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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:178-185.)
© 1999 American Heart Association, Inc.

Original Contributions

The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C- and Protein Kinase C-

Presented in part at the Annual Meeting of the American Society of Nephrology, San Diego, California, November 5–7, 1995.

Maren Wellner; Christian Maasch; Christine Kupprion; Carsten Lindschau; Friedrich C. Luft; Hermann Haller

From the Franz Volhard Clinic and Max Delbrück Center, Virchow Klinikum, Humboldt University, Berlin, Germany.

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Str 50, 13122 Berlin, Germany. E-mail haller{at}orion.rz.mdc-berlin.de

	Abstract

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Abstract—The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca²⁺]_i regulation and [Ca²⁺]_i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca²⁺]_i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca²⁺]_i followed by a sustained elevation. The sustained [Ca²⁺]_i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms , , , and were identified in endothelial cells. VEGF induced a translocation of PKC- and PKC- toward the nucleus and the perinuclear area, whereas cellular distribution of PKC- and PKC- was not influenced. Cell exposure to TPA led to a down-regulation of PKC- and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC- and PKC- with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca²⁺ influx. VEGF leads to the translocation of the [Ca²⁺]_i-sensitive PKC isoform and the atypical PKC isoform . Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms and are important for VEGF's angiogenic effects.

Key Words: VEGF • protein kinase C • isoforms • endothelial cells • cytosolic calcium • cell proliferation

	Introduction

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Vascular endothelial growth factor (VEGF) is a homodimeric 45-kDa glycoprotein that exerts proliferative effects on endothelial cells, induces angiogenesis, and is able to induce vascular permeability.^{1 2 3 4} Multiple VEGF isoforms with similar bioactivities, which are generated by differential mRNA splicing, have been described.² VEGF binds specifically to 2 receptors on endothelial cells, namely VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2).^{5 6 7} Targeted homozygous null mutations of VEGF receptors have demonstrated that VEGFR-1 (also known as flt-1) is mostly responsible for endothelial proliferation and angiogenesis.⁸ VEGFR-2 (also known as flk-1), on the other hand, plays a role in the initial differentiation of endothelial cells.⁹ The VEGF receptors belong to the family of membrane-bound tyrosine kinase receptors such as the PDGF and the fibroblast growth factor (FGF) receptor; viz, their intracellular signaling is mediated by tyrosine phosphorylation of several intracellular and membrane-bound proteins.¹⁰ Intracellular tyrosine phosphorylation is involved in VEGF-induced signaling.^{11 12} However, VEGF also induces an increase in intracellular free calcium concentration [Ca²⁺]_i.¹³ An effect of tyrosine kinase receptors on Ca²⁺ signaling also has been reported for the EGF and PDGF receptors.^{10 14} Kohn et al¹⁵ demonstrated that the [Ca²⁺]_i increase is important for FGF-induced endothelial cell proliferation. We characterized the VEGF-induced Ca²⁺ influx and investigated the hypothesis that a Ca²⁺-dependent signaling mechanism plays a role in the proliferative response to VEGF.

A distal step in the Ca²⁺ signaling pathway is the activation of the Ca²⁺ and phospholipid-dependent kinase, protein kinase C (PKC). PKC is activated by an increase in [Ca²⁺]_i and the generation of diacylglycerol (DAG).¹⁶ Activation of PKC plays a prominent role in the growth response of endothelial cells.¹⁷ Hu and Fan¹⁸ also have reported previously that incubation with PKC inhibitors can significantly decrease the proliferation of endothelial cells.

PKC is not a single entity but instead consists of several distinct isoforms with different regulatory and biochemical properties.^{19 20} These isoforms are expressed on separate genes and play different roles in cell signaling and cell function.¹⁹ They are grouped in 3 different categories according to their regulatory domains.²⁰ We recently found that the PKC isoforms , , , , and are present in endothelial cells.²¹ We therefore investigated the intracellular distribution of the PKC isoforms and tested the hypothesis that VEGF-induced activation of specific PKC isoforms is important for its proliferative effect.

