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Immunology |
B Activation in Hematopoietic Progenitor Cells1
The Vanderbilt Cancer Center and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6838 [M. M. D., T. O., T. T., K. T., T. S., B. E., Y. A., S. N., T. D., D. P. C.], and Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University of Chicago, Maywood, Illinois 60153, [P. C., D. I. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B) in hematopoietic progenitor cells (HPCs), and this is associated with alterations in the development of multiple lineages of hematopoietic cells and defective immune induction in tumor-bearing animals. Antibodies to VEGF have been shown to abrogate this effect. The mechanism by which VEGF antagonizes the induction of NF-B was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor 
(TNF-) induced the nuclear translocation and DNA binding of p65-containing complexes, VEGF alone induced nuclear translocation and DNA binding of the complexes containing RelB. These results were confirmed by immunofluorescence confocal microscopy. VEGF effectively blocked TNF--induced NF-B activation in HPCs from RelB-/- mice, however, similar to the effect observed in HPCs obtained from RelB+/- and RelB+/+ mice. This suggests that RelB is not required for VEGF to inhibit NF-B activation. However, although TNF- induced rapid activation of IB kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of IB phosphorylation and degradation of IB and IB
in HPCs. VEGF alone, however, did not have any effect on phosphorylation of IB or degradation of IB and other inhibitory molecules IBß, IB, or Bcl-3. SU5416, a potent inhibitor of the VEGF receptor 1 (VEGFR1) and VEGFR2 receptor tyrosine kinases, did not abolish the inhibitory effect of VEGF, indicating that the VEGF effect is mediated by a mechanism unrelated to VEGFR1 or VEGFR2 tyrosine kinase activity. Thus, VEGF appears to inhibit TNF--induced NF-B activation by VEGFR kinase-independent inhibition of IKK. Therapeutic strategies aimed at overcoming VEGF-mediated defects in immune induction in tumor-bearing hosts will need to target this kinase-independent pathway. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ineffective antigen presentation is one of the critical factors involved in tumor evasion from immune system control and plays an important role in immunological abnormalities observed in other conditions associated with hyperproduction of VEGF. Therefore, the effects of VEGF on hematopoiesis could be an important factor in the pathophysiology of these diseases as well. The significant molecular consequences of VEGFR1 activation by VEGF in HPCs are unclear. One possible mechanism is suggested by our recent demonstration that the effects of VEGF are closely associated with the inhibition of TNF--inducible activation of the transcription factor NF-B in HPCs (10)
. This DNA-binding transcription factor is composed of five subunits: p65 (RelA), p50, p52, c-Rel, and RelB (13)
. These different family members can associate in various homo- or heterodimers through a highly conserved NH2-terminal Rel homology domain. In the cytoplasm of quiescent cells, they are associated with inhibitory molecules of the IB family. Cell activation by various stimuli, including TNF-, lipopolysaccharide, interleukin 1, CD40, and others, results in serine phosphorylation and degradation of IkB with subsequent nuclear translocation and specific DNA binding of NF-B dimers (14)
. It has also been suggested that tyrosine phosphorylation of IB can activate NF-B without degradation (15)
. NF-B plays a critical role in the differentiation of dendritic cells and B cells, and its involvement in T-cell development has been also demonstrated recently (16, 17, 18, 19, 20, 21)
. These data suggest that inhibition of NF-B could be responsible for the observed effects of VEGF on HPCs. However, it is unclear how VEGF might affect NF-B activation. It is also unclear whether this mechanism is restricted to HPCs or might apply to other tissue targets of VEGF action. In this study, we investigated the mechanism of VEGF action on both HPCs and vascular endothelial cells. We showed that VEGF inhibited NF-B activation in HPCs, but not in endothelial cells. This effect was associated with the ability of VEGF to induce translocation in HPCs of RelB-containing NF-B complexes, but that RelB is not required for VEGF to inhibit NF-B activation by TNF-. Instead, VEGF works through a mechanism independent of VEGFR kinase to inhibit the activation of IKK, thus preventing serine phosphorylation and proteolytic degradation of the NF-B inhibitory subunits IB and IB in HPCs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Reagents.
