VEGF-A Stimulates ADAM17-Dependent Shedding of VEGFR2 and Crosstalk Between VEGFR2 and ERK Signaling
Vascular endothelial growth factor (VEGF)-A and the VEGF receptors are critical for regulating angiogenesis during development and homeostasis and in pathological conditions, such as cancer and proliferative retinopathies. Most effects of VEGF-A are mediated by the VEGFR2 and its coreceptor, neuropilin (NRP)-1. Here, we show that VEGFR2 is shed from cells by the metalloprotease disintegrin ADAM17, whereas NRP-1 is released by ADAM10. VEGF-A enhances VEGFR2 shedding by ADAM17 but not shedding of NRP-1 by ADAM10. VEGF-A activates ADAM17 via the extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase pathways, thereby also triggering shedding of other ADAM17 substrates, including tumor necrosis factor α, transforming growth factor α, heparin-binding epidermal growth factor–like growth factor, and Tie-2. Interestingly, an ADAM17-selective inhibitor shortens the duration of VEGF-A–stimulated ERK phosphorylation in human umbilical vein endothelial cells, providing evidence for an ADAM17-dependent crosstalk between the VEGFR2 and ERK signaling. Targeting the sheddases of VEGFR2 or NRP-1 might offer new opportunities to modulate VEGF-A signaling, an already-established target for treatment of pathological neovascularization.
Vascular endothelial growth factor (VEGF)-A and its receptors VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR), and neuropilin (NRP)-1 play critical roles in angiogenesis.1 The tyrosine kinase VEGFR2 is a principal coordinator of adult neoangiogenesis that affects endothelial cell migration, capillary formation, and vascular permeability.1 By contrast, a soluble splice variant of VEGFR1 can sequester VEGF-A, thereby negatively regulating angiogenesis.2 Recently, soluble VEGFR2 was identified in supernatants of murine tumor cells and human plasma.3 Given the importance of VEGFR2 and its coreceptor NRP-14 in angiogenesis, and because protein ectodomain shedding has emerged as a key posttranslational regulator of receptor function,5 we sought to address whether these 2 VEGF-A receptors are shed, identify the responsible sheddases, and determine how shedding might affect VEGF signaling.
Materials and Methods
Please see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org, for a description of cell lines, culture conditions, growth factors, inhibitors, and antibodies used in this study. Moreover, the generation of alkaline phosphatase tagged substrates, as well as the shedding assay for their detection is described.
Results and Discussion
When alkaline phosphatase (AP)-tagged VEGFR2 was expressed in COS-7 cells, its ectodomain was shed, and its shedding could be stimulated by phorbol-12-myristate-13-acetate (PMA) and the calcium ionophore ionomycin (IM) (Figure 1A) and blocked by the metalloproteinase inhibitor marimastat (MM) (Figure 1A). Identical experiments with NRP-1–AP showed constitutive shedding with only weak stimulation by PMA but strong stimulation by IM via an MM-sensitive activity (Figure 1B). Similar results were obtained with untagged full length VEGFR2 or NRP-1 transfected into COS-7 cells (Figure I in the online data supplement).
The PMA stimulation of VEGFR2 shedding indicated a possible role for ADAM17 (a disintegrin and metalloprotease 17), whereas the stimulation of the NRP-1–sheddase by IM, but not PMA, matched the properties of ADAM10.6 Shedding of VEGFR2-AP from wild-type or Adam10−/− mouse embryonic fibroblasts (mEFs) was stimulated by PMA, whereas less constitutive and almost no PMA-stimulated VEGFR2 shedding was seen in Adam17−/− mEFs (Figure 1C). VEGFR2 shedding from Adam17−/− mEFs could be rescued by ADAM17, but not by the inactive ADAM17 E>A (supplemental Figure IIA). Similar experiments with NRP-1–AP showed IM-stimulated shedding from wild-type and Adam17−/− mEFs but only little IM-stimulated shedding from Adam10−/− mEFs (Figure 1D). Shedding of NRP-1–AP from Adam10−/− mEFs was enhanced by ADAM10 but not by the inactive ADAM10E>A (supplemental Figure IIB).
