Our data reveals that anti-angiogenic signaling initiated by the interaction of TSP1 and related ligands with CD36 results at least in part from inhibition of its fatty acid translocase activity. Those CD36 ligands that exhibit anti-angiogenic activity inhibit myristate uptake and myristate-dependent NO signaling and downstream cGMP-mediated effects on endothelial cell adhesion (). TSP1 is less potent for inhibiting fatty acid uptake than for inhibiting cGMP signaling via CD47, but this is consistent with the differential responses of murine CD36 null and wild type vascular cells to TSP1 (22
). CD47-dependent anti-angiogenic signaling in the context of NO stimulation requires only picomolar concentrations of TSP1. Engaging CD47 does not inhibit myristate uptake but can inhibit myristate signaling downstream of NOS. An anti-angiogenic peptide derived from the CD36-binding type 1 repeats of TSP1, however, is a potent antagonist of fatty acid transport via CD36. This peptide inhibits NO/cGMP/cGK signaling in a NOS-dependent manner and so acts upstream of sGC, which is the major apparent target of CD47-mediated inhibition by native TSP1 (22
). Therefore, TSP1 can inhibit two different steps in NO signaling by engaging CD36 versus CD47 on endothelial cells.
Model for CD36-dependent activities of TSP1 and other CD36 ligands
Our data suggest an alternate mechanism for the recently described connection between endogenous nitric oxide production and CD36 (26
). Zhu and coworkers inferred that activation of NOS is mediated by binding of myristic acid and, to a lesser extent, palmitic acid to CD36 without internalization. We now show that agents that inhibit myristate uptake via CD36 prevent NOS-dependent cGMP synthesis. Therefore, uptake of myristate is probably involved in the previously reported NOS activation by this fatty acid. These new results also suggest a mechanistic basis for our previous observations that CD36 ligation modulates several NO-stimulated vascular cell responses (17
). In support of this hypothesis, we found that basal HUVEC adhesion to collagen in growth medium containing only FAF BSA is significantly increased upon addition of myristate. Involvement of NO signaling in stimulation of endothelial cell adhesion by myristate was confirmed by its inhibition in the presence of the nonselective NOS inhibitor L-NAME, the sGC inhibitor ODQ, and the cGK inhibitor Rp-8-p
CPT-cGMPs. Taken together these results suggest that the stimulatory effects of myristate on endothelial cell adhesion to type I collagen requires the generation of endogenous NO, which in turn activates sGC and leads to cGK activation via cGMP ().
In addition to regulating NO signaling, myristic acid modifies the function of a number of proteins via N-myristoylation (35
), and exogenous myristate has been documented to be efficiently utilized for protein acylation by other cell types (46
). One myristoylated protein that was previously implicated in TSP signaling via CD36 is Fyn (12
). Although Fyn is necessary for induction of apoptosis and inhibition of corneal angiogenesis by TSP1 (12
), other investigators have concluded that Fyn mediates pro-angiogenic signaling. Fyn is not required for the vascular permeability activity of VEGF (47
) but is required for stimulation of mitogenesis and tube formation by VEGF (48
). Fyn also contributes to endothelial tube formation and migration stimulated by FGF2 and angiopoietin-2 (49
). Conversely, the angiogenesis inhibitor pigment epithelium-derived growth factor specifically down regulated FGF2-stimulated Fyn activity via Fes (50
We now show that the positive effects of myristate on endothelial cell signaling and function are associated with increased membrane translocation of Fyn and functional activation of Src family kinases as assessed by Tyr416
phosphorylation. The rapid increase in Fyn translocation following addition of exogenous myristate that we observed is consistent with the previously reported rapid translocation of this Src kinase following myristoylation (51
). Src localization and activation may also be regulated via CD36-mediated myristate uptake because, unlike Fyn, Src is exclusively tethered to membrane via myristoylation (52
Fyn and other Src family kinases are known to co-precipitate with CD36 in lysates from platelets and endothelial cells (14
), but given their mutual association with lipid raft microdomains this is not proof of their direct interaction. Indeed, a more detailed examination failed to detect direct interaction of Fyn with CD36 (16
). Our results show that independent of any potential physical coupling, the fatty acid translocase activity of CD36 can modulate Fyn function by altering its cellular localization and functional activation. Therefore, regulation of Fyn by CD36 probably does not require any physical interaction.
It was surprising that exogenous myristic acid would be required for protein myristoylation given that myristic acid is an abundant component of cellular phospholipids. Presumably, the free fatty acid pool must be limiting for synthesis of myristoyl-CoA in HUVEC deprived of serum. Additional studies are required to determine whether CD36 globally limits protein myristoylation or acts specifically to regulate trafficking of certain targets such as Fyn. We previously found that TSP1 also inhibits signaling downstream of cGMP (17
). Inhibiting myristoylation was previously shown to prevent membrane localization of cGMP-dependent protein kinase II (53
). This, therefore, is a potential target for the latter activity of TSP1.
The results of Zhu (26
) and our results, showing L-NAME inhibition of myristate-induced cGMP signaling, indicate that exposure of serum-deprived HUVEC to exogenous myristate rapidly activates eNOS. This could occur by regulation of eNOS myristoylation, which is essential for its membrane localization and function (39
) However, the efficient cotranslational myristoylation of eNOS coupled with further palmitoylation may make this target less sensitive to limiting the myristic acid pool (41
). Furthermore, the ~20 h half life of eNOS may preclude detecting a significant shift in distribution of eNOS within the 1 h period of our assay. Because the activity of eNOS is regulated by exogenous myristate within this time frame, translocation of other myristoylated proteins that control eNOS activation, such as the phosphatase PP2B and certain myristoylated peptides (55
) should be considered. Future studies will examine which myristoylated proteins are responsive to CD36-mediated myristate uptake within the time frame of eNOS activation.
Our observations that CD36 limits myristate uptake and the myristate/CD36-dependent translocation of at least one important signaling protein suggest a common basis for the activities of CD36- and methionine aminopeptidase (MetAP2)-targeted drugs as angiogenesis inhibitors (). MetAP2 inhibitors such as fumagillin, ovalicin, and TNP-470 prevent cleavage of the N-terminal Met from proteins destined to be myristoylated (58
). CD36 transports myristic acid that can be activated by acyl-CoA synthetases to become the substrate for N-myristoyl transferases. Although myristic acid is abundant in membrane phospholipids, our data and that of Zhu (26
) indicates that the availability of free myristic acid is limiting for regulation of eNOS in serum starved endothelial cells.
This common mechanism is relevant to development of therapeutic angiogenesis inhibitors. Irreversible MetAP2 inhibitors such as TNP470 have toxic side effects that have prompted efforts to develop reversible inhibitors of MetAP2 (59
). Our data suggests that the CD36-directed drug ABT-510 may show better efficacy by reversibly blocking access to the myristoyl-CoA needed for tethering Fyn and other signaling proteins to membranes subsequent to MetAP2 cleavage of the terminal Met.
TNP-470 shares with ABT-510 the property of synergizing with radiation to inhibit tumor angiogenesis (60
). Similar synergism with cytotoxic agents was also noted for TNP-470 and ABT-510 (63
). The convergent effects of these two drugs on protein myristoylation may explain these similarities. Furthermore, the myristoylation pathway may play a more general role in tumor growth independent of angiogenesis. N-myristoyl transferases have been considered as potential targets for anti-neoplastic drugs (65
), and MetAP2 is elevated in colon carcinoma (66
). Therefore, drugs that target CD36, such as ABT-510, may also have anti-tumor activities through inhibiting myristate uptake that are independent of angiogenesis.