RMCs were prepared from male Wistar rats as described (22)
. Cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) and were used between passages 5 and 10. HMEC-1 cells were grown in MCDB131 medium (Sigma Chemical Co., St. Louis, MO) containing 15% fetal bovine serum (Hyclone Laboratories, Logan UT), 10 ng/ml epidermal growth factor (Collaborative Biomedical Products; Becton Dickinson, Bedford, MA), and 1 mg/ml hydrocortisone (Sigma; Ref. 23
). Growth medium was supplemented with 1 mM L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). HUVECs were purchased from BioWhittaker/Clonetics (Walkersville, MD) and cultured in EGM-2 medium (Clonetics). Passages four to six were used in experiments.
The following antibody-producing hybridomas were obtained from American Type Culture Collection (Rockville, MD) and used as culture supernatants: anti-CD4 (L3T4; TIB-207); anti-CD8 (Lyt-2.2; TIB-210); and anti-MHC class II (TIB-120). hGM-CSF, mGM-CSF, TNF-, and VEGF165 were obtained from R&D Systems (Minneapolis, MN); polyclonal antimouse immunoglobulin was obtained from Sigma.
All anti-NF-B antibodies used for EMSA (anti-p50, p52, c-Rel, Rel-A, and Rel-B antisera) as well as anti-IB antiserum were generous gifts from Dr. N. Rice (ABL-Basic Research Program, National Cancer Institute, NIH). Anti-IB, -IBß, -IB
, -VEGFR2 (Flk1), and -IKK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine (4G10) was from New England Biolabs, and anti-Ser-36 IB was from Upstate Biotechnology, Inc.
Portions of mutant and wild-type IB (amino acids 1–51) were amplified by PCR from pBS(SK-)-IB containing full-length wild-type or mutant IB. In mutant IB, two serines in positions 32 and 36 were substituted by two alanines. PCR products were cloned to pGEX vector, and the constructs were confirmed by sequencing in both directions. GST-IB fusion proteins were made according to standard manufacturer’s protocol (Amersham).
Cell Separation.
Human CD34+ cells were isolated from umbilical cord blood or bone marrow from the discarded filter sets used for clinical marrow collections by a positive selection technique with CD34 immunomagnetic beads (Dynal, Oslo, Norway) according to the manufacturer’s protocol. The purity of CD34+ cells was >90% as estimated by flow cytometry with an anti-CD34+ antibody (Becton Dickinson). Cells were cultured overnight in complete culture medium (RPMI-1640; Life Technologies; with 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 1 x 10-5 M 2-mercaptoethanol, and 10% fetal calf serum; HyClone, Logan, UT) supplemented with 3 ng/ml recombinant human GM-CSF. After that time, cells were collected, washed in serum-free RPMI 1640, and used for further study.
mHPCs were obtained from bone marrow. Briefly, bone marrow was obtained from the femurs and tibias of mice. T and B cells, macrophages, and dendritic cells were depleted by incubation with a mixture of antibodies: TIB-207, TIB-210, TIB-120, antimouse immunoglobulin, and complement (Low-Tox-Guinea Pig Complement; Cedarline, Hornby, Ontario, Canada). Dead cells were removed by centrifugation over a LympholyteM gradient (Cedarline). Cells were cultured overnight with 3 ng/ml recombinant mouse GM-CSF in complete medium, washed, and used in further experiments.
EMSA.
Double-stranded oligonucleotide probes were prepared by annealing the appropriate single-stranded oligonucleotides at 65°C for 10 min in 10 mM Tris, 1 mM EDTA, and 10 mM NaCl, followed by slow cooling to room temperature. The probes were end-labeled with [32P]-dCTP by filling in the 5' overhangs with Klenow fragment. Two different B probes were used:
For the murine intronic -chain KB site: wild type (5'-AGTTGAGGGGACTTTCCCAGG) and mutant B site (5'-AGTTGAGGCGACTTTCCCAGG; and for the palindromic B probe: wild type (5'-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA) and mutant (5'-GATCCAACGGCAGATCTATTCCCCTCTCCTTA).