	Methods

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Materials
Phorbol ester TPA, histone type III-S, DEAE-cellulose, and all other materials, if not stated otherwise, were purchased from Sigma. ³[H]thymidine was obtained from Amersham. The fluorescent probe fura-2 AM was purchased from Serva. The fluorescent probe fluo-3 was purchased from Molecular Probes. Recombinant VEGF was obtained from Seromed.

Preparation of Endothelial Cells
Human umbilical vein endothelial cells were isolated from umbilical cords by chymotrypsin treatment as previously described.^{21 22} The cords were cleaned with isotonic NaCl buffer at room temperature and incubated for 25 minutes at 37°C with 1% chymotrypsin in PBS (Seromed). Endothelial cells then were removed by centrifugation (400g for 10 minutes), and the pellet was resuspended in M-199 (Seromed) with 20% FCS, 1% L-glutamine, 1% nonessential amino acids (Seromed), 1% HEPES (Gibco), 1% Na-pyruvate, 1% Schutzmedium (Seromed), as well as streptomycin, and penicillin. Primarily cultured cells were grown for 3 to 4 days and then subcultured. Subcultures 1 to 2 were used for the experiments.

Thymidine Uptake
Proliferation was assessed by thymidine uptake. Endothelial cells were incubated with recombinant VEGF (10 ng/mL) for 18 hours, then ³[H]thymidine (0.5 µCi) was added and cells were incubated for an additional 6 hours. Cells then were washed several times and lysed in 1% SDS, and the incorporated radioactivity was determined via liquid scintillation counting.

Measurement of [Ca²⁺]_i in Single Cells
SPEX Fluorometry
For [Ca²⁺]_i measurements, endothelial cells were sowed on cover slips and incubated at 37°C for 1 to 2 days. Measurements of [Ca²⁺]_i in single cells were carried out as described previously.^{22 23} The [Ca²⁺]_i measurements were performed using a Spex DM 3000 CM spectrofluorometer, which was connected to a Nikon diaphot microscope and a variable-aperture photometer for isolating individual cells on the microscope stage (Spex Industries Inc). Cultured endothelial cells were loaded with fura-2 with a 20-minute incubation in PBS containing 5 µmol/L fura-2-AM (added from a 5-mmol/L stock solution in DMSO). Fluorescence of calcium-bound and unbound fura-2 was determined by rapidly alternating (0.1 s) the exciting radiation between 340 and 380 nm and electronically separating the resulting emission signals at 505 nm. The maximum fluorescence ratio (R_max) was determined by adding 40 µmol/L ionomycin (using a 0.01-M stock solution in DMSO). The minimal fluorescence (R_min) was obtained by adding 0.25 M EGTA at pH 7.8. The ratio of the 2 signals was used to calculate [Ca²⁺]_i as described elsewhere.²³ All experiments were carried out at room temperature.

[Ca²⁺]_i Imaging With Confocal Microscopy
For [Ca²⁺]_i imaging, endothelial cells grown on cover slips were incubated at 37°C for 1 to 2 days.^{21 24} The cells then were loaded with fluo-3 by a 20-minute incubation in PBS containing 5 µmol/L fluo-3-AM (added from a 5-mmol/L stock solution in DMSO) at 37°C. Before the measurements, cells were rinsed 3 times with PBS and mounted on a Nikon Diaphot inverted microscope. Measurements were performed with a Biorad MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon laser. We used a Nikon Plan Apo 100 objective with a numerical aperture of 1.4. Because the optical section depth depends on the confocal aperture, we selected a small aperture, 1.8 mm (range of opening width, 0.7 to 8 mm). We then assessed the Z-width half-maximum for this pinhole setting by measuring the distance between maximal and half-maximal intensity of a fluo-3 solution. The measured Z-width half-maximum was 0.45 mm.

