Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dikov, M. M.
Right arrow Articles by Carbone, D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dikov, M. M.
Right arrow Articles by Carbone, D. P.
[Cancer Research 61, 2015-2021, March 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Vascular Endothelial Growth Factor Effects on Nuclear Factor-{kappa}B Activation in Hematopoietic Progenitor Cells1

Mikhail M. Dikov2, Tsunehiro Oyama2, Pingyan Cheng, Takamune Takahashi, Keiko Takahashi, Tanya Sepetavec, Byard Edwards, Yasushi Adachi, Sorena Nadaf, Thomas Daniel, Dmitry I. Gabrilovich and David P. Carbone3

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.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth factor (VEGF) inhibits of the activation of transcription factor nuclear factor-{kappa}B (NF-{kappa}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-{kappa}B was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor {alpha} (TNF-{alpha}) 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-{alpha}-induced NF-{kappa}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-{kappa}B activation. However, although TNF-{alpha} induced rapid activation of I{kappa}B kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of I{kappa}B{alpha} phosphorylation and degradation of I{kappa}B{alpha} and I{kappa}B{epsilon} in HPCs. VEGF alone, however, did not have any effect on phosphorylation of I{kappa}B{alpha} or degradation of I{kappa}B{alpha} and other inhibitory molecules I{kappa}Bß, I{kappa}B{epsilon}, 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-{alpha}-induced NF-{kappa}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
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VEGF4 is a Mr 34,000–43,000 protein that stimulates endothelial cell growth and plays a critical role in angiogenesis (1, 2, 3) . VEGF production is dramatically increased in many pathological conditions, especially local hypoxia (4, 5, 6) . VEGF plays an especially important role in cancer. Almost all tumor cells produce VEGF, and elevated levels are frequently detected in sera from cancer patients. VEGF production is closely associated with a poor prognosis (6, 7, 8, 9) . We have demonstrated previously that, in addition to its effects on angiogenesis, VEGF also binds to hematopoietic progenitor cells through the VEGF receptor 1 (Flt1; Ref. 10 ). This results in inhibition of the maturation of dendritic cells, the professional antigen-presenting cells, in vitro and in vivo, and dramatically affects the maturation of other hematopoietic cells (11 , 12) .

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-{alpha}-inducible activation of the transcription factor NF-{kappa}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 I{kappa}B family. Cell activation by various stimuli, including TNF-{alpha}, lipopolysaccharide, interleukin 1, CD40, and others, results in serine phosphorylation and degradation of IkB with subsequent nuclear translocation and specific DNA binding of NF-{kappa}B dimers (14) . It has also been suggested that tyrosine phosphorylation of I{kappa}B{alpha} can activate NF-{kappa}B without degradation (15) . NF-{kappa}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-{kappa}B could be responsible for the observed effects of VEGF on HPCs. However, it is unclear how VEGF might affect NF-{kappa}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-{kappa}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-{kappa}B complexes, but that RelB is not required for VEGF to inhibit NF-{kappa}B activation by TNF-{alpha}. 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-{kappa}B inhibitory subunits I{kappa}B{alpha} and I{kappa}B{epsilon} in HPCs.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Female BALB/c and C57BL/6 mice, 6–8 weeks of age, were purchased from Harlan, Inc. (Indianapolis, IN) and were housed in specific pathogen-free units of the Division of Animal Care at Vanderbilt University Medical Center. RelB-deficient mice were obtained from Dr. D. Lo (The Scripps Research Institute, La Jolla, CA). These mice were generated on an inbred C57BL/6J background and were described in detail elsewhere (19) .

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-{alpha}, and VEGF165 were obtained from R&D Systems (Minneapolis, MN); polyclonal antimouse immunoglobulin was obtained from Sigma.

All anti-NF-{kappa}B antibodies used for EMSA (anti-p50, p52, c-Rel, Rel-A, and Rel-B antisera) as well as anti-I{kappa}B{epsilon} antiserum were generous gifts from Dr. N. Rice (ABL-Basic Research Program, National Cancer Institute, NIH). Anti-I{kappa}B{alpha}, -I{kappa}Bß, -I{kappa}B{gamma}, -VEGFR2 (Flk1), and -IKK{alpha} antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine (4G10) was from New England Biolabs, and anti-Ser-36 I{kappa}B{alpha} was from Upstate Biotechnology, Inc.