Nuclear extract was obtained from the cells as described (24) . Ten µg of nuclear extract were incubated for 20 min with labeled probe (50,000 cpm) in the presence of 4 µg of poly(deoxyinorinic-deoxycytidylic acid) (Pharmacia) in binding buffer (20 mM HEPES, 5% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, and 5 mM MgCl2). Competition assays were performed with a 100-fold excess of unlabeled probes. The DNA-protein complexes were separated on 4% polyacrylamide gels and visualized and analyzed on a Phosphorimager (Molecular Dynamics). For supershifts with antibodies, the proteins and the antibodies were incubated on ice for 20 min in incubation buffer plus poly(deoxyinorinic-deoxycytidylic acid) before the probe was added.
Western Blotting of IB.
Cells were treated as indicated in the text. Cell lysates were prepared as described (25)
. The protein concentration was estimated by the Bradford method (Bio-Rad). Lysates (20–40 µg) were separated by 10% SDS-PAGE and immunoblotted onto polyvinylidene Hybond-P membrane (Amersham, Arlington Heights, IL). Western blot analysis was performed using the indicated antibodies, and bands were visualized by ECL (Amersham).
Immunofluorescent Staining.
The cell pellet of 105 CD34+ cells isolated from cord blood was embedded in OCT compound and snap-frozen in a dry ice-acetone bath. Cryostat sections (4 µm) were fixed in acetone at -20°C for 10 min, washed in PBS, and preadsorbed with avidin-biotin blocking reagents (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Sections were washed with PBS, blocked with 5% goat serum, and incubated with anti-p65 or anti-Rel B antisera for 60 min at room temperature. After washing, slides were incubated with biotinylated antirat antibody (Vector Laboratories) for 60 min at room temperature, followed by a 30-min incubation at room temperature with 4 µg/ml FITC-conjugated streptavidin (Pierce). Coverslips were mounted (Vectashield Vector) and analyzed by confocal microscopy (Zeiss LSM410).
In Vitro Kinase Assay.
To determine IKK activity, the whole-cell lysates were obtained in immunoprecipitation buffer [50 mM HEPES (pH 7.6), 250 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% NP40, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitor tablets (Boehringer Mannheim)] according to Devalaraja et al. (26)
. The cell lysates (100–150 µg) were cleared and immunoprecipitated by adding anti-IKK antibodies (Santa Cruz Biotechnology) and protein A/G-agarose (Pierce), with overnight incubation at 4°C. In vitro kinase assay was performed by incubating immunoprecipitates with 4 µg of GST-IB (amino acids 1–51) fusion protein in 20 µl of kinase buffer containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 0.5 mM DTT, 100 µM ATP, and 5 µCi of [-32P]ATP for 30 min at 30°C (27)
. Samples were separated by 10% SDS-PAGE, immunoblotted onto polyvinylidene difluoride membrane (Millipore), and analyzed by autoradiography. Mutant GST-IµB substrate was used to confirm the specificity of reaction. Membrane was also probed with anti-IKK antibodies to check for the equality of loading.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B Activation in Different Types of Cells.-dependent NF-B activation in these cells (10)
. We asked whether VEGF effects on NF-B were restricted to HPCs or could also be observed in other types of cells expressing the VEGFR1. In this study, we investigated the effect of VEGF on three distinct types of cells: hHPCs, and mHPCs, HMEC-1 cells, and RMCs. All of these cells express mRNA for both TNF- receptor and VEGFR1 (data not shown). TNF- induced strong NF-B activation in all tested types of cells (Fig. 1
, Lanes 2 and 3 versus Lane 1). VEGF alone also activated NF-B in both HPCs and HPCs and RMCs (Fig. 1
, Lanes 4 and 5 versus Lane 1); however, this effect was weaker than the effect of TNF-. No VEGF-inducible NF-B activation was detected in HMEC-1 cells. In agreement with our data reported previously, VEGF prevented TNF--dependent NF-B activation in human HPCs. The same effect was observed in mHPCs and RMCs (Fig. 1
, Lane 6 versus Lane 3). This effect was observed when VEGF was added at the same time with TNF- or within 10 min before TNF-. Addition of VEGF 10 min after TNF- did not affect NF-B activation (data not shown). No effect of VEGF on TNF--inducible NF-B activation was observed in HMEC-1 cells. These data indicate that the effects of VEGF on NF-B are not restricted to HPCs.
