Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a tightly regulated process controlled by the balance between positive and negative stimuli in the environment of endothelial cells (Bouck et al., 1996; Hanahan and Folkman, 1996). Identification of the molecules that regulate this complex process has been the first step towards understanding the mechanisms controlling angiogenesis (Carmeliet, 2000). Additional insight comes from the discovery that a large proportion of known inhibitors of angiogenesis, including thrombospondin-1, angiostatin, endostatin, 2-methoxyestradiol and canstatin, have the ability to induce apoptosis in endothelial cells thereby precluding the cells from responding to a wide variety of pro-angiogenic factors that signal through different pathways (Yue et al., 1997; Yeh et al., 1998; Claesson-Welsh et al., 1998; Lucas et al., 1998; Dhanabal et al., 1999; Jiménez et al., 2000; Kamphaus et al., 2000). In their turn, inducers of angiogenesis, besides being able to activate endothelial cell proliferation, migration and capillary morphogenesis, promote endothelial cell survival (Tran et al., 1999; Fujio and Walsh, 1999; Nor et al., 1999; O'Connor et al., 2000). Thus the balance between inducers and the apoptosis-inducing inhibitors of angiogenesis seems to be interpreted within the endothelial cell as a balance between survival and apoptosis pathways and to be critical for the regulation of angiogenesis (Jiménez et al., 2000). Defining the signaling pathways within the cell that are triggered by the molecules that regulate angiogenesis will be essential if these mechanisms are to be understood on a molecular level.
We have recently shown that the anti-angiogenic activity of TSP-1 requires the sequential activation of CD36, p59fyn, caspase-3-like proteases and p38 mitogen-activated protein kinases (Jiménez et al., 2000). Here we have investigated the contribution of c-Jun N-terminal kinase-1 (JNK-1) and p42/p44 mitogen-activated protein kinases (MAPKs) to the inhibition of neovascularization by TSP-1.
Results and discussion
Several inducers of angiogenesis, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and tumor necrosis factor α (TNFα), are known to rely on activation of p42 and p44 MAPK in order to transduce their stimulatory effects within the endothelial cell (D'Angelo et al., 1995; Modur et al., 1996; Pedram et al., 1998). The activation of p42 and p44 by bFGF (Figure 1a), VEGF, and PDGF (data not shown) in capillary endothelial cells was sustained for over 60 min, far exceeding the rapid and transient activation of these kinases that is commonly associated with the induction of proliferative responses (Marshall, 1995). This prolonged activation suggests that these kinases may also contribute to other essential components of the complex angiogenic response of endothelial cells, including migration and survival.
The activation of MAPK was indeed critical for these growth factors to induce endothelial cell migration. PD98059, a peptide inhibitor that blocks MEK (Dudley et al., 1995) effectively inhibited MAPK activation in endothelial cells (Figure 1b). Pretreatment with PD98059 was sufficient to block endothelial cell migration towards multiple inducers of angiogenesis including VEGF, PDGF and bFGF (Figure 1c). As might be expected, PD98056 did not block migration towards TGFβ, which signals through a MAPK- independent pathway (Wrana, 2000).
The inhibitory effect of TSP-1 was not due to interference with the prolonged, inducer-dependent activation of MAPK. TSP-1 was not able to block p42/p44 MAPK activation by bFGF, nor was it able to independently activate MAPKs as measured both by mobility shift assay (EMSA) (Figure 1d) and by the more sensitive and quantitative immunocomplex kinase assay (Figure 1e). A similar lack of interference was seen when MAPK was activated by either VEGF or PDGF instead of bFGF (data not shown). This observation is in keeping with our previous results where we have demonstrated that the ability of TSP-1 to induce apoptosis in endothelial cells is also independent of either p42 or p44 MAPKs (Jiménez et al., 2000).
In contrast to the non-essential MAPKs, the stress-activated kinases are crucial to the inhibitory activity of TSP-1. We have shown previously that p38 MAPK is activated by TSP-1 and is essential for TSP-1 anti-angiogenic activity both in vitro and in vivo (Jiménez et al., 2000). Here we demonstrate that TSP-1 can also activate a second stress activated kinase, c-Jun N-terminal kinase (JNK). When used at doses that are inhibitory in the endothelial cell migration assay (Tolsma et al., 1993), TSP-1 stimulated JNK with a rapid kinetics typical for an early step in the signaling cascade (Figure 2a).
