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Identification of CD36 molecular features required for its in vitro angiostatic activity

Luca Primo^*,1, Chiara Ferrandi^*, Cristina Roca^*, Serena Marchiò^*, Laura di Blasio^*, Massimo Alessio and Federico Bussolino^*

^* Institute for Cancer Research and Treatment, Candiolo, and School of Medicine, University of Torino, Italy; and
Proteomics Laboratory, DIBIT, San Raffaele Scientific Institute, Milano, Italy

¹ Correspondence: I.R.C.C.-Sp.142, Km 3.95-10060 Candiolo, Italy. E-mail: luca.primo{at}ircc.it

SPECIFIC AIMS

To investigate the mechanisms by which CD36 exerts its angiostatic activity and the molecular features of its C-terminal cytoplasmic tail necessary to mediate the inhibitory effects of TSP-1.

PRINCIPAL FINDINGS

1. TSP-1 inhibits VEGF-A₁₆₅-induced migration and in vitro angiogenesis
CD36 is expressed on microvascular ECs but not on ECs from large vessels, thus providing a useful tool to assess CD36-dependent cellular functions. We infected human ECs from umbilical cord veins at high efficiency, using a retroviral vector carrying gfp and CD36, respectively, under the control of CMV and LTR promoters. When tested in chemotaxis assay, EC-CD36 were sensitive to a gradient of VEGF-A₁₆₅ and their motility was inhibited by TSP-1 at 10 nM. In contrast, EC-Pinco responded to VEGF-A₁₆₅ but not to TSP-1. The agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A₁₆₅ induced-migration of EC-CD36 but not that of EC-Pinco (Fig. 1 A).

View larger version (29K): [in this window] [in a new window]   Figure 1. Inhibition of EC migration and sprouting by TSP-1 in CD36 expressing cells. A) VEGF-A165 induced migration of EC-Pinco (white bar) and EC-CD36 (black bar) in Boyden chamber assay. TSP-1 (10 nM), agonist mAb SMO (1 µg/mL), and oxLDL (2 µg/mL) were tested for ability to inhibit EC-Pinco and EC-CD36 migration (mean±SD n=3) *P < 0.001 and P < 0.005 vs. VEGF-A165-stimulated cells. B) EC-Pinco (white bar) and EC-CD36 (black bar) were cultured in the absence of serum for 24 h (SF) or the presence of VEGF-A (20 ng/mL), with or without TSP-1 (10 nM). Cell apoptosis was quantified by measuring DNA fragmentation as oligonucleosomes OD (absorbance A405nm-A490nm). Values are means ± SD of 3 separate experiments performed in triplicate. C) Spheroids of ECs, embedded in collagen gel, give rise to radial outgrowing capillary sprouts in the presence of VEGF-A165. The presence of TSP-1 (10 nM) or MoAb SMO (1 µg/mL) blocks the cell sprouting from spheroids of EC-CD36 (x200). All photographs are representative of 3 experiments with similar results.

The effect of CD36 expression in ECs was also evaluated in an in vitro angiogenesis assay where spheroids of ECs were embedded in collagen gel. In the presence of VEGF-A₁₆₅, EC-Pinco and EC-CD36 sprout and invade the surrounding collagen gel forming a ring of capillary-like structures. The addition of TSP-1 (10 nM) completely inhibited EC-CD36 sprouting without affecting EC-Pinco (Fig. 1C ). Similar results were obtained by adding anti-CD36 MoAb SMO to the collagen gel (Fig. 1C ).

2. Point mutations of C-terminal CD36 intracellular tail affect TSP-1 inhibition of EC migration and in vitro angiogenesis
To elucidate the cytosolic molecular determinants of CD36 relevant for its angiostatic activities, ECs were engineered with stable retroviral expression vector bearing different CD36 constructs, with point, nonconservative mutations of C464, C466, R467, and K469. We wanted to look at the effects of TSP-1 on migration and differentiation into capillary-like structures of ECs engineered with different CD36 constructs. When tested in Boyden’s chamber assay in the presence of a VEGF-A₁₆₅ gradient, CD36 mutants C464S and K469A were not sensitive to TSP-1 inhibition. In contrast, TSP-1 was able to inhibit the VEGF-A₁₆₅ activity in ECs expressing CD36 mutants on C466 (C466S) and R467 (R467G) (Fig. 2 A).