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on Nuclear Translocation and DNA Binding of NF-B Subunits. induced translocation of the same members of the NF-B family in HPCs. Using supershift EMSA, we found striking differences in the translocation and DNA binding of two members of the NF-B family, p65 (RelA) and RelB, in response to treatment with VEGF or TNF-. Significant levels of p65-containing complexes were detected in hHPCs after treatment with TNF- but not with VEGF (Fig. 2
, compare Lanes 2 and 5 in p65 supershift). By contrast, treatment of hHPCs with VEGF resulted in a significant increase in the translocation and DNA binding of complexes containing RelB (Fig. 2
, Lanes 2 and 5 in RelB supershift). No differences in the level of p50, p52, or cRel were detected (data not shown). In endothelial cells, TNF- also induced translocation of predominantly p65-containing complexes, whereas no effect of VEGF was observed (data not shown). Pretreatment of HPCs with VEGF reduced nuclear translocation and DNA binding of complexes containing p65 (Fig. 2
, compare Lanes 2 and 3 in the p65 supershift).
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B was visualized using immunofluorescent confocal microscopy. As shown in Fig. 3
, treatment of hHPCs with TNF- resulted in the accumulation of p65 but not RelB proteins in the nuclei of hHPCs. However, the treatment of these cells with VEGF resulted in nuclear translocation of RelB but not p65 (Fig. 3)
. To quantitatively analyze these differences, the total number of cells with nuclear localization of the proteins was evaluated in two independently performed experiments using double-blinded observers. Less than 15% of untreated cells demonstrated nuclear localization of p65 or RelB. After treatment of hHPCs with TNF-, nuclear localization of p65 was found in 95% of cells, whereas nuclear localization of RelB was found only in 35% of cells. Seventy percent of cells treated with VEGF demonstrated nuclear localization of RelB, whereas only 35% of cells had nuclear localization of p65. These data confirm the results obtained by EMSA. Taken together, these data suggest that RelB nuclear localization could be directly involved in the inhibition of TNF--mediated NF-B activation by VEGF, perhaps by blocking p65 access to promoter sites. To test this hypothesis, HPCs were isolated from the bone marrow of RelB knockout mice (RelB-/-), and heterozygous (RelB+/-) mice served as controls. Cells were treated with TNF- and VEGF, and NF-B-specific DNA binding was analyzed using EMSA. VEGF blocked TNF--induced NF-B activation in mHPCs isolated from RelB+/- mice (Fig. 4)
, very similar to its effect in the control RelB+/+ mice used in the experiments described in Fig. 1
. The same effect was observed in RelB-/- mice (Fig. 4)
. These data indicate that RelB does not play a direct role in the inhibition of NF-B activation by VEGF.
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B Inhibitory Subunits IB.B is bound in cytoplasm to inhibitor proteins IB. Serine phosphorylation of these molecules in response to various signals results in their ubiquitin-mediated degradation and the consequent release of NF-B dimers. NF-B dimers are then able to translocate to the nucleus and bind DNA. This is the best understood mechanism of regulation of NF-B activity. In the next group of experiments, we studied the effect of VEGF on these natural inhibitors of NF-B. TNF- is a very potent inducer of IB serine phosphorylation by IKK, resulting in its degradation within minutes of exposure. VEGF alone did not induce IB degradation in any of the studied types of cells. However, pretreatment of HPCs or RMCs, but not HMEC-1, with VEGF substantially inhibited TNF--induced IB degradation (Fig. 5)
. We have tested the effect of TNF- and VEGF on other known inhibitors of NF-B IB, IBß, and Bcl-3 in hHPCs. VEGF alone did not induce degradation of any of these proteins (data not shown). Treatment of hHPCs with TNF- alone resulted in the degradation of IB but not of IkBß or Bcl-3 (Fig. 6)
. VEGF prevented TNF--inducible degradation of IkB (Fig. 6)
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B. VEGF reduced the level of TNF--inducible Ser-32 phosphorylation in both hHPCs and mHPCs as was determined by anti-phosphoserine antibodies (Fig. 7)
. It has been reported that site-specific tyrosine phosphorylation of IB may inhibit the inducible serine phosphorylation and degradation of IB (28)
. We therefore tested whether VEGF might induce tyrosine phosphorylation of IB. hHPCs were treated with VEGF for various periods of time (5–60 min). IB was then immunoprecipitated from cell lysates with anti-IB antibody. The complex was then subjected to electrophoresis, transferred to membranes, and then probed with anti-phosphotyrosine antibodies. Cells treated with the phosphatase inhibitor pervanadate were used as a positive control. No VEGF-inducible tyrosine phosphorylation of IB was detected (data not shown).