PKC Activity
PKC activity was measured in cultured confluent cells. Cell cultures (in 90-mm dishes) were incubated in control medium (5% glucose) or with VEGF (10 ng/mL) for the described time periods. The cells were harvested, homogenized by sonification in 20 mmol/L Tris-HCl (pH 7.5), and incubated with 100 µL reaction buffer solution containing a pseudosubstrate and various phospholipids from a commercially available kit (PepTaq-Nonradioactive PKC activity kit, Promega). Samples were incubated for 30 minutes with the pseudosubstrate, the reaction terminated by heat and separated into phosphorylated and nonphosphorylated substrates on a 0.9% agarose gel. For quantification, bands were made visible by UV light and cut out, and absorbency was measured at 570 nm. The results are expressed in OD units.

Western Blotting
Western blot analysis was carried out as described previously.^{24 25} After the experiments, the cultured endothelial cells were treated with ice-cold homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 250 mmol/L sucrose, 7.5 mmol/L EGTA, 1 mmol/L EDTA, 10 mmol/L mercaptoethanol, 1 mmol/L phenylmethane-sulfonyl fluoride, and 50 µmol/L leupeptin). The homogenate then was spun in a TLA 100-2 rotor (Beckman) at 100 000 rpm for 10 minutes, and the supernatant was used as the cytosolic fraction. The pellet was resuspended in buffer containing 1.0% Triton X-100 and shaken at 4°C for 30 minutes. The homogenate then was diluted with buffer to a final concentration of 0.5% Triton X-100 and centrifuged at 100 000 rpm for an additional 10 minutes. The supernatant was used as the particulate fraction. Both PKC-containing fractions then underwent chromatography using 10% SDS-PAGE. Protein (10 to 30 µg) was loaded into each lane. The fractions then were electroblotted by the semi-dry technique onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). The membranes were incubated successively, first with blocking buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 10% nonfat dry milk powder (Merck), 0.2% (vol/vol) Tween-20, and 0.02% NaN₃ for 120 minutes at room temperature. The next incubation was conducted in affinity-purified, isoenzyme-specific antibody diluted in incubation buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 1% BSA at room temperature. We used highly specific polyclonal antibodies directed against peptide sequences of PKC that reacted specifically with the , , and subspecies of PKC (antibodies were from Gibco; 1:80 to 1:100); the antibody against PKC- was monoclonal and from UBI (1:200). A final incubation was carried out in TBS with alkaline phosphatase–conjugated anti-rabbit or anti-mouse IgG (Oncogene Science). The membranes were washed thoroughly after each incubation with a buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 0.2% (vol/vol) Tween-20. Immunoreactive bands were visualized by the enhanced chemiluminescence method (Amersham).

Immunocytochemistry
The techniques for confocal microscopy were as described previously.^{24 25} The cells were fixed with 3% paraformaldehyde and permeabilized with 80% methanol at -20°C. After incubation with 3% skim milk in PBS (SM/PBS) for 60 minutes, the preparation was incubated for 1 hour at room temperature with the PKC antibodies (see above) diluted in PBS with 0.1% BSA (1:80), washed 3 times with PBS, and then exposed to the secondary antibody (FITC-conjugated anti-rabbit or anti-mouse IgG, at 1:100, 1% BSA/PBS; Pierce Chemicals) for 60 minutes. The preparation was mounted with 50% glycerol under a glass coverslip on a Nikon-Diaphot microscope. A Biorad MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon laser was used. At least 10 to 18 cells from each of at least 7 experiments were examined under each experimental condition. The results were reproduced by 2 separate investigators, and multiple experiments were done. The observers were unaware of the experimental design and antibodies used.

Oligonucleotides
Phosphorothioate ODNs were purchased (TIB Molbiol). The antisense sequence used for PKC- (5'-GTTCTCGCTGGTGAGTTTCA-3') was derived from the 3' untranslated region from the human PKC- sequence. The sense ODN sequence (5'-TGAAACTCACCAGCGAGAAC-3') was used as a control. For human PKC-, the antisense oligonucleotide was directed against the AUG start codon of the human PKC- (5'-GCCGCTCCCTTCCAT-3'), and the corresponding sense ODN sequence (5'-ATCGAAGGGAGCGGC-3') was used as a control. To enhance the ODN uptake, we incubated the cells with lipofectin (10 µg/mL) and ODN (1 µmol/l) in the absence of FCS at 37°C for 4 hours. Afterward, the medium was changed back to 10% FCS for 24 hours.

