VEGF receptors on PC12 cells mediate transient activation of ERK1/2 and Akt: comparison of nerve growth factor and vascular endothelial growth factor
© Berger et al; licensee BioMed Central Ltd. 2006
Received: 04 February 2006
Accepted: 01 June 2006
Published: 01 June 2006
Vascular endothelial growth factor (VEGF) and endostatin are angiogenic and anti-angiogenic molecules, respectively, that have been implicated in neurogenesis and neuronal survival. Using alkaline phosphatase fusion proteins, we show that the PC12 neuronal cell line contains cell membrane receptors for VEGF but not for endostatin and the collagen XV endostatin homologue. Immunocytochemistry confirmed that proliferating and differentiated PC12 cells express VEGF receptors 1, 2 and neuropilin-1. While no functional effects of VEGF on PC12 cell proliferation and differentiation could be observed, a slight VEGF-induced reduction of caspase-3 activity in differentiated apoptotic PC12 cells was paralleled by transient activation of ERK1/2 and Akt. In direct comparison, nerve growth factor proved to be a strikingly more potent neuroprotective agent than VEGF.
VEGF, VEGF receptor antagonists, and the C-terminal collagen XVIII fragment endostatin, an inhibitor of angiogenesis and tumor growth , have been tested for use in long-term therapies to enhance or reduce vascularisation . Therefore, knowledge of VEGF and endostatin receptor expression patterns as well as of their non-endothelial cell functions is important. VEGF was originally identified as a vascular permeability factor  which turned out to be crucial for vasculo- and angiogenesis . Later, non-endothelial VEGF target cells have been described in a variety of organs . More recently, autocrine and paracrine functions have been observed in neurogenesis and neuronal survival in vitro and in vivo, both in the central nervous system and the peripheral nervous system . Endostatin was implicated in neuronal cell migration and axon guidance in Caenorhabditis elegans . Fc-endostatin dimers were also reported to have motogenic activity on rat pheochromocytoma PC12 cells cultured on Matrigel , an extracellular matrix preparation used for differentiation of endothelial cells into tube-like structures. NGF-treated PC12 cells are an established model for analysis of neuronal differentiation, neuronal survival and neurotrophin signal transduction . Finally, increased neuronal and paracellular endostatin deposits were found in patients with Alzheimer's disease .
VEGF exerts its anti-apoptotic effect on hypoxic neurons via VEGF receptor 2 (VEGFR-2), neuropilin-1 (NRP1), the Ras/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathways [11–13] as in VEGFR-2-dependent endothelial survival . Ras/MAPK and PI3K/Akt are also involved in PC12 cell survival signaling stimulated by nerve growth factor (NGF) [15, 16]. Since VEGF has also been suggested to act as a neurotrophin in motoneuron degeneration , we intended to evaluate the effects of VEGF and endostatins on neuronal differentiation and survival in direct comparison with the prototypic neurotrophin NGF. PC12 cells were first probed with dimeric fusion proteins composed of the human placental isozyme of alkaline phosphatase (AP) at the N-terminus and murine (m) VEGF164 or endostatins at the C-terminus. While the endostatin affinity probes did not react with PC12 cells, AP-mVEGF164 strongly bound to proliferating and differentiated PC12 cells. Although PC12 cells were subsequently shown to express VEGF receptors 1, 2 and neuropilin-1, only a minor neuroprotective effect was observed for VEGF when compared to NGF.
Materials and methods
PC12 cells were a gift from Drs. M. Sendtner and S. Wiese (Department of Neurology, University of Wuerzburg, Germany). Cow pulmonary artery endothelial (CPAE) cells were purchased from ATCC (CCL-209). PC12 cells were cultured in DMEM with glutamax-I (Gibco) supplemented with 10% horse serum, 5% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin (Gibco) in 5% CO2 at 37°C. For differentiation experiments, PC12 cells were plated on poly-L-ornithine coated tissue culture dishes and allowed to adhere over night (o/n). After one wash with serum-free DMEM, the cells were differentiated in serum-free DMEM containing 50 ng/ml human recombinant NGF (PAN Biotech) for 3 days . Although Fc-endostatin dimer application induced the formation of multicellular PC12 aggregates on Matrigel , Matrigel was not chosen for the current study since it is an extracellular matrix preparation generally used for endothelial tube formation assays.
Alkaline phosphatase staining of PC12 cells
For construction and expression of AP fusion proteins see . PC12 cells were either grown to 80% confluence or differentiated in 6-well plates, and AP staining was performed as described in . Staining was monitored with a Nikon Eclipse TE2000-U inverted microscope and documented using the Spot Insight QE Color imaging software (Visitron). Quantitative measurement of AP fusion protein binding to proliferating PC12 cells was carried out as described previously .