TSP is known to bind a number of cellular receptors (Roberts, 1996) but it is CD36 that mediates its anti-angiogenic effects both in vitro (Dawson et al., 1997) and in vivo (Jiménez et al., 2000). To link JNK activation more tightly to the anti-angiogenic activity of TSP-1, its dependence on CD36 was tested. JNK activation by TSP-1 was blocked in the presence of FA6 (Figure 2b), an antibody that interferes with TSP-1 access to CD36 and prevents its acting as an anti-angiogenic agent (Dawson et al., 1997). The FA6 inhibitory effect was specific since TNFα activation of JNK was unaffected by the presence of these antibodies (Figure 2c). Additionally, the anti-CD36 monoclonal antibody SMΦ that is agonistic for CD36 and able to mimic TSP-1 activity on endothelial cells (Dawson et al., 1997), also caused activation of JNK in microvascular endothelial cells (data not shown).
The anti-angiogenic activity of TSP-1 resides in the 50 kDa central stalk of the molecule (Tolsma et al., 1993), where two independent anti-angiogenic sub-regions have been identified. From these regions two peptides were derived, Mal III and Col overlap, that share a common motif and act directly on endothelial cells via the CD36 receptors to mimic the anti-angiogenic properties of the whole TSP-1 molecule (Tolsma et al., 1993; Dawson et al., 1997). Both Mal III and Col overlap were able to activate JNK to the same extent as TSP-1 (Figure 2d). For both peptides this activation was effectively blocked by FA6 monoclonal antibodies, supporting the essential role of CD36 signaling for JNK activation by Mal III and Col overlap, and therefore by TSP-1.
To further link JNK activation by TSP-1 to the inhibition of angiogenesis a corneal neovascularization assay was performed on mice null for JNK-1 (JNK-1−/−). Null mice (Sabapathy et al., 1999b) were verified by Western blots of the whole cell extracts of embryonic fibroblasts derived from E 13.3 embryos (Figure 3a, upper panel). As a loading control the membrane was re-probed with an antibody that recognizes both isoforms of JNK-1 and JNK-2 (Figure 3a, lower panel). Null animals were bred to wild-type C56/Bl6J, the F1 backcrossed to the nulls, and the null F2 mice identified by their lack of the 46 kDa isomer of JNK-1. Progeny of these animals were used for cornea assays. JNK-1−/− mice showed normal response to inducers of angiogenesis, while their response to the inhibitory action of TSP-1 in the corneal neovascularization assay was substantially impaired (Figure 3 and Table 1). This result was further confirmed in an in vitro assay where endothelial cells were induced to migrate out of corneal explants in the presence of VEGF. For this assay, corneas of JNK-1−/− and C57Bl6 control mice were excised and placed in Matrigel depleted of most growth factors. Three days later active cell migration from the explants and capillary morphogenesis was found in the presence of VEGF (Figure 4). Migrating cells stained positively for the endothelial marker CD31 (T Ferguson, Washington University, St. Louis, MD, USA, personal communication). TSP-1 was able to halt the induction of cord formation by this corneal endothelium when it was derived from wild-type animals, but it was ineffective when applied to the corneas from JNK-1 −/− mice (Figure 4).
These results demonstrate that JNK-1 is required for the anti-angiogenic effect of TSP-1. Studies of mice that lack distinct members of the JNK family, JNK-1, JNK-2, JNK-3 reveal that although these kinases belong to a common family, they can play highly specific roles in T cell differentiation (Dong et al., 1998; Yang et al., 1998; Chu et al., 1999 and Sabapathy et al., 1999a) and apoptosis (Kuan et al., 1999). Data presented in this work support that JNK-1 requirement for TSP-1 anti-angiogenic action cannot be adequately compensated for by other MAPK family members. This suggests that independent targets exist in endothelial cells for each of the JNK kinases, and that JNK-1 plays a critical role in the inhibition of neovascularization by TSP-1. Thus two stress-activated kinases, JNK-1 and p38 MAPK seem to be integral parts of the signaling network that leads from CD36 to the apoptosis-dependent inhibition of angiogenesis by TSP-1.