View larger version (31K): [in this window] [in a new window]   Figure 2. Inhibition of migration and sprouting of ECs expressing CD36 and CD36 mutants by TSP-1. A) Migration of ECs induced by vehicle (white bar), VEGF-A165 (black bar), VEGF-A165 in the presence of TSP-1 (10 nM) (hatched bar), and VEGF-A165 in the presence of abSMO (1 µg/mL) (gray bar). Mean ± SD of 3 experiments. *P < 0.005 vs. VEGF-A-treated cells. B) Spheroids of ECs, embedded in collagen gel give rise to radial outgrowing capillary sprouts in the presence of VEGF-A165 (back bar). The addition of TSP-1 (10 nM) (hatched bar) and SMO (1 µg/mL) (gray bar) blocks the sprouting of endothelial cells from spheroids of EC-CD36 and EC-CD36C466S. Control spheroids were maintained in the presence of serum and without VEGF-A165 (white bar). Sprouts length were measured by using Image Proplus 4.0 analysis software. Mean ± SD of 3 experiments. P < 0.005 vs. VEGF-A-treated spheroids.

Similar results were obtained when the effect of TSP-1 was evaluated in sprouting assay. All cell mutants examined responded to VEGF-A₁₆₅ as EC-Pinco or EC-CD36. Addition of TSP-1 blocked the capillary sprouting from spheroid of EC-CD36 and EC-CD36^C466S. In contrast, EC-CD36^C464S, EC-CD36^R467G, and EC-CD36^K469A formed tubular structures in the presence of TSP-1, suggesting that these amino acids are required for CD36-mediated intracellular signal (Fig. 2B ).

3. CD36 down-regulates VEGFR-2 phosphorylation and associates with ß1 integrin
To understand the mechanisms by which CD36 negatively modulates VEGF-A₁₆₅ signaling, we evaluated whether CD36 interfered directly with the VEGFR-2, which is the most important receptor transducing VEGF-A signals. Co-immunoprecipitation experiments between CD36 and VEGFR-2, showed that CD36 did not associate with VEGFR-2 even in the presence of TSP-1 or VEGF-A₁₆₅ (data not shown). VEGFR-2 is a tyrosine kinase receptor that dimerizes upon ligand binding and autophosphorylates. When EC-CD36 were stimulated with VEGF-A₁₆₅, the tyrosine phosphorylation of VEGFR-2 was comparable to that of EC-Pinco. Preincubation of EC-CD36 for 15 min with TSP-1 down-regulated the tyrosine phosphorylation of VEGFR-2 stimulated by VEGF-A₁₆₅ but did not change the phosphorylation of VEGFR-2 in EC-Pinco.

EC-CD36^C466S exhibited a pattern of VEGFR-2 phosphorylation similar to that of EC-CD36 with a decreased phosphorylation in the presence of TSP-1. In contrast, EC-CD36^C464S did not exhibit any alterations in VEGFR-2 phosphorylation when stimulated by TSP-1.

Therefore we looked at the association between CD36 and ß1 integrin in EC-CD36, EC-CD36^C464S, or EC-Pinco. By co-immunoprecipitation experiments, we demonstrated that CD36 constitutively associated with ß1 integrin; this interaction required C464 as inferred by the reduced association in EC-CD36^C464S.