PC12 cells plated on poly-L-ornithine coated glass coverslips were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde at room temperature for 20 min, washed three times in prewarmed tris-buffered saline (TBS) for 5 min, and incubated in blocking buffer (TBS with 10% goat serum) at room temperature for 1 h. After washing once with prewarmed TBS, the cells were incubated with anti-Flt-1 (sc-316), anti-Flk-1 (sc-504) or anti-Neuropilin (sc-5541) antibodies (2 μg/ml, Santa Cruz Biotechnology) in blocking buffer at 4°C o/n. Unbound primary antibodies were removed by washing, and the cells were incubated in 20 mM ammonium chloride solution for 30 min to reduce autofluorescence. Cells were stained for 1 h at 37°C with a secondary Cy3-conjugated goat anti-rabbit antibody (1:500, Dianova) in blocking buffer. After washing, the coverslips were mounted in Kaiser's glycerol gelatine (Merck). Fluorescent preparations of proliferating PC12 cells were documented at 600-fold magnification (Nikon Eclipse TE2000-U). Image acquisition of differentiated PC12 cells was performed with a Zeiss Axiophot at 1000-fold magnification.
Cell proliferation and cell death analyses
PC12 cell proliferation upon stimulation with 50 and 100 ng/ml VEGF165 (R&D Systems) was assayed after 24 h, 48 h, and 72 h using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). These assays were performed using two different cell densities (2 × 104 cells/cm2 and 6.7 × 104 cells/cm2) and full serum as well as serum-deprived conditions (0.1% and 0.4% horse serum). In addition, increasing VEGF165 concentrations from 0.2 to 400 ng/ml were added to PC12 cells cultured in 5% fetal bovine serum, and [3H]thymidine incorporation was measured 72 hours after onset of stimulation. The fluorometric CaspACE™ Assay System and western blot analyses with an anti-cleaved caspase-3 antibody (1:1000, Cell Signaling, #9664) were used to monitor apoptosis of differentiated PC12 cells (see below). All experiments were performed in triplicate.
SDS-PAGE/Western blot analyses of differentiated apoptotic PC12 cell lysates
PC12 cells were grown to 50% confluence in 10 cm tissue culture dishes and NGF-differentiated for 72 h in serum-free DMEM. For induction of apoptosis, cells were washed three times with serum-free DMEM and incubated for 7.5 h under serum-deprived conditions in NGF-free DMEM. After addition of exogenous recombinant human VEGF165 (R&D Systems) for the indicated time frames, PC12 cells were washed once with ice-cold PBS containing 100 μM sodium orthovanadate, followed by centrifugation at 5000 rpm for 5 min at 4°C. The cells were lysed in 200 μl of ice-cold lysis-buffer (HEPES, pH 7.8, 150 mM KOAc, 50 mM ß-glycerolphosphate, 25 mM NaF, 10 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0,05% (v/v) ß-mercaptoethanol, 1 μg/ml aprotinin, 6 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate). Supernatants were collected after centrifugation at 14 000 rpm for 10 min at 4°C. Standardized samples containing 50 μg of whole protein (Bradford assay) were separated using 10–20% gradient gels. Proteins were wetblotted onto nitrocellulose and probed with anti-phospho-ERK1/2 (1:2000, Sigma, M8159) or anti-phospho-Akt-Ser473 (1:1000, Cell Signaling, #9271) antibodies. The blots were stripped and reprobed with antibodies detecting the respective non-phosphorylated proteins (anti-ERK1, 1:1000, Santa Cruz Biotechnology, sc-94; anti-Akt, 1:1000, Cell Signaling, #9272). Horseradish peroxidase-conjugated secondary antibodies (1:2000, Dianova) were visualized by enhanced chemiluminescence detection (Western Lightning™ Chemiluminescence Reagent System, PerkinElmer). Experiments were performed in triplicate.