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-dependent Activation of IKK.B by TNF- involves successive activation of NIK and the IKK complex (29
, 30)
. Serine phosphorylation of the IKKß subunit by NIK induces its activity and allows it to phosphorylate IB proteins. We studied the effect of VEGF on IKK activity by in vitro kinase assays using GST-IB fusion protein as a substrate. In both hHPCs and mHPCs, VEGF remarkably inhibited TNF--induced activation of IKK but did not show any effect when it was added alone (Fig. 8)
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(Fig. 9)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inducible activation of transcription factor NF-B (10)
. Previous data suggested that VEGF might exert its effect via inhibition of NF-B activation, which is essential for dendritic cell differentiation. In this study, we have tried to elucidate the mechanism of VEGF effects on NF-B.
We first asked whether the observed effects of VEGF on NF-B activation were restricted to HPCs or whether they could be observed in other types of cells known to express the receptors, particularly endothelial cells, commonly assumed to be the primary targets for VEGF action. Endothelial cells express all major specific VEGFRs and actively proliferate in response to VEGF. VEGF does not induce appreciable proliferation of other cell types, including HPCs (1
, 2
, 11
, 38
, 39)
. RMCs were also used in these studies. These cells are derived from smooth muscle cell precursors and are a component of the renal glomerulus. We compared the effects of VEGF on HPCs (mouse and human), endothelial cells, and RMCs. All of these cells express both TNF- (40)
and VEGFR1 receptors (41)
. Not surprisingly, TNF- induced NF-B activation in all of these types of cells. VEGF alone also showed some activation of NF-B DNA binding in HPCs and RMCs but not in endothelial cells. Using mHPCs and hHPCs, we confirmed our previous observation that VEGF prevented TNF--inducible NF-B activation in human HPCs (10)
. The same effect was observed in RMCs but not in endothelial cells. Thus, the effects of VEGF on NF-B signaling are dependent on the cellular context.
In an attempt to investigate the mechanism of VEGF effects on HPCs, we compared the profile of NF-B subunit activation induced by VEGF or TNF- using a supershift EMSA assay. TNF- induced nuclear translocation and DNA binding of the complexes containing predominantly p65. Preferential induction of p65 by TNF- has been described previously in many reports (42, 43, 44)
. NF-B complexes induced by VEGF alone also bound to DNA but predominantly contained RelB and not p65, a finding that was confirmed in intact cells by direct nuclear translocation studies.
Our initial hypothesis was that these RelB complexes could be directly involved in the inhibition of p65-containing NF-B complexes after combined stimulation with TNF- and VEGF. It was possible that transcriptionally inactive complexes containing RelB could compete for promoter sites normally occupied by p65-containing complexes. Alternatively, RelB release and translocation could be a "bystander phenomenon" associated with failure of TNF- to induce p65-containing NF-B complexes in the presence of VEGF. A direct effect of RelB on dendritic cell differentiation was suggested by previous work showing that RelB knockout mice lacked myeloid dendritic cells. However, in this case the lack of RelB was associated with defective dendritic cell differentiation rather than the inappropriate induction of RelB-containing complexes as observed in this study. To determine whether RelB is directly involved in reduction of NF-B activation by VEGF, we used HPCs obtained from RelB knockout mice. Our experiments demonstrated that VEGF was able to inhibit TNF--inducible NF-B activation in RelB-/- mice similar to the effect observed in RelB+/+ and RelB+/- mice. These data thus argue against a direct inhibitory role for RelB on NF-B stimulation in this system and imply the existence of other inhibitory mechanisms. Not formally ruled out, however, is the possibility that the lack of RelB has resulted in a loss of a crucial subset of early myeloid committed HPCs in which RelB plays an important role.