Portions of mutant and wild-type I{kappa}B{alpha} (amino acids 1–51) were amplified by PCR from pBS(SK-)-I{kappa}B{alpha} containing full-length wild-type or mutant I{kappa}B{alpha}. In mutant I{kappa}B{alpha}, 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-I{kappa}B{alpha} 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 {kappa}B probes were used:

For the murine intronic {kappa}-chain KB site: wild type (5'-AGTTGAGGGGACTTTCCCAGG) and mutant {kappa}B site (5'-AGTTGAGGCGACTTTCCCAGG; and for the palindromic {kappa}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 I{kappa}B.
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{alpha} 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-I{kappa}B{alpha} (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 [{gamma}-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{alpha} substrate was used to confirm the specificity of reaction. Membrane was also probed with anti-IKK{alpha} antibodies to check for the equality of loading.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF Effects on NF-{kappa}B Activation in Different Types of Cells.
Previously, we have reported VEGF binding to one of its specific receptors, VEGFR1, on the surface of hHPCs. This resulted in the inhibition of TNF-{alpha}-dependent NF-{kappa}B activation in these cells (10) . We asked whether VEGF effects on NF-{kappa}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-{alpha} receptor and VEGFR1 (data not shown). TNF-{alpha} induced strong NF-{kappa}B activation in all tested types of cells (Fig. 1Citation , Lanes 2 and 3 versus Lane 1). VEGF alone also activated NF-{kappa}B in both HPCs and HPCs and RMCs (Fig. 1Citation , Lanes 4 and 5 versus Lane 1); however, this effect was weaker than the effect of TNF-{alpha}. No VEGF-inducible NF-{kappa}B activation was detected in HMEC-1 cells. In agreement with our data reported previously, VEGF prevented TNF-{alpha}-dependent NF-{kappa}B activation in human HPCs. The same effect was observed in mHPCs and RMCs (Fig. 1Citation , Lane 6 versus Lane 3). This effect was observed when VEGF was added at the same time with TNF-{alpha} or within 10 min before TNF-{alpha}. Addition of VEGF 10 min after TNF-{alpha} did not affect NF-{kappa}B activation (data not shown). No effect of VEGF on TNF-{alpha}-inducible NF-{kappa}B activation was observed in HMEC-1 cells. These data indicate that the effects of VEGF on NF-{kappa}B are not restricted to HPCs.



View larger version (45K):
[in this window]
[in a new window]
  		Fig. 1. VEGF effects on NF-{kappa}B are dependent on cell type. Nuclear extracts were obtained from the cell types indicated. EMSA was performed as described in "Materials and Methods." The palindromic {kappa}B probe was used in experiments with hHPCs and HMEC-1 cells, and the murine intronic {kappa}B probe was used in experiments with HPCs and RMCs. Human recombinant TNF-{alpha} and human recombinant VEGF were used in experiments with hHPCs and HMEC-1; murine recombinant TNF-{alpha} and VEGF were used in experiments with mHPECs and RMCs. Lane C, competition with 100-fold excess of cold probe; Lane 1, control untreated cells; Lane 2, cells treated with 10 ng/ml TNF-{alpha} for 10 min; Lane 3, cells treated with 10 ng/ml TNF-{alpha} for 20 min; Lane 4, treatment with 100 ng/ml VEGF for 10 min; Lane 5, treatment with 100 ng/ml VEGF for 20 min; Lane 6, 20 min of combined treatment with 100 ng/ml VEGF and 10 ng/ml TNF-{alpha} added simultaneously; Lane 7, 20 min of combined treatment with 100 ng/ml VEGF and 10 ng/ml TNF-{alpha} (VEGF was added 10 min before TNF-{alpha}). At least four experiments with similar results were performed for each cell type.

 
Effects of VEGF and TNF-{alpha} on Nuclear Translocation and DNA Binding of NF-{kappa}B Subunits.
We asked whether VEGF and TNF-{alpha} induced translocation of the same members of the NF-{kappa}B family in HPCs. Using supershift EMSA, we found striking differences in the translocation and DNA binding of two members of the NF-{kappa}B family, p65 (RelA) and RelB, in response to treatment with VEGF or TNF-{alpha}. Significant levels of p65-containing complexes were detected in hHPCs after treatment with TNF-{alpha} but not with VEGF (Fig. 2Citation , 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. 2Citation , 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-{alpha} 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. 2Citation , compare Lanes 2 and 3 in the p65 supershift).



View larger version (57K):
[in this window]
[in a new window]
  		Fig. 2. VEGF and TNF-{alpha} induce the nuclear translocation of distinct NF-{kappa}B subunits in hHPCs. Nuclear extracts were obtained from hHPCs, and supershift EMSA was performed as described in "Materials and Methods." p65 and RelB depict the type of antibody used in supershift. For each antibody: Lane 1, control untreated cells; Lane 2, cells treated with 10 ng/ml TNF-{alpha} for 20 min; Lane 3, 100 ng/ml VEGF was added 10 min before TNF-{alpha}; Lane 4, 100 ng/ml VEGF was added 10 min after TNF-{alpha}; Lane 5, cells were treated with 100 ng/ml VEGF alone for 20 min. Three independent experiments with similar results were performed.