Statistics
Statistical analysis was performed on a Macintosh computer (Apple Inc) using a commercially available statistics program (Statview, Cricket Software Inc). Because the data feature substantial variability and are not uniformly distributed, we used nonparametric statistical tests, such as the sign test and Mann Whitney test to analyze the data from the 7 to 10 separate experiments. A P value <0.05 was accepted as significant. References to increases or decreases in Results are only stated if statistically significant.

	Results

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We first investigated the effects of VEGF on [Ca²⁺]_i as shown in Figure 1. Shown are representative experiments from n12. VEGF induced a rapid increase of [Ca²⁺]_i followed by a sustained plateau phase for several minutes (Figure 1A). Preincubation with EGTA (10 mmol/L) for 1 minute (Figure 1B) did not influence the rapid [Ca²⁺]_i increase but abolished the sustained plateau phase, indicating that the plateau was generated by Ca²⁺ influx from the extracellular space. Preincubation of the cells with pertussis toxin (10^-5 M) also abolished the sustained plateau phase (Figure 1C). Thapsigargin abolished the initial rise in [Ca²⁺]_i (Figure 1D). However, the plateau phase continued to develop.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effects of VEGF (10 ng/mL) on the intracellular free calcium concentration [Ca2+]i in isolated human umbilical vein endothelial cells in control medium (A), after preincubation with EGTA (10 mmol/L) (B), after preincubation with pertussis toxin (10-5 M) (C), and after preincubation with thapsigargin (10-5 M) (D). The experiments demonstrate that the [Ca2+]i plateau phase is dependent on G protein–coupled Ca2+ entry, whereas the initial [Ca2+]i surge is related to Ca2+ release from stores.

Figure 2 shows a confocal micrograph of VEGF-stimulated fluo-3-AM–loaded endothelial cells at 0, 20, 40, and 60 s. Shown are representative experiments from n=20. A prompt [Ca²⁺]_i signal is visible in the cytoplasm and in the nucleus. At 40 s, the nuclear calcium signal is still visible, whereas at 60 s, the [Ca²⁺]_i signal has dissipated largely. We then measured PKC activity in the endothelial cells after exposure to VEGF as shown in Figure 3 (n=3). Total PKC activity was increased at 5 minutes by VEGF as shown in Figure 3 (P<0.05) and remained significantly increased at 10 minutes (P<0.05).

View larger version (61K): [in this window] [in a new window]   Figure 2. Confocal microscopy of the VEGF-induced changes in intracellular distribution of [Ca2+]i at 0, 20, 40, and 60 s in endothelial cells. [Ca2+]i is not only visible in the cytoplasm but is also prominent within the nucleus.

View larger version (37K): [in this window] [in a new window]   Figure 3. Effect of VEGF (10 ng/mL) on PKC activity in endothelial cells at 0, 5, and 10 minutes. VEGF induced a rapid increase in PKC activity.

We next fractionated endothelial cells exposed to VEGF (10 minutes, 10 ng/mL) into cytosolic and particulate fractions and examined the PKC isoforms , , , and by Western blot. The blots are shown in Figure 4 and revealed that VEGF markedly increased PKC- expression in the particulate fraction. PKC- and PKC- expression was not affected. PKC-, on the other hand, was increased in the particulate fraction, similar to the effect on PKC-.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of VEGF on the intracellular distribution of PKC isoforms , , , and in endothelial cells by Western blot. The cells underwent immediate cell fractionation by ultracentrifugation. The cytosolic (c) and particulate (p) fractions were run on a 10% SDS gel and stained with the respective PKC isoform antibodies. VEGF induced a translocation of PKC- and PKC- from the cytosolic to the particulate fraction, whereas other PKC isoforms were not affected.

We then followed the translocation of the PKC isoforms with confocal microscopy. Figure 5 shows the VEGF-induced changes in the intracellular distribution of PKC isoforms , , , and . VEGF induced a shift of the PKC isoforms and toward the perinuclear and nuclear area, which corresponds to the appearance of these isoforms in the particulate cell fractions as observed by Western blot.