What then is the mechanism by which VEGF inhibits the induction of p65-containing complexes? Here we have shown that the VEGF effect is mediated via inhibition of IKK activation in response to TNF-. Decreased IKK activity prevents efficient serine phosphorylation of IB and its ubiquitin proteasome-dependent degradation. The large Mr 
700,000 IKK complex that phosphorylates IB proteins in response to proinflammatory signals consists of two catalytic subunits, IKK and IKKß, and a regulatory subunit IKK (45
, 46)
. Upstream kinases, NIK and mitogen-activated protein kinase kinase kinase 1, are able to phosphorylate both IKK catalytic subunits (27
, 47)
; however, only IKKß phosphorylation contributes to IKK activation by TNF- (29)
. The proposed mechanism of IKK function suggests that the IKKß subunit contains sites for both positive and negative regulation. IKKß is rapidly activated through phosphorylation of its "activation loop" (T loop) by upstream kinases, and this is followed by progressive autophosphorylation at a COOH-terminal serine cluster, which in turn inhibits catalytic activity (29)
. VEGF may exert its inhibitory action by blocking the initial phosphorylation of serines in the T loop of IKKß or by causing its rapid dephosphorylation. This mechanism would imply participation of VEGF-activated phosphatase(s). Phosphatases SHP1 and SHP2 (48)
and HCPTPA (49)
have been proposed to be an integral part of the VEGF receptor signaling pathway, playing a role in the down-regulation of the VEGF signal. It is possible that VEGFR-related phosphatases may interfere with other signaling pathways, in particular with that of NF-B. The potential involvement of phosphatases in the regulation of NF-B signaling was demonstrated recently by association of the c-Rel subunit of NF-B with serine/threonine protein phosphatase 4 (also known as PPX; Ref. 50
). Alternatively, VEGF may enhance subsequent phosphorylation of the COOH-terminal serine cluster, leading to IKK deactivation. Upstream NIK kinase activity may also be a target for VEGF effect. With the current data, it is not possible to discriminate between these possibilities, and these hypotheses are under investigation.
It is interesting to note that although VEGF induces DNA binding of RelB containing dimers, we were unable to demonstrate its effect on IB proteins, especially IB and IB, which are readily degraded upon TNF- stimulation. One possible explanation is that IB has high affinity for p65-containing complexes (14)
but much less affinity for RelB-containing complexes, which are also less susceptible to inhibition by IB (51
, 52)
. VEGF could also induce tyrosine phosphorylation of IB or IB and dissociation of NF-B complexes without IB degradation, the mechanism described recently by Imbert et al. (15)
. However, we could not detect tyrosine phosphorylation of IB induced by VEGF. Not excluded is the possibility that in HPCs, RelB is predominantly associated with another, as yet unknown, member of the IB family, resulting in RelB release upon stimulation with VEGF but not TNF-.
VEGFR2 exercises its important functions through autophosphorylation and activation of its receptor tyrosine kinase domain upon ligand binding (53
, 54)
. Less is known about the VEGFR1 signal transduction pathway. However, data suggest that the kinase domain of this receptor plays a much less important role, and its function is realized rather through other mechanisms. Thus, although VEGFR1 knockout mice die in early embryonic development because of disorganization of blood vessels, VEGFR1 tyrosine kinase-deficient homozygous mice survive without developing an observed phenotype (53)
. To study the role of receptor tyrosine kinase activity in VEGF-induced effects on HPCs, we used VEGFR kinase inhibitor SU5416. This inhibitor potently prevents phosphorylation of both VEGFR1 and VEGFR2, activation of downstream signaling from VEGFR2, proliferation of endothelial cells, and angiogenesis in vivo (35
, 36)
. In our control experiments, SU5416 completely blocked VEGFR2 autophosphorylation in HUVECs, as expected. However, at the same concentration, it was ineffective in reversing the VEGF inhibitory effect on IKK activity. This indicates that activity of the VEGFR tyrosine kinases is not involved in the observed effects on NF-B. It is most likely mediated by alternative mechanisms linked to VEGFR1 in agreement with our previous data (10)
or an as yet undescribed receptor.