Stimulation of the VEGFR2 activates signaling pathways such as the p42/44 and p38 mitogen-activated protein kinases, which also activate ADAM17;7 therefore, we tested whether VEGF-A could stimulate VEGFR2 shedding. When full-length VEGFR2 and VEGFR2-AP were cotransfected into COS-7 cells, addition of murine VEGF-A stimulated VEGFR-AP shedding by ≈50% compared to unstimulated controls, or to cells expressing only VEGFR2-AP, which cannot bind VEGF (Figure 2A). Moreover, VEGF-A–stimulated VEGFR2-AP shedding from Adam17−/− mEF cells was only observed after cotransfections of VEGFR2 and ADAM17, but not ADAM17 E>A (Figure 2B). Finally, VEGF-A–stimulated shedding of other ADAM17 substrates from COS-7 cells cotransfected with VEGFR2 (Figure 2C), including intercellular adhesion molecule-1, CD40, transforming growth factor-α, amphiregulin,8,9 tumor necrosis factor-α,8 and Tie-210 (see also supplemental Figure III). Thus, stimulation of VEGFR2 by VEGF-A activates shedding of VEGFR2 and several other substrates of ADAM17. However, we found no evidence for a role of NRP-1 in regulating VEGF-dependent activation of ADAM10 or -17, at least under the conditions tested here (supplemental Figure IV).
To extend our analysis to endothelial cells, porcine aortic endothelial cells expressing human VEGFR2/KDR (PAE-KDR cells) were treated with PMA or VEGF-A, both of which stimulated shedding of VEGFR2 (Figure 2D). VEGF-A–stimulated shedding was sensitive to MM and an ADAM17-selective inhibitor (IN17) (Figure 2D),11 and was blocked by the MEK1/2 inhibitor U0126, the Akt inhibitor LY 294002, and the p38 mitogen-activated protein kinase inhibitor SB202190 (Figure 2E). Treatment of human umbilical vein endothelial cells (HUVECs) with PMA or VEGF-A also enhanced shedding of endogenous VEGFR2, and VEGF-A–stimulated shedding was sensitive to MM and IN17 (Figure 2F). The response of the VEGFR2-sheddase in PAE-KDR and HUVECs to PMA and VEGF-A and its inhibition by 2 μmol/L IN17 strongly implicates ADAM17 as the principal VEGFR2-sheddase in these endothelial cells, which both express ADAM17 (supplemental Figure V), although a contribution of other enzymes sensitive to 2 μmol/L IN17 cannot be ruled out.
To explore how shedding affects VEGFR2 signaling, HUVECs were stimulated with VEGF-A, with or without MM (Figure 3A) or IN17 (supplemental Figure VI). Western blot revealed strong initial phosphorylation of ERK1/2 at 5 and 15 minutes after adding VEGF-A with or without MM or IN17. However, after 30 and 60 minutes, phosphorylation of ERK1/2 was reduced by MM (n=6) or IN17 (n=4) compared to controls. To test whether VEGF-A/VEGFR2 stimulated shedding of other ADAM17 substrates, such as ErbB-ligands, leads to prolonged ERK activation, alkaline phosphatase-tagged ErbB ligands known to be expressed in endothelial cells (heparin-binding EGF-like growth factor [HB-EGF], neuregulin1, and epidermal growth factor [EGF]12) were transfected into PAE-KDR cells. VEGF-A enhanced the shedding of the ADAM17 substrates HB-EGF and neuregulin 1β1 and -1β2 but not of the ADAM10 substrate EGF (Figure 3B and 3C).9,13 Moreover, U0126 and SB202190 blocked VEGF-A–stimulated shedding of HB-EGF (Figure 3C). These results suggest that VEGFR2 stimulates the release of ErbB ligands via activation of ADAM17, providing a plausible mechanism for the prolonged ERK activation in response to VEGF-A, although other membrane anchored signaling molecules released by ADAM17 could also contribute to VEGFR2/ERK crosstalk.