 
To confirm these differences, nuclear translocation of p65 and RelB subunits of NF-{kappa}B was visualized using immunofluorescent confocal microscopy. As shown in Fig. 3Citation , treatment of hHPCs with TNF-{alpha} 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)Citation . 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-{alpha}, 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-{alpha}-mediated NF-{kappa}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-{alpha} and VEGF, and NF-{kappa}B-specific DNA binding was analyzed using EMSA. VEGF blocked TNF-{alpha}-induced NF-{kappa}B activation in mHPCs isolated from RelB+/- mice (Fig. 4)Citation , very similar to its effect in the control RelB+/+ mice used in the experiments described in Fig. 1Citation . The same effect was observed in RelB-/- mice (Fig. 4)Citation . These data indicate that RelB does not play a direct role in the inhibition of NF-{kappa}B activation by VEGF.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Nuclear translocation of p65 and RelB in response to treatment with TNF-{alpha} or VEGF. hHPCs were obtained as described in "Materials and Methods." Cells were treated for 20 min with either 10 ng/ml TNF-{alpha} or with 100 ng/ml VEGF. Cells were fixed, stained, and analyzed using confocal microscopy as described in "Materials and Methods." Anti-p65 antibody panels, cells stained with anti-p65 antibody; Anti-RelB antibody panels, cells stained with anti-RelB antibody. TNF-{alpha} and VEGF in each panel showed cells treated with either of these factors. Two independent experiments with the same results were performed.

 


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 4. VEGF inhibits NF-{kappa}B activation in HPC from RelB-/- mice. HPCs were isolated from the bone marrow of RelB+/- and RelB-/- mice. Cells were either untreated (Lane 1); treated with 10 ng/ml TNF-{alpha} for 20 min (Lane 2); or treated with 100 ng/ml VEGF and TNF-{alpha} (Lane 3). VEGF and TNF-{alpha} were added simultaneously. M, mutant {kappa}B probe. Nuclear extracts were obtained, and EMSA was performed as described in "Materials and Methods." Three experiments with similar results were performed for each cell type.

 
VEGF Effects on NF-{kappa}B Inhibitory Subunits I{kappa}B.
In quiescent cells, NF-{kappa}B is bound in cytoplasm to inhibitor proteins I{kappa}B. Serine phosphorylation of these molecules in response to various signals results in their ubiquitin-mediated degradation and the consequent release of NF-{kappa}B dimers. NF-{kappa}B dimers are then able to translocate to the nucleus and bind DNA. This is the best understood mechanism of regulation of NF-{kappa}B activity. In the next group of experiments, we studied the effect of VEGF on these natural inhibitors of NF-{kappa}B. TNF-{alpha} is a very potent inducer of I{kappa}B{alpha} serine phosphorylation by IKK, resulting in its degradation within minutes of exposure. VEGF alone did not induce I{kappa}B{alpha} degradation in any of the studied types of cells. However, pretreatment of HPCs or RMCs, but not HMEC-1, with VEGF substantially inhibited TNF-{alpha}-induced I{kappa}B{alpha} degradation (Fig. 5)Citation . We have tested the effect of TNF-{alpha} and VEGF on other known inhibitors of NF-{kappa}B I{kappa}B{epsilon}, I{kappa}Bß, and Bcl-3 in hHPCs. VEGF alone did not induce degradation of any of these proteins (data not shown). Treatment of hHPCs with TNF-{alpha} alone resulted in the degradation of I{kappa}B{epsilon} but not of IkBß or Bcl-3 (Fig. 6)Citation . VEGF prevented TNF-{alpha}-inducible degradation of IkB{epsilon} (Fig. 6)Citation .



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 5. VEGF inhibits I{kappa}B{alpha} degradation. Cells were obtained as described in "Materials and Methods." The cells were treated with 10 ng/ml TNF-{alpha} or 100 ng/ml VEGF for the indicated amount of time (min). -10, VEGF was added 10 min before TNF-{alpha}; -1, VEGF was added together with TNF-{alpha}. Cells were lysed, and Western blots were performed as described in "Materials and Methods." Membranes were probed with anti-I{kappa}B{alpha} antibody and visualized using the ECL system. At least four experiments with similar results were performed for each cell type.

 


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6. VEGF effect on other members of I{kappa}B family. hHPCs were obtained and treated as described above. C, control untreated cells; T, cells treated with TNF-{alpha} alone (10 ng/ml) for 20 min; T + V, cells were treated with 100 ng/ml and TNF-{alpha}. -10, VEGF was added 10 min before TNF-{alpha}; -1, VEGF was added together with TNF-{alpha}. Cellular extract was prepared as described in "Materials and Methods." The membranes were probed with the appropriate antibodies. A representative example of three similar experiments is shown.