Bornstein P, Sage EH . 1994 Methods Enzymol. 245: 62–84
Bouck N, Stellmach V, Hsu S . 1996 Adv. Cancer Res. 69: 135–174
Carmeliet P . 2000 Nat. Med. 6: 389-395
Chu WM, Ostertag D, Li ZW, Chang L, Chen Y, Hu Y, Williams B, Perrault J, Karin M . 1999 Immunity 11: 721–31
Claesson-Welsh L, Welsh M, Ito N, Anand-Apte B, Soker S, Zetter B, O'Reilly MS, Folkman J . 1998 Proc. Natl. Acad. Sci. USA 95: 5579–5583
D'Angelo G, Struman I, Maltral J, Weiner R . 1995 Proc. Natl. Acad. Sci. USA 92: 6374–6378
Dhanabal M, Ramchandran R, Waterman MJ, Knebelmann B, Segal M, Sukhatme VP . 1999 J. Biol. Chem. 274: 11721–11726
Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP . 1997 J. Cell Biol. 138: 707–717
Dong C, Yang DD, Wysk M, Whitmarsh AJ, Davis RJ, Flavell RA . 1998 Science 282: 2093–2095
Dudley DT, Pang L. Decker SJ, Bridges AJ, Saltiel AR . 1995 Proc. Natl. Acad. Sci. USA 92: 7686–7689
Fujio Y, Walsh K . 1999 J. Biol. Chem. 274: 16349–16354
Hanahan D, Folkman J . 1996 Cell 86: 353–364
Hibi M, Lin A, Smeal T, Minden A, Karin A . 1993 Genes Dev. 7: 2135–2148
Jiménez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N . 2000 Nat. Med. 6: 41–48
Kamphaus GD, Colorado PC, Panka DJ, Hopfer H, Ramchandran R, Torre A, Maeshima Y, Mier JW, Sukkatma VP, Kalluri R . 2000 J. Biol. Chem. 275: 1209–1215
Kuan CY, Yang DD, Samanta-Roy DR, Davis RJ, Rakia P, Flavell RA . 1999 Neuron 22: 667–676
Loo D, Cotman C . 1994 Cell Biology: a laboratory handbook. Celis JE (ed). San Diego, New York, Boston, London, Sydney, Tokyo, Toronto pp. 45–53
Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota SJ, Borlat F, Sim BK, Wu Z, Grau GE, Shing Y, Soff GA, Bouck N, Pepper MS . 1998 Blood 92: 4730–4741
Marshall CJ . 1995 Cell 80: 179–185
Modur V, Zimmerman GA, Precott JM, McIntyre TM . 1996 J. Biol. Chem. 271: 13094–13102
Nor JE, Christensen J, Mooney DJ, Polverini PJ . 1999 Am. J. Pathol. 154: 375–384
O'Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, Nath AK, Pober JS, Altieri DC . 2000 Am. J. Pathol. 156: 393–398
Pedram A, Razandi M, Levin ER . 1998 J. Biol. Chem. 273: 26722–26728
Roberts DD . 1996 FASEB J. 10: 1183–1191
Sabapathy K, Hu Y, Kallunki T, Schreiber M, David JP, Jochum W, Wagner EF, Karin M . 1999a Curr. Biol. 9: 116–125
Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, Wagner EF . 1999b Mech. Dev. 89: 1115–1124
Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N . 1993 J. Cell Biol. 122: 497–511
Tran J, Rak J, Sheehan C, Saibil SD, LaCasse E, Korneluk RG, Kerbel RS . 1999 Biochem. Biophys. Res. Commun. 264: 781–788
Volpert OV, Lawler J, Bouck NP . 1998 Proc. Natl. Acad. Sci. USA 95: 6343–6348
Wrana JL . 2000 Cell 100: 189–192
Yang DD, Conze D, Whitmarsh AJ, Barret T, Davis RJ, Rincon M, Flavell RA . 1998 Immunity 9: 575–585
Yeh CH, Peug HC, Huang TF . 1998 Blood 92: 3268–3276
Yue TL, Wang X, Louden CS, Gupta S. Pilarisetti K, Gu JL, Hart TK, Lysko PG, Feuerstein GZ . 1997 Mol. Pharmacol. 51: 951–962
This work was funded by NIH grants CA52750 and CA64239 to N Bouck and by American Heart Association grant AHA SGD 0030023N to OV Volbert and by Plan Nacional de I+D grant SAF 98-0060 to A Muñoz and Comunidad de Madrid 08.1/0010/2000 to B Jiménez. L Chang was supported by a fellowship from Bank of America-Ginnini Foundation. M Karin is the Frank and Else Schilling American Cancer Society Research Professor.
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