View larger version (90K): [in this window] [in a new window]   Figure 5. Confocal microscopy of the time course of VEGF-induced changes in the distribution of PKC isoforms , , , and . PKC induced a shift of the PKC isoforms and toward the perinuclear and nuclear area.

We then analyzed the effects of VEGF on endothelial cell growth in culture. As shown in Figure 6, VEGF induced endothelial cells to proliferate. This proliferative effect was markedly reduced almost to control level by the PKC inhibitors staurosporine (5x10^-8 M) and calphostin C (10^-7 M). We then down-regulated PKC by pretreating endothelial cells with the phorbol ester TPA (100 nmol/L) for 24 hours before administering VEGF. This down-regulation led to a decrease in PKC- protein levels, whereas PKC- expression was not affected (data not shown). The 24-hour exposure to TPA attenuated the VEGF-induced effects on endothelial cell proliferation.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of the PKC inhibitors staurosporine (10-8 M), calphostin C (10-5 M) and a 24-hour pretreatment with TPA (100 nmol/L) on the VEGF-induced thymidine uptake of endothelial cells. Staurosporine and calphostin C inhibited the VEGF-induced thymidine uptake almost to control values. Down-regulation of PKC after 24-hour pretreatment with TPA also had an inhibitory effect. *P<0.05 vs control.

To investigate the specific role of PKC- and PKC-, respectively, we applied antisense ODN to specifically suppress expression of the respective PKC isoform. Antisense ODN led to a down-regulation of PKC- to 28% versus control (n=3). In contrast, protein levels of PKC- were not affected by exposure of the endothelial cells to antisense ODN against PKC-. Lipofectin alone had no effect on PKC- expression levels. Antisense ODN for PKC- led to a down-regulation of PKC- to 43% versus control (n=3). In contrast, protein levels of PKC- were not affected by exposure of the endothelial cells to antisense ODN against PKC- (data not shown).

Using antisense ODN against the 3' untranslated region of PKC- and the starting region of PKC-, we investigated whether the specific down-regulation of PKC- and PKC- with antisense ODN influenced the VEGF-induced increase in endothelial cell thymidine uptake-monitored proliferation as shown in Figure 7 (n=4). Endothelial cells were incubated with lipofectin (10 mg/mL) alone, antisense ODN, and sense ODN against PKC- or PKC-, respectively, before exposure to VEGF (10 ng/mL). Antisense ODN for PKC- almost completely inhibited the increase in VEGF-induced endothelial cell proliferation. Sense ODN for PKC- had no effect on the VEGF-induced proliferation. The antisense ODN against PKC- also reduced the VEGF-induced cell growth significantly, whereas the sense control had no effect on VEGF-induced proliferation.

View larger version (35K): [in this window] [in a new window]   Figure 7. Effect of antisense ODN, sense ODN against PKC-, or PKC- on VEGF-induced endothelial cell proliferation. Endothelial cells were exposed to ODN with lipofectin (10 mg/mL) 24 hours before exposure to VEGF (n=4). Antisense ODN against PKC- significantly reduced the VEGF-induced thymidine uptake (P<0.05), whereas the control sense ODN or lipofectin alone had no significant effect. Antisense ODN against PKC- reduced the VEGF-induced increase in endothelial cell thymidine uptake, whereas sense ODN had no significant effect (n=4). *P<0.05 vs control ODN.

	Discussion

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We tested the hypothesis that the activation of distinct PKC isoforms plays a role in the VEGF-induced proliferation of endothelial cells. We demonstrated that VEGF leads to an increase in [Ca²⁺]_i, which is mostly dependent on transmembranous Ca²⁺ influx from the extracellular space. The effect of VEGF on [Ca²⁺]_i is mediated via pertussis-sensitive G proteins. With confocal microscopy, we found that VEGF leads not only to an increase in cytosolic [Ca²⁺]_i, but also to an increase in [Ca²⁺]_i within the cell nucleus. VEGF led to an activation of PKC. The PKC isoforms and were translocated from the cytosolic to the particulate fraction. These findings were confirmed in the confocal micrographs. We were able to demonstrate an increase in nuclear immunoreactivity for both PKC isoforms. PKC inhibitors and down-regulation of phorbol ester–sensitive PKC isoforms led to an inhibition of VEGF-induced endothelial cell proliferation. Using specific antisense ODN for PKC- and PKC-, we observed an inhibition of VEGF-induced endothelial cell proliferation. These experiments suggest that the proliferative response of VEGF is, at least partially, mediated by the activation of PKC- and PKC-.