 
To study the mechanism of this inhibition, we evaluated the level of serine and tyrosine phosphorylation of I{kappa}B{alpha}. VEGF reduced the level of TNF-{alpha}-inducible Ser-32 phosphorylation in both hHPCs and mHPCs as was determined by anti-phosphoserine antibodies (Fig. 7)Citation . It has been reported that site-specific tyrosine phosphorylation of I{kappa}B{alpha} may inhibit the inducible serine phosphorylation and degradation of I{kappa}B{alpha} (28) . We therefore tested whether VEGF might induce tyrosine phosphorylation of I{kappa}B{alpha}. hHPCs were treated with VEGF for various periods of time (5–60 min). I{kappa}B{alpha} was then immunoprecipitated from cell lysates with anti-I{kappa}B{alpha} 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 I{kappa}B{alpha} was detected (data not shown).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7. VEGF inhibits serine phosphorylation of I{kappa}B{alpha} in HPCs. Cells were treated with 10 ng/ml TNF-{alpha} (T), 100 ng/ml VEGF (V), or both together (T + V) for 10 min. C, control untreated cells. Cells were lysed, and Western blots were performed as described in "Materials and Methods." Membranes were probed with anti-Ser-32 I{kappa}B{alpha} antibody and visualized using ECL. Two experiments with the similar results were performed for each cell type.

 
VEGF Inhibits TNF-{alpha}-dependent Activation of IKK.
The activation of NF-{kappa}B by TNF-{alpha} 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 I{kappa}B proteins. We studied the effect of VEGF on IKK activity by in vitro kinase assays using GST-I{kappa}B{alpha} fusion protein as a substrate. In both hHPCs and mHPCs, VEGF remarkably inhibited TNF-{alpha}-induced activation of IKK but did not show any effect when it was added alone (Fig. 8)Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 8. VEGF inhibits IKK activation in HPCs. Cells were treated with 10 ng/ml TNF-{alpha} (T), 100 ng/ml VEGF (V), or both together (T + V) for 15 min. Cells were lysed, and an in vitro IKK assay was performed as described in "Materials and Methods." C, control untreated cells; M, mutant GST-I{kappa}B{alpha} substrate was used with TNF-{alpha}-treated sample. The representative of three experiments is shown.

 
VEGFR2 Tyrosine Kinase Inhibition Does Not Block the Inhibitory Effect of VEGF on IKK Activation.
We have shown the presence of VEGFR1 mRNA transcripts in human and mouse progenitor cells and specific binding of VEGF to the surface of HPCs (10 , 11) . Others have detected VEGFR2 mRNA on hematopoietic cells (31) . Recently, using antibodies to the VEGFR extracellular domains, distinct populations of HPCs expressing each of the receptors were identified (32, 33, 34) . To determine whether the effect of VEGF on IKK activation was mediated via the tyrosine kinase activity of VEGFR, we used SU5416, a potent inhibitor of the tyrosine kinase of VEGFR (35 , 36) . SU5416 completely blocked tyrosine autophosphorylation of VEGFR2 in HUVECs stimulated by VEGF but did not abolish the inhibitory effects of VEGF on IKK activation by TNF-{alpha} (Fig. 9)Citation .



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 9. The VEGFR tyrosine kinase inhibitor SU5416 prevents phosphorylation of VEGFR2 but does not abolish the inhibitory effect of VEGF on IKK activation. Cells were cultured with or without 4 µM VEGF tyrosine kinase inhibitor SU5416 in serum-free medium for 1 h. mHPCs then were treated with 10 ng/ml TNF-{alpha} (T), 100 ng/ml VEGF (V), or both together (T + V) for 15 min and lysed. In vitro IKK assay was performed as described in "Materials and Methods." HUVECs were treated with 100 ng/ml VEGF for 5 min and lysed, and Western blots were performed as described in "Materials and Methods." Membranes were probed with anti-pTyr antibodies or anti-VEGFR2. C, control untreated cells. Two experiments with similar results were performed for each cell type.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
VEGF plays an essential role in both blood vessel formation and hematopoiesis during normal embryonic development. In adult animals, it is induced by local hypoxia and may play a role in wound repair and tissue remodeling. However, increased production of VEGF has been found in many pathological conditions (4, 5, 6, 7) . An especially important role is attributed to VEGF in cancer. VEGF is produced by the majority of tumors and plays a critical role in the development of tumor neovasculature. Elevated levels of VEGF in the plasma of cancer patients are closely associated with an adverse prognosis (8 , 9) . Recently, we have demonstrated that VEGF produced by tumors may also have effects on hematopoiesis and in particular result in the inhibition of functional maturation of dendritic cells, the professional antigen-presenting cells (11 , 12) . We have demonstrated that a close correlation exists between increased levels of VEGF produced by breast tumor cells and the decreased development of functional dendritic cells. It has been reported recently that elevated levels of VEGF in the vicinity of tumors is associated with decreased tumor infiltration by dendritic cells in patients with gastric cancer (37) . Chronic infusion of recombinant VEGF into non-tumor-bearing animals results in dramatic changes in the development of other hematopoietic lineages as well as dendritic cells (12) . Thus, VEGF could be an important factor in the abnormal regulation of hematopoietic cell differentiation in a variety of pathological conditions. Understanding the mechanism by which VEGF affects the HPCs could give insights into both normal and pathological hematopoiesis. VEGF is thought to bind to HPCs via the specific receptors VEGFR1 and VEGFR2. This results in inhibition of TNF-{alpha}-inducible activation of transcription factor NF-{kappa}B (10) . Previous data suggested that VEGF might exert its effect via inhibition of NF-{kappa}B activation, which is essential for dendritic cell differentiation. In this study, we have tried to elucidate the mechanism of VEGF effects on NF-{kappa}B.