CONCLUSIONS AND SIGNIFICANCE

TSP-1, a natural inhibitor of vascularization in vitro and in vivo, makes activated-ECs unable to form new vessels and so slows the growth of tumors in experimental animals. We demonstrate that the activation of CD36 by TSP-1 is sufficient to inhibit some biological properties of VEGF-A, a pivotal molecule of vasculogenesis and angiogenesis in embryo and adult life. To exclude the possibility that TSP-1 inhibits VEGF-A₁₆₅-induced angiogenesis by using different mechanisms rather than activation of CD36, we transduced CD36 in ECs lacking this receptor. In fact, ECs isolated from large vessels do not express CD36 and are insensitive to TSP1 or CD36 agonist mAb SM. In contrast, EC-CD36 become sensitive to exogenous TSP-1 in chemotaxis and sprouting and do not respond to the migratory and differentiative stimulus exerted by VEGF-A_165. These observations demonstrate that CD36 activates specific intracellular pathways that interfere with some downstream effectors of tyrosine kinase receptors.

We investigated how CD36 modulates EC migration and sprouting, and mutated putative key amino acids in the C-terminal cytosolic tail to gain a better understanding of the molecular determinants of CD36 involved. In the C-terminal tail of CD36, specific protein domains or identification sequences have not been clearly identified, so we mutated cysteines and basic amino acids that could be potentially involved in protein-protein interactions. Our results indicate that C464 and K469 of CD36 are required for the angiostatic activities of TSP-1. Both point mutations affect CD36 functions and ECs carrying these mutations are refractory to the inhibitory activity of TSP-1 lasting sensitive to the chemotactic and differentiative activity of VEGF-A₁₆₅. The TSP-1-dependent inhibition of VEGFR-2 activation was negligible in ECs carrying C464S mutant but not in those with CD36^C466S supporting the concept that polar amino acid residues in the cytoplasmic tail of CD36 may have essential roles in the angiostatic activity of this receptor.

We wanted to see whether they could single out a consensus sequence present in others transmembrane proteins and how this consensus could interact with intracellular transducers. By aligning sequences beginning from the transmembrane domain, we emphasized a consensus sequence that include C646 and R467. These two residues are highly conserved within CD36 and the tetraspanin family proteins, which are characterized by four transmembrane domains and a short C-terminal tail. Tetraspanin proteins are widely expressed transmembrane proteins involved in cellular migration, proliferation, and differentiation. They appear to function by regulating the activity of other receptor systems, including integrins. Our results demonstrate that integrin ß1 associates with CD36 on EC. The CD36 mutant C464S that loses the ability to inhibit EC migration showed only a residual ability to associate with integrin ß1. We therefore suggest that this interaction could be an important regulatory mechanism for CD36-mediated inhibition of migration. Exactly how CD36 regulates integrin function remains to be determined but we may hypothesize that CD36 residues close to intracellular plasma membrane regulate its lateral migration on cell surface promoting the association with integrins or recruiting adjacent integrins into membrane microdomains. Moreover, because integrins are largely involved in angiogenesis and modulate the activation of VEGFR-2, it is intriguing to speculate that CD36 could act as tetraspanin proteins or could make supramolecular complexes with integrins and tetraspanins regulating the activation of VEGFR-2. The finding that TSP-1 reduces VEGFR-2 phosphorylation in EC expressing CD36 but not in EC-CD36^C464S suggests that the complex CD36-integrin ß1 could be involved in the function regulation of other membrane proteins (Fig. 3 ). A recent report showed a similar mechanism for the anti-angiogenic effect of TIMP-2: TIMP-2 induced a decreased tyrosine phosphorylation of VEGFR-2 and FGFR-1, and these effects require 3ß1 integrin-mediated binding of TIMP-2 to endothelial cells.

View larger version (18K): [in this window] [in a new window]   Figure 3. A proposed model of TSP1-mediated inhibition of EC migration and sprouting involves the association of CD36 with integrin b1 and a reduced tyrosine phosphorylation of VEGFR-2. The cysteine 464 located in intracellular tail of CD36 is required for transducing the TSP-1 inhibitory effect on migration and sprouting. The same mutant loses the ability to associate with integrin b1 and to reduce VEGFR-2 phosphorylation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3697fje;

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