VEGF induced a biphasic [Ca²⁺]_i response in endothelial cells with a rapid initial increase followed by a sustained plateau phase. This biphasic [Ca²⁺]_i response indicates that VEGF induces an initial release of Ca²⁺ from intracellular stores and a subsequent transmembranous Ca²⁺ influx. This assumption is supported by our experiments using EGTA and thapsigargin, respectively. An interesting observation in our experiments is the finding that pertussis toxin abrogates the VEGF-induced calcium influx. The finding suggests that VEGF receptor stimulation leads to the activation of a pertussis toxin–sensitive G protein. No such mechanism has been described for VEGF receptors thus far; however, Okamoto et al²⁶ observed a G protein activation after stimulation of another membrane-bound tyrosine kinase, namely the insulin-like growth factor receptor. The relationship between the VEGF receptor(s) and G protein activation warrants further investigation.

Our observations support an activation of phospholipase C by VEGF receptor stimulation. Such an effect has been described by others.^{12 27} The binding of other growth factors to tyrosine kinase receptors has been shown to stimulate the phospholipase C–mediated breakdown of inositol lipids, with subsequent [Ca²⁺]_i mobilization and activation of PKC.^{14 25} However, Waltenberger et al⁷ did not find an increase in the receptor-associated activity of phosphatidyl inositol 3'-kinase or tyrosine phosphorylation of phospholipase C after stimulation of the VEGF receptors flt-1 or flk-1. The fact that VEGF induced a translocation of PKC- also suggests that other signaling events, in addition to the activation of phospholipase C , are involved. Presently, we can only speculate about the underlying mechanism. Possibly, sphingomyelin hydrolysis and the generation of ceramide play a role in the signal transduction of VEGF receptors.²⁸ Similar observations have been made by Nanberg et al²⁹ for another endothelial cell growth factor which binds to a tyrosine kinase receptor, namely FGF. They hypothesized that bFGF stimulates PKC through a signal transduction pathway distinct from inositol phospholipid turnover. However, Nanberg et al²⁹ observed no [Ca²⁺]_i mobilization by bFGF. Thus, VEGF signaling is distinct from FGF signaling by virtue of a [Ca²⁺]_i signal.

We observed a stimulatory effect of VEGF on PKC isoforms and . Two other PKC isoforms, and , were not translocated by VEGF, and their intracellular distribution did not change. PKC isoform , which also is expressed in endothelial cells, was not investigated in this study.²¹ We observed an increase in perinuclear and nuclear immunoreactivity for PKC- after stimulation by VEGF. This observation could indicate that VEGF induces a translocation of PKC- into the nucleus. Nuclear translocation of this isoform by tyrosine kinase receptor signaling has been reported previously by us^{21 30 31} and others.^{32 33 34} Which signal directs PKC isoforms to the nucleus is presently unclear. However, it is generally assumed that the translocation of PKC- is dependent on DAG and an increase in [Ca²⁺]_i.¹⁶ Our data suggest that an increase in [Ca²⁺]_i is sufficient for this process. Preliminary experiments using Ca²⁺ ionophores support this assumption (H. Haller, unpublished data, 1996). It is also possible that the increased nuclear PKC immunoreactivity after hormonal stimulation of endothelial cells could be due to activation of PKC, which is already present in the nucleus. Activators of nuclear PKC could be DAG and/or Ca²⁺ ions. Several reports have described the presence of phospholipids and components of phosphoinositide metabolism in the nucleus.³⁵ Furthermore, Divecha et al³⁶ have demonstrated that extracellular stimuli lead to rapid changes in nuclear DAG content, which may promote PKC- activation. Increases in [Ca²⁺]_i in the nucleus after hormonal stimulation, as observed after VEGF, also could contribute to the activation of nuclear PKC. In support of this hypothesis, we have shown recently that thrombin elicits a substantial increase in nuclear [Ca²⁺]_i in endothelial cells.²² The exact function of PKC- in the nucleus is unknown. Nuclear PKC- is involved in ligand-dependent transcription of a retinoic acid–inducible promoter. PKC- presumably increases the DNA-binding activity of complexes containing the human retinoic acid receptor .³⁷ We recently showed that retinoic acid–induced differentiation of vascular smooth muscle cells is associated with increased expression of PKC- and that this isoform can induce differentiation in these cells.³⁰ Others have suggested that PKC- may be among the most likely mediators of G1/S inhibition in endothelial cells.^{38 39 40}