Differential cell binding of VEGF and endostatins
PC12 cells express high-affinity VEGF receptors
VEGF induces transient activation of ERK1/2 and Akt kinase in differentiated apoptotic PC12 cells
We here report that endostatin affinity probes derived from collagens XVIII and XV do not bind to PC12 cells indicating that these cells do not express endostatin cell membrane receptors. This observation is consistent with absent effects of recombinant endostatins on neurite outgrowth (data not shown) and lack of binding of AP-mESXVIII and AP-mESXV to murine embryonal nerve tissues . AP-mESXVIII predominantly labelled blood vessels while AP-mESXV binding was restricted to the lense capsule  which correlates with the current results that only AP-mESXVIII, but not AP-mESXV, strongly stained CPAE cells. As opposed to the endostatin affinity probes, AP-mVEGF164 showed strong binding to PC12 cells. Undifferentiated PC12 cells were known to express VEGF to stimulate angiogenesis . We now demonstrate that proliferating and differentiated PC12 cells also express VEGFR-1 and -2 and NRP1. NRP1 acts as an isoform-specific VEGF co-receptor which only binds VEGF165 . Since C-terminally deleted AP-mVEGF110 did not bind to PC12 cells, NRP1 appears to be required for the interaction of VEGF165 with PC12 cells.
Despite prominent expression of VEGF receptors on PC12 cells, exogenous VEGF165 had no effect on PC12 cell proliferation and neurite formation. One reason might be endogenous VEGF-expression of proliferating PC12 cells which is downregulated only 48 h after induction of differentiation with NGF . This would also explain the slight anti-apoptotic effect of VEGF165 on PC12 cells that had been differentiated for three days prior to VEGF165 stimulation. However, only an insignificant decrease of cell proliferation could be observed upon treatment with an antibody against rat VEGF164 (data not shown). Thus, our data are in line with the observation that VEGFR-1-expressing cells show a poor mitogenic response to VEGF stimulation . Analysis of VEGF165-induced signal transduction in differentiated apoptotic PC12 cells demonstrated activation of ERK1/2 and Akt. The transient nature of VEGF165-triggered ERK1/2 phosphorylation in PC12 cells provides a further explanation for the observation that VEGF165 was not able to induce PC12 cell differentiation which is known to require sustained activation of the MAPK cascade . The inefficient rescue of PC12 cells from apoptosis through VEGF165 is likely also due to its short-lived and relatively small effect on ERK1/2 and Akt.
VEGF-induced neuroprotective signaling via VEGFR-2, NRP1 and the two above-mentioned signaling cascades was shown in hypoxic and glucose-deprived hippocampal neuron × neuroblastoma (HN33) hybrid cells , in rat primary hippocampal neurons that had been exposed to glutamate , and in hypoxic murine primary cortical neurons . In these in vitro model systems of cerebral ischemia, no comparison of VEGF and NGF activation kinetics was performed. Our results on growth factor stimulated PC12 cells show that both the anti-apoptotic effect and the activation of ERK1/2 and Akt were transient and minor when compared to NGF. It remains to be clarified whether this is a consequence of experimental conditions, cell line-specific or a general feature of VEGF-induced neuroprotection.
Based on experiments using growth factor deprivation, the present study suggests that NGF protects neuronal cells from cell death much more efficiently than VEGF165. The significant NGF-induced reduction of caspase-3 activity in differentiated apoptotic PC12 cells correlates with a much more pronounced and prolonged activation of downstream effectors when compared to VEGF165. Thus, the angiogenic compound VEGF165 may only be a minor player in neurogenesis and neuronal survival and may only have little therapeutic and side effects on neuronal cells.
This work was supported by an Emmy Noether-grant from the Deutsche Forschungsgemeinschaft (Fe 432/6-4). Sonja Stahl receives a stipend from the Graduiertenkolleg 1048.