We have therefore identified two unique effects of VEGF on NF-B activation: nuclear translocation and DNA binding of complexes containing predominantly RelB, and receptor tyrosine kinase-independent inhibition of TNF--dependent activation of IKK, leading to a decrease in IB serine phosphorylation and degradation of IB and IB. These studies may have identified a new mechanism for the regulation of NF-B and for VEGF effects on NF-B in hematopoietic progenitor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by NIH Grant CA 76321 (to D.P.C.). 
2 The first two authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Vanderbilt-Ingram Cancer Center, 648 Preston Building, Nashville, TN 37232-6838. Phone: (615) 936-3321; Fax: (615) 936-3322; E-mail: d.carbone{at}vanderbilt.edu
4 The abbreviations used: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; HPC, hematopoietic progenitor cell; hHPC, human HPC; mHPC, mouse HPC; HMEC, human dermal microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; IKK, IB kinase; NIK, IB-inducing kinase; EMSA, electrophoretic mobility shift assay; GM-CSF, granulocyte/macrophage-colony stimulating factor; RMC, rat glomerular mesangial cell; GST, glutathione S-transferase.
Received 8/ 8/00. Accepted 1/ 4/01.
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Z. F. Yang, R. T. Poon, Y. Luo, C. K. Cheung, D. W. Ho, C. M. Lo, and S. T. Fan Up-Regulation of Vascular Endothelial Growth Factor (VEGF) in Small-for-Size Liver Grafts Enhances Macrophage Activities through VEGF Receptor 2-Dependent Pathway J. Immunol., August 15, 2004; 173(4): 2507 - 2515. [Abstract] [Full Text] [PDF]
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P. Monti, B. E. Leone, A. Zerbi, G. Balzano, S. Cainarca, V. Sordi, M. Pontillo, A. Mercalli, V. Di Carlo, P. Allavena, et al. Tumor-Derived MUC1 Mucins Interact with Differentiating Monocytes and Induce IL-10highIL-12low Regulatory Dendritic Cell J. Immunol., June 15, 2004; 172(12): 7341 - 7349. [Abstract] [Full Text] [PDF]
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 Y. Wang, J. Chang, Y.-C. Li, Y.-S. Li, J. Y.-J. Shyy, and S. Chien Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H685 - H692. [Abstract] [Full Text] [PDF]
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Y. Nefedova, M. Huang, S. Kusmartsev, R. Bhattacharya, P. Cheng, R. Salup, R. Jove, and D. Gabrilovich Hyperactivation of STAT3 Is Involved in Abnormal Differentiation of Dendritic Cells in Cancer J. Immunol., January 1, 2004; 172(1): 464 - 474. [Abstract] [Full Text] [PDF]
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P. Cheng, Y. Nefedova, L. Miele, B. A. Osborne, and D. Gabrilovich Notch signaling is necessary but not sufficient for differentiation of dendritic cells Blood, December 1, 2003; 102(12): 3980 - 3988. [Abstract] [Full Text] [PDF]
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 D. K. Biswas, S.-C. Dai, A. Cruz, B. Weiser, E. Graner, and A. B. Pardee The nuclear factor kappa B (NF-kappa B): A potential therapeutic target for estrogen receptor negative breast cancers PNAS, August 17, 2001; (2001) 151257998. [Abstract] [Full Text] [PDF]
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D. K. Biswas, S.-C. Dai, A. Cruz, B. Weiser, E. Graner, and A. B. Pardee The nuclear factor kappa B (NF-kappa B): A potential therapeutic target for estrogen receptor negative breast cancers PNAS, August 28, 2001; 98(18): 10386 - 10391. [Abstract] [Full Text] [PDF]
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