In summary, this study demonstrates that VEGFR2 and NRP-1 are shed from various cell types, including endothelial cells and identifies the responsible sheddases. The release of soluble VEGFR2 or NRP-1 should decrease their availability on cells and simultaneously generate soluble decoys that could intercept VEGF-A, similar to soluble VEGFR1.2 Moreover, VEGF-A/VEGFR2 stimulate ADAM17, resulting in shedding of VEGFR2 and other ADAM17 substrates, including ErbB-ligands. These results provide the first evidence, to our knowledge, for an ADAM17-dependent crosstalk between the VEGFR2 and ERK signaling, which extends the duration of VEGF-dependent ERK signaling, with likely consequences for migration and proliferation of endothelial and perivascular cells. Thus, the sheddases for VEGFR2 and NRP-1 could be novel targets for treatment of pathological neovascularization in cancer, rheumatoid arthritis, and proliferative retinopathies.
We thank Dr Paul Saftig for Adam10−/− cells and murine ADAM10 cDNA.
Sources of Funding
Supported by NIH Eye Institute grant RO1 015719 (to C.P.B.) and the Howard Hughes Medical Institute and NIH Heart, Lung, and Blood Institute (to S.R.).
Original received February 1, 2008; resubmission received July 30, 2008; revised resubmission received September 16, 2008; accepted September 17, 2008.
Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, Albuquerque RJ, Richter E, Sakurai E, Newcomb MT, Kleinman ME, Caldwell RB, Lin Q, Ogura Y, Orecchia A, Samuelson DA, Agnew DW, St Leger J, Green WR, Mahasreshti PJ, Curiel DT, Kwan D, Marsh H, Ikeda S, Leiper LJ, Collinson JM, Bogdanovich S, Khurana TS, Shibuya M, Baldwin ME, Ferrara N, Gerber HP, De Falco S, Witta J, Baffi JZ, Raisler BJ, Ambati J. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006; 443: 993–997.
Ebos JM, Bocci G, Man S, Thorpe PE, Hicklin DJ, Zhou D, Jia X, Kerbel RS. A naturally occurring soluble form of vascular endothelial growth factor receptor 2 detected in mouse and human plasma. Mol Cancer Res. 2004; 2: 315–326.
Horiuchi K, Le Gall S, Schulte M, Yamaguchi T, Reiss K, Murphy G, Toyama Y, Hartmann D, Saftig P, Blobel C. Substrate selectivity of EGF-receptor ligand sheddases and their regulation by phorbol esters and calcium influx. Mol Biol Cell. 2007; 18: 176–188.
Weskamp G, Schlöndorff J, Lum L, Saftig P, Hartmann D, Becherer D, Murphy G, Blobel CP. Evidence for a critical role of the TNFa convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR). J Biol Chem. 2004; 279: 4241–4249.
Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russel WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, Black RA. An essential role for ectodomain shedding in mammalian development. Science. 1998; 282: 1281–1284.
Sahin U, Weskamp G, Zhou HM, Higashiyama S, Peschon JJ, Hartmann D, Saftig P, Blobel CP. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR-ligands. J Cell Biol. 2004; 164: 769–779.
Findley CM, Cudmore MJ, Ahmed A, Kontos CD. VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling. Arterioscler Thromb Vasc Biol. 2007; 27: 2619–2626.
Zhou BB, Peyton M, He B, Liu C, Girard L, Caudler E, Lo Y, Baribaud F, Mikami I, Reguart N, Yang G, Li Y, Yao W, Vaddi K, Gazdar AF, Friedman SM, Jablons DM, Newton RC, Fridman JS, Minna JD, Scherle PA. Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell. 2006; 10: 39–50.
Iivanainen E, Nelimarkka L, Elenius V, Heikkinen SM, Junttila TT, Sihombing L, Sundvall M, Maatta JA, Laine VJ, Yla-Herttuala S, Higashiyama S, Alitalo K, Elenius K. Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor. FASEB J. 2003; 17: 1609–1621.