VEGF also induced a translocation of PKC-, and the confocal micrographs show an increased immunoreactivity in the cytosol and in the nucleus. PKC- is not stimulated by [Ca²⁺]_i, phorbol esters, or DAG.¹⁹ Recently, Lozano et al²⁸ suggested that PKC- is activated by ceramide, which is produced by stimulation of sphingomyelin hydrolysis. We therefore can speculate that VEGF transmits signals via this pathway. PKC- seems to play a role in mitogenic signaling. Using a dominant kinase–defective PKC- mutant, Berra et al⁴¹ recently showed that PKC- is required for mitogenic activation in oocytes and fibroblasts. PKC- in the nucleus could lead to the activation of transcription factors. Several recent reports support the notion that PKC- plays a decisive role in NF-B regulation in mammalian cells.²⁸ The function of PKC- in endothelial cells remains to be determined.^{38 39 40} An interesting speculation is that PKC- may play a role in the induction of metalloproteinases. Sanz et al⁴² recently showed that PKC- induces stromelysin gene expression via a promoter region with the palindromic sequence ACTAGT. This observation links the proliferative effects of VEGF with a possible local increase in proteinase activity mediated by PKC-.

Our findings are in accordance with the recent observation by Friedländer and coworkers⁴³ that PKC activation is 1 of 2 distinct signaling pathways for angiogenesis. However, Xia et al⁴⁴ recently described that antisense PKC- oligonucleotides enhance VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC- protein content. The different results in their study compared with ours are not easy to understand. One possible explanation is that these investigators have used bovine aortic endothelial cells versus human umbilical vein endothelial cells in our study. Because we have observed marked differences in the expression of PKC isoforms both during differentiation of endothelial cells²¹ and in endothelial cells from different vascular beds (M. Wellner, unpublished observation, 1998), it is conceivable that PKC isoforms have different functions in the endothelium.

In summary, we investigated [Ca²⁺]_i and PKC isoforms in endothelial cells and tested the hypothesis that the proliferative effects of VEGF is mediated via the activation of specific PKC isoforms. We showed that VEGF induced biphasic increase in [Ca²⁺]_i. The Ca²⁺ influx is mediated via a pertussis toxin–sensitive G protein. VEGF induced an activation of PKC. Analysis of the PKC isoforms showed that PKC- and PKC- are translocated by VEGF. Confocal microscopy demonstrated that both isoforms are translocated in the nucleus. The mitogenic effects of VEGF were decreased significantly by PKC inhibitors and, partially, by PKC down-regulation. Finally, we demonstrated that specific down-regulation of PKC- and PKC- by antisense ODN prevented the VEGF-induced endothelial cell proliferation. We conclude that the mitogenic effects of VEGF on endothelial cells are mediated at least partially by PKC- and PKC-.

	Acknowledgments

 
The authors thank Dora Fiedler, Jana Czychi, and Petra Quass for their excellent technical assistance. We also thank Yvette Neuenfeld for providing the human umbilical vein endothelial cells. The project was supported by a grant-in-aid from the Deutsche Forschungsgemeinschaft (H.H.).

Received December 23, 1997; accepted April 24, 1998.

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