- O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J: Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997, 88: 277-285. 10.1016/S0092-8674(00)81848-6.View ArticlePubMedGoogle Scholar
- Cross MJ, Dixelius J, Matsumoto T, Claesson-Welsh L: VEGF-receptor signal transduction. Trends Biochem Sci. 2003, 28: 488-494. 10.1016/S0968-0004(03)00193-2.View ArticlePubMedGoogle Scholar
- Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF: Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983, 219: 983-985.View ArticlePubMedGoogle Scholar
- Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med. 2003, 9: 669-676. 10.1038/nm0603-669.View ArticlePubMedGoogle Scholar
- Matsumoto T, Claesson-Welsh L: VEGF receptor signal transduction. Sci STKE. 2001, 2001 (112): RE21-PubMedGoogle Scholar
- Storkebaum E, Lambrechts D, Carmeliet P: VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays. 2004, 26: 943-954. 10.1002/bies.20092.View ArticlePubMedGoogle Scholar
- Ackley BD, Crew JR, Elamaa H, Pihlajaniemi T, Kuo CJ, Kramer JM: The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. J Cell Biol. 2001, 152: 1219-1232. 10.1083/jcb.152.6.1219.View ArticlePubMedPubMed CentralGoogle Scholar
- Kuo CJ, LaMontagne KRJ, Garcia-Cardena G, Ackley BD, Kalman D, Park S, Christofferson R, Kamihara J, Ding YH, Lo KM, Gillies S, Folkman J, Mulligan RC, Javaherian K: Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain. J Cell Biol. 2001, 152: 1233-1246. 10.1083/jcb.152.6.1233.View ArticlePubMedPubMed CentralGoogle Scholar
- Kaplan DR, Miller FD: Signal transduction by the neurotrophin receptors. Curr Opin Cell Biol. 1997, 9: 213-221. 10.1016/S0955-0674(97)80065-8.View ArticlePubMedGoogle Scholar
- Deininger MH, Fimmen BA, Thal DR, Schluesener HJ, Meyermann R: Aberrant neuronal and paracellular deposition of endostatin in brains of patients with Alzheimer's disease. J Neurosci. 2002, 22: 10621-10626.PubMedGoogle Scholar
- Jin KL, Mao XO, Greenberg DA: Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc Natl Acad Sci U S A. 2000, 97: 10242-10247. 10.1073/pnas.97.18.10242.View ArticlePubMedPubMed CentralGoogle Scholar
- Matsuzaki H, Tamatani M, Yamaguchi A, Namikawa K, Kiyama H, Vitek MP, Mitsuda N, Tohyama M: Vascular endothelial growth factor rescues hippocampal neurons from glutamate-induced toxicity: signal transduction cascades. FASEB J. 2001, 15: 1218-1220.PubMedGoogle Scholar
- Ogunshola OO, Antic A, Donoghue MJ, Fan SY, Kim H, Stewart WB, Madri JA, Ment LR: Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system. J Biol Chem. 2002, 277: 11410-11415. 10.1074/jbc.M111085200.View ArticlePubMedGoogle Scholar
- Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N: Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem. 1998, 273: 30336-30343. 10.1074/jbc.273.46.30336.View ArticlePubMedGoogle Scholar
- Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME: Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995, 270: 1326-1331.View ArticlePubMedGoogle Scholar
- Wert MM, Palfrey HC: Divergence in the anti-apoptotic signalling pathways used by nerve growth factor and basic fibroblast growth factor (bFGF) in PC12 cells: rescue by bFGF involves protein kinase C delta. Biochem J. 2000, 352 Pt 1: 175-182. 10.1042/0264-6021:3520175.View ArticlePubMedGoogle Scholar
- Sopher BL, Thomas PSJ, LaFevre-Bernt MA, Holm IE, Wilke SA, Ware CB, Jin LW, Libby RT, Ellerby LM, La Spada AR: Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration. Neuron. 2004, 41: 687-699. 10.1016/S0896-6273(04)00082-0.View ArticlePubMedGoogle Scholar
- Gotz R, Karch C, Digby MR, Troppmair J, Rapp UR, Sendtner M: The neuronal apoptosis inhibitory protein suppresses neuronal differentiation and apoptosis in PC12 cells. Hum Mol Genet. 2000, 9: 2479-2489. 10.1093/hmg/9.17.2479.View ArticlePubMedGoogle Scholar
- Rychkova N, Stahl S, Gaetzner S, Felbor U: Non-heparan sulfate-binding interactions of endostatin/collagen XVIII in murine development. Dev Dyn. 2005, 232: 399-407. 10.1002/dvdy.20222.View ArticlePubMedGoogle Scholar
- Flanagan JG, Cheng HJ, Feldheim DA, Hattori M, Lu Q, Vanderhaeghen P: Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues, and embryos. Methods Enzymol. 2000, 327: 19-35.View ArticlePubMedGoogle Scholar
- Stahl S, Gaetzner S, Mueller TD, Felbor U: Endostatin phenylalanines 31 and 34 define a receptor binding site. Genes to Cells. 2005, 10 (9): 929-39. 10.1111/j.1365-2443.2005.00890.x.View ArticlePubMedGoogle Scholar
- Gaetzner S, Deckers MM, Stahl S, Lowik C, Olsen BR, Felbor U: Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants. Matrix Biol. 2005, 23: 557-561. 10.1016/j.matbio.2004.10.001.View ArticlePubMedGoogle Scholar
- Claffey KP, Wilkison WO, Spiegelman BM: Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways. J Biol Chem. 1992, 267: 16317-16322.PubMedGoogle Scholar
- Soker S, Fidder H, Neufeld G, Klagsbrun M: Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. J Biol Chem. 1996, 271: 5761-5767. 10.1074/jbc.271.10.5761.View ArticlePubMedGoogle Scholar
- Traverse S, Gomez N, Paterson H, Marshall C, Cohen P: Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J. 1992, 288 ( Pt 2): 351-355.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.