We first asked whether the observed effects of VEGF on NF-{kappa}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-{alpha} (40) and VEGFR1 receptors (41) . Not surprisingly, TNF-{alpha} induced NF-{kappa}B activation in all of these types of cells. VEGF alone also showed some activation of NF-{kappa}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-{alpha}-inducible NF-{kappa}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-{kappa}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-{kappa}B subunit activation induced by VEGF or TNF-{alpha} using a supershift EMSA assay. TNF-{alpha} induced nuclear translocation and DNA binding of the complexes containing predominantly p65. Preferential induction of p65 by TNF-{alpha} has been described previously in many reports (42, 43, 44) . NF-{kappa}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-{kappa}B complexes after combined stimulation with TNF-{alpha} 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-{alpha} to induce p65-containing NF-{kappa}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-{kappa}B activation by VEGF, we used HPCs obtained from RelB knockout mice. Our experiments demonstrated that VEGF was able to inhibit TNF-{alpha}-inducible NF-{kappa}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-{kappa}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-{alpha}. Decreased IKK activity prevents efficient serine phosphorylation of I{kappa}B and its ubiquitin proteasome-dependent degradation. The large Mr ~700,000 IKK complex that phosphorylates I{kappa}B proteins in response to proinflammatory signals consists of two catalytic subunits, IKK{alpha} and IKKß, and a regulatory subunit IKK{gamma} (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-{alpha} (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-{kappa}B. The potential involvement of phosphatases in the regulation of NF-{kappa}B signaling was demonstrated recently by association of the c-Rel subunit of NF-{kappa}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 I{kappa}B proteins, especially I{kappa}B{alpha} and I{kappa}B{epsilon}, which are readily degraded upon TNF-{alpha} stimulation. One possible explanation is that I{kappa}B{alpha} has high affinity for p65-containing complexes (14) but much less affinity for RelB-containing complexes, which are also less susceptible to inhibition by I{kappa}B{alpha} (51 , 52) . VEGF could also induce tyrosine phosphorylation of I{kappa}B{alpha} or I{kappa}B{epsilon} and dissociation of NF-{kappa}B complexes without I{kappa}B degradation, the mechanism described recently by Imbert et al. (15) . However, we could not detect tyrosine phosphorylation of I{kappa}B{alpha} induced by VEGF. Not excluded is the possibility that in HPCs, RelB is predominantly associated with another, as yet unknown, member of the I{kappa}B family, resulting in RelB release upon stimulation with VEGF but not TNF-{alpha}.

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-{kappa}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-{kappa}B activation: nuclear translocation and DNA binding of complexes containing predominantly RelB, and receptor tyrosine kinase-independent inhibition of TNF-{alpha}-dependent activation of IKK, leading to a decrease in I{kappa}B{alpha} serine phosphorylation and degradation of I{kappa}B{alpha} and I{kappa}B{epsilon}. These studies may have identified a new mechanism for the regulation of NF-{kappa}B and for VEGF effects on NF-{kappa}B in hematopoietic progenitor cells.


   ACKNOWLEDGMENTS
 
We thank Dr. D. Lo for providing us with RelB knockout mice, and we thank Sugen for providing the SU5416.


