similar pathway, whereas other tested stimuli like lysophosphatidic acid and hepatocyte growth factor do not (not shown). Although the detailed mechanisms are still unclear, our results suggest that KLEIP directs ECT2 to a location within the cell that is essential for its proper function or that KLEIP promotes the formation and stabilizes the maintenance of the active conformation of ECT2.37 Nevertheless, the mechanisms by which the VEGF and bFGF pathways activate ECT2 await further analyses. Because many BTB-domain-containing proteins regulate the stability of certain molecules,38–40 we have also tested whether the BTB-kelch protein KLEIP regulates the stability of ECT2. However, coexpression studies using KLEIP and ECT2 did not provide evidence that KLEIP regulates the amount of ECT2 within the cell (T.G.N. and J.K., unpublished data, 2006).
KLEIP has been identified in this study as a hypoxia-regulated gene that leads to an enhancement of endothelial activity. This is remarkable for 2 reasons. Firstly, hypoxia is an important stimulator of RhoGTPases expression and function.41 Therefore, upregulation of KLEIP under hypoxia could be a functional consequence that ensures proper function of the Rho protein family machinery during hypoxic episodes. In accordance with such a scenario, return to normal oxygen conditions lead to a normalization of KLEIP expression, which indicates a rapid turn over of KLEIP with subsequent inactivation of KLEIP-mediated Rho protein signaling in ECs. Secondly, our observation that hypoxia induces endothelial sprouting independently of KLEIP, whereas VEGF/bFGF-induced sprouting is dependent on KLEIP, further supports the specificity of KLEIP for VEGF/bFGF-induced endothelial function. It also suggests that hypoxia and VEGF may use different signal-transduction pathways to activate ECs.
In summary, these functional KLEIP experiments establish specific roles of KLEIP in controlling the VEGF-induced migratory and invasive response program of ECs. The BTB-kelch protein KLEIP is an essential downstream regulator of VEGF/bFGF-induced migration and sprouting angiogenesis, and its characterization sheds further light into pathways controlling VEGF-driven functions on ECs. Further investigations are in progress to uncover the vascular functions of KLEIP during physiological and pathological angiogenesis in vivo as well as during mouse development.
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Acknowledgments |
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We thank Dr Susanne Lutz (University of Heidelberg, Germany) for assistance in performing the RhoA assay.
Sources of Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (KR1887/4-1 and KR1887/4-3 to J.K.) and Sonderforschungsbereichs/TransRegio 23 subprojects A3 (to H.G.A.) and B6 (to T.W.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received June 12, 2006; first resubmission received November 30, 2006; second resubmission received February 9, 2007; revised second resubmission received March 6, 2007; accepted March 20, 2007.
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