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

1 This study was supported by NIH Grant CA 76321 (to D.P.C.). Back

2 The first two authors contributed equally to this work. Back

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 Back

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, I{kappa}B kinase; NIK, I{kappa}B-inducing kinase; EMSA, electrophoretic mobility shift assay; GM-CSF, granulocyte/macrophage-colony stimulating factor; RMC, rat glomerular mesangial cell; GST, glutathione S-transferase. Back

Received 8/ 8/00. Accepted 1/ 4/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Leung D. W., Cachianes G., Kuang W.-J., Goeddel D. V., Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (Washington DC), 246: 1306-1309, 1989.[Abstract/Free Full Text]
  2. Connolly D. T., Heuvelman D. M., Nelson R., Olander J. V., Eppley B. L., Delfino J. J., Siegel N. R., Leimgruber R. M., Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J. Clin. Investig., 84: 1470-1478, 1989.
  3. Takeshita S., Zheng L. P., Brogi E., Kearney M., Pu L-Q., Bunting S., Ferrara N., Symes J. F., Isner J. M. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J. Clin. Investig., 93: 662-670, 1994.
  4. Shweiki D., Itin A., Soffer D., Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature (Lond.), 359: 843-845, 1992.[Medline]
  5. Tuder R. M., Flook B. E., Voelkel N. F. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. Modulation of gene expression by nitric oxide. J. Clin. Investig., 95: 1798-1807, 1995.
  6. Ferrara N., Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr. Rev., 18: 4-25, 1997.[Abstract/Free Full Text]
  7. Toi M., Taniguchi T., Yamamoto Y., Kurisaki T., Suzuki H., Tominaga T. Clinical significance of the determination of angiogenic factors. Eur. J. Cancer, 32A: 2513-2519, 1996.
  8. Ellis L. M., Liu W., Wilson M. Down-regulation of vascular endothelial growth factor in human colon carcinoma cell lines by antisense transfection decreases endothelial cell proliferation. Surgery, 120: 871-878, 1996.[Medline]
  9. Takano S., Yoshii Y., Kondo S., Suzuki H., Maruno T., Shirai S., Nose T. Concentration of vascular endothelial growth factor in the serum and tumor tissue of brain tumor patients. Cancer Res., 56: 2185-2190, 1996.[Abstract/Free Full Text]
  10. Oyama T., Ran S., Ishida T., Nadaf S., Kerr L., Carbone D. P., Gabrilovich D. I. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-{kappa}B activation in hemopoietic progenitor cells. J. Immunol., 160: 1224-1232, 1998.[Abstract/Free Full Text]
  11. Gabrilovich D. I., Chen H. L., Girgis K. R., Cunningham T., Meny G. M., Nadaf S., Kavanaugh D., Carbone D. P. Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat. Med., 2: 1096-1103, 1996.[Medline]
  12. Gabrilovich D., Ishida T., Oyama T., Ran S., Kravtsov V., Nadaf S., Carbone D. P. Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood, 92: 4150-4166, 1998.[Abstract/Free Full Text]
  13. Liou H. C., Baltimore D. Regulation of the NF-{kappa}B/rel transcription factor and I{kappa}B inhibitor system. Curr. Opin. Cell Biol., 5: 477-487, 1993.[Medline]
  14. Verma I. M., Stevenson J. K., Schwarz E. M., Van Antwerp D., Miyamoto S. Rel/NF-{kappa}B/I{kappa}B family: intimate tales of association and dissociation. Genes Dev., 9: 2723-2735, 1995.[Free Full Text]
  15. Imbert V., Rupec R. A., Livolsi A., Pahl H. L., Traenckner E. B., Mueller-Dieckmann C., Farahifar D., Rossi B., Auberger P., Baeuerle P. A., Peyron J. F. Tyrosine phosphorylation of I{kappa}B{alpha} activates NF-{kappa}B without proteolytic degradation of I{kappa}B{alpha}. Cell, 86: 787-798, 1996.[Medline]
  16. Caamano J. H., Rizzo C. A., Durham S. K., Barton D. S., Raventos-Suarez C., Snapper C. M., Bravo R. Nuclear factor (NF)-{kappa}-B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med., 187: 185-196, 1998.[Abstract/Free Full Text]
  17. Franzoso G., Bours V., Park S., Tomita-Yamaguchi M., Kelly K., Siebenlist U. The candidate oncoprotein Bcl-3 is an antagonist of p50/NF-{kappa}B-mediated inhibition. Nature (Lond.), 359: 339-342, 1992.[Medline]
  18. Carrasco D., Ryseck R. P., Bravo R. Expression of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells. Development (Camb.), 118: 1221-1231, 1993.[Abstract]
  19. Burkly L., Hession C., Ogata L., Reilly C., Marconi L. A., Olson D., Tizard R., Cate R., Lo D. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature (Lond.), 373: 531-536, 1995.[Medline]
  20. Boehmelt G., Madruga J., Dorfler P., Briegel K., Schwarz H., Enrietto P. J., Zenke M. Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-RelER. Cell, 80: 341-352, 1995.[Medline]
  21. Esslinger C. W., Wilson A., Sordat B., Beermann F., Jongeneel C. V. Abnormal T lymphocyte development induced by targeted overexpression of I{kappa}B{alpha}. J. Immunol., 158: 5075-5078, 1997.[Abstract]
  22. Kitamura M., Mitarai T., Maruyama N., Nagasawa R., Yoshida H., Sakai O. Mesangial cell behavior in a three-dimensional extracellular matrix. Kidney Int., 40: 653-661, 1991.[Medline]
  23. Ades E. W., Candal F. J., Swerlick R. A., George V. G., Summers S., Bosse D. C., Lawley T. J. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J. Investig. Dermatol., 99: 683-690, 1992.[Medline]
  24. Osborn L., Kunkel S., Nabel G. J. Tumor necrosis factor {alpha} and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor {kappa}B. Proc. Natl. Acad. Sci. USA, 86: 2336-2340, 1989.[Abstract/Free Full Text]
  25. Chen H. L., Gabrilovich D., Tampe R., Girgis K. R., Nadaf S., Carbone D. P. A functionally defective allele of TAP1 results in loss of MHC class I antigen presentation in a human lung cancer. Nat. Genet., 13: 210-213, 1996.[Medline]
  26. Devalaraja M. N., Wang D. Z., Ballard D. W., Richmond A. Elevated constitutive I{kappa}B kinase activity and I{kappa}B{alpha} phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-{alpha} transcription. Cancer Res., 59: 1372-1377, 1999.[Abstract/Free Full Text]
  27. Ling L., Cao Z., Goeddel D. V. NF-{kappa}B-inducing kinase activates IKK-{alpha} by phosphorylation of Ser-176. Proc. Natl. Acad. Sci. USA, 95: 3792-3797, 1998.[Abstract/Free Full Text]
  28. Singh S., Darnay B. G., Aggarwal B. B. Site-specific tyrosine phosphorylation of I{kappa}B{alpha} negatively regulates its inducible phosphorylation and degradation. J. Biol. Chem., 271: 31049-31054, 1996.[Abstract/Free Full Text]
  29. Delhase M., Hayakawa M., Chen Y., Karin M. Positive and negative regulation of I{kappa}B kinase activity through IKKß subunit phosphorylation [see comments]. Science (Washington DC), 284: 309-313, 1999.[Abstract/Free Full Text]
  30. Li Z. W., Chu W., Hu Y., Delhase M., Deerinck T., Ellisman M., Johnson R., Karin M. The IKKß subunit of I{kappa}B kinase (IKK) is essential for nuclear factor {kappa}B activation and prevention of apoptosis. J. Exp. Med., 189: 1839-1845, 1999.[Abstract/Free Full Text]
  31. Broxmeyer H. E., Cooper S., Li Z. H., Lu L., Song H-Y., Kwon B. S., Warren R. E., Donner D. B. Myeloid progenitor cell regulatory effects of vascular endothelial cell growth factor. Int. J. Hematol., 62: 203-215, 1995.[Medline]
  32. Ziegler B. L., Valtieri M., Porada G. A., De Maria R., Muller R., Masella B., Gabbianelli M., Casella I., Pelosi E., Bock T., Zanjani E. D., Peschle C. KDR receptor: a key marker defining hematopoietic stem cells. Science (Washington DC), 285: 1553-1558, 1999.[Abstract/Free Full Text]
  33. Peichev M., Naiyer A. J., Pereira D., Zhu Z., Lane W. J., Williams M., Oz M. C., Hicklin D. J., Witte L., Moore M. A., Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood, 95: 952-958, 2000.[Abstract/Free Full Text]
  34. Buhring, H. J., Seiffert, M., Bock, T. A., Scheding, S., Thiel, A., Scheffold, A., Kanz, L., and Brugger, W. Expression of novel surface antigens on early hematopoietic cells. Ann. NY Acad. Sci., 872: 25-38; discussion 38-39, 1999.
  35. Holmgren L., O’Reilly M. S., Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression[see comments]. Nat. Med., 1: 149-153, 1995.[Medline]
  36. Fong T. A., Shawver L. K., Sun L., Tang C., App H., Powell T. J., Kim Y. H., Schreck R., Wang X., Risau W., Ullrich A., Hirth K. P., McMahon G. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res., 59: 99-106, 1999.[Abstract/Free Full Text]
  37. Saito H., Tsujitani S., Ikeguchi M., Maeta M., Kaibara N. Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue. Br. J. Cancer, 78: 1573-1577, 1998.[Medline]
  38. Pepper M. S., Wasi S., Ferrara N., Orci L., Montesano R. In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells. Exp. Cell Res., 210: 298-305, 1994.[Medline]
  39. Keck P. J., Hauser S. D., Krivi G., Sanzo K., Warren T., Feder J., Connolly D. T. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (Washington DC), 246: 1309-1312, 1989.[Abstract/Free Full Text]
  40. Pai R., Bassa B., Kirschenbaum M. A., Kamanna V. S. TNF-{alpha} stimulates monocyte adhesion to glomerular mesangial cells. The role of intercellular adhesion molecule-1 gene expression and protein kinases. J. Immunol., 156: 2571-2579, 1996.[Abstract]
  41. Takahashi T., Shirasawa T., Miyake K., Yahagi Y., Maruyama N., Kasahara N., Kawamura T., Matsumura O., Mitarai T., Sakai O. Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells: Flt-1 and VEGF expression in renal mesangial cells. Biochem. Biophys. Res. Commun., 209: 218-226, 1995.[Medline]
  42. Munoz E., Blazquez M. V., Ortiz C., Gomez-Diaz C., Navas P. Role of ascorbate in the activation of NF-{kappa}B by tumour necrosis factor-{alpha} in T-cells. Biochem. J., 325: 23-28, 1997.
  43. Steffan N. M., Bren G. D., Frantz B., Tocci M. J., O’Neill E. A., Paya C. V. Regulation of I{kappa}B{alpha} phosphorylation by PKC- and Ca2+-dependent signal transduction pathways. J. Immunol., 155: 4685-46891, 1995.[Abstract]
  44. Menon S. D., Guy G. R., Tan Y. H. Involvement of a putative protein-tyrosine phosphatase and I{kappa}B{alpha} serine phosphorylation in nuclear factor {kappa}B activation by tumor necrosis factor. J. Biol. Chem., 270: 18881-18887, 1995.[Abstract/Free Full Text]
  45. Rothwarf D. M., Zandi E., Natoli G., Karin M. IKK{gamma} is an essential regulatory subunit of the I{kappa}B kinase complex [see comments]. Nature (Lond.), 395: 297-300, 1998.[Medline]
  46. Yamaoka S., Courtois G., Bessia C., Whiteside S. T., Weil R., Agou F., Kirk H. E., Kay R. J., Israel A. Complementation cloning of NEMO, a component of the I{kappa}B kinase complex essential for NF-{kappa}B activation. Cell, 93: 1231-1240, 1998.[Medline]
  47. Nakano H., Shindo M., Sakon S., Nishinaka S., Mihara M., Yagita H., Okumura K. Differential regulation of I{kappa}B kinase {alpha} and ß by two upstream kinases, NF-{kappa}B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. J. Proc. Natl. Acad. Sci. USA, 95: 3537-3542, 1998.[Abstract/Free Full Text]
  48. Kroll J., Waltenberger J. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J. Biol. Chem., 272: 32521-32527, 1997.[Abstract/Free Full Text]
  49. Huang L., Sankar S., Lin C., Kontos C. D., Schroff A. D., Cha E. H., Feng S. M., Li S. F., Yu Z., Van Etten R. L., Blanar M. A., Peters K. G. HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity. J. Biol. Chem., 274: 38183-38188, 1999.[Abstract/Free Full Text]
  50. Hu M. C., Tang-Oxley Q., Qiu W. R., Wang Y. P., Mihindukulasuriya K. A., Afshar R., Tan T. H. Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor {kappa}B activity. J. Biol. Chem., 273: 33561-33565, 1998.[Abstract/Free Full Text]
  51. Dobrzanski P., Ryseck R. P., Bravo R. Differential interactions of Rel-NF-{kappa}B complexes with I{kappa}B{alpha} determine pools of constitutive and inducible NF-{kappa}B activity. EMBO J., 13: 4608-4616, 1994.[Medline]
  52. Lernbecher T., Kistler B., Wirth T. Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. EMBO J., 13: 4060-4069, 1994.[Medline]
  53. Hiratsuka S., Minowa O., Kuno J., Noda T., Shibuya M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc. Natl. Acad. Sci. USA, 95: 9349-9354, 1998.[Abstract/Free Full Text]
  54. Mukhopadhyay D., Nagy J. A., Manseau E. J., Dvorak H. F. Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium. Cancer Res., 58: 1278-1284, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
Y. Huang, X. Chen, M. M. Dikov, S. V. Novitskiy, C. A. Mosse, L. Yang, and D. P. Carbone
Distinct roles of VEGFR-1 and VEGFR-2 in the aberrant hematopoiesis associated with elevated levels of VEGF
Blood, July 15, 2007; 110(2): 624 - 631.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Yang, Z. Cai, Y. Zhang, W. H. Yutzy IV, K. F. Roby, and R. B.S. Roden
CD80 in Immune Suppression by Mouse Ovarian Carcinoma-Associated Gr-1+CD11b+ Myeloid Cells.
Cancer Res., July 1, 2006; 66(13): 6807 - 6815.
[Abstract] [Full Text] [PDF]