We have demonstrated that ephrinA1, present on the cell membrane as a GPI-anchored protein, is cleaved at three positions within the C terminus, releasing three forms of the protein, eA1-(19–175), -(19–177), and -(19–178) (). Our findings also reveal that ephrinA1 is a substrate for cleavage by multiple MMPs which are likely responsible for proteolysis of membrane-bound ephrinA1 from cancer cells. These findings strongly suggest, for the first time, the mechanism of release and the forms of soluble ephrinA1.
Fig 11 Schematic of ephrinA1 arrival on the cell membrane and subsequent cleavage. The signal peptide (amino acids [aa] 1 to 18) and GPI linkage sequences (aa 183 to 205) are removed in the endoplasmic reticulum and ephrinA1 is transported through the Golgi (more ...)
In support of previous studies revealing the functionality of a soluble form of ephrinA1 cleaved from cancer cells (2
), the current study demonstrates downregulation of EphA2 and alteration of cell morphology in response to exogenous monomeric ephrinA1 treatment. Notably, the ephrinA1 protein used in those experiments is the same form as was used for crystallography studies in which it was shown that ephrinA1 and EphA2 bind in a 1:1 ratio (27
). Functionality of soluble ephrinA2 has also been documented, and interaction between soluble ligand and receptor influences osteoclast differentiation (31
). Interestingly, while we and others have documented functional soluble ephrinA1 activity (2
), clustered ephrinA1 was shown to suppress Slit2-mediated angiogenesis in endothelial cells, while the soluble form of ephrinA1 abrogated this effect (15
). This seemingly contradictory role for ephrinA1 may highlight the importance of cell type and context in ephrin and Eph function.
We investigated the protease(s) possibly responsible for ephrinA1 cleavage using broad-spectrum inhibitors and determined that members of the MMP family are likely the responsible proteases. Inhibition of serine proteases also led to a decrease in ephrinA1 release likely due to serine proteases activating the MMP(s) (1
). We demonstrated that monomeric ephrinA1 is released from cancer cells via cleavage by at least four different MMPs: MMP-1, -2, -9, and -13 (). In addition, mass spectrometry analysis of cleaved ephrinA1 purified from the medium of ephrinA1-transfected cells revealed three forms of ephrinA1, which is not a surprising finding considering the broad specificity of MMP cleavage and the functional overlap within the MMP family (35
MMPs are a family of zinc-dependent endopeptidases known to play fundamental roles in tumor progression (20
). Multiple MMPs are expressed highly in GBM and other human malignancies. Extensive research on the gelatinase family of MMPs (MMP-2 and MMP-9) has identified them as critical players in various stages of cancer growth, metastasis, and angiogenesis (34
). MMP-2 and MMP-9 are overexpressed in GBM (10
). In fact, the expression pattern of MMP-2 and MMP-9 correlates with increased invasion in vitro
, and in vivo
studies verified that increased MMP-2 and MMP-9 expression correlates with increasing tumor grade (40
). Additionally, expression of collagenases, such as MMP-1 and MMP-13, is deregulated in various cancers (9
). MMP-1 expression has been documented to occur in GBM cells (4
), and increased expression correlates with increased tumor grade (58
) and tumorigenicity (52
), and decreased survival in GBM patients (71
). Moreover, increased MMP-13 production in glioma cells in response to various stimuli leads to an increase in migration and invasion (43
). Also of note, MMP-13 is a critical player in the activation of other MMPs (39
Not only do MMPs cleave components of the extracellular matrix to facilitate cancer cell migration and invasion, but also they are responsible for the shedding of multiple membrane-bound proteins (20
). In fact, interaction of ephrinB1 with EphB receptors leads to reverse signaling within the ephrin-expressing cell, causing an increase in MMP-8 secretion and cleavage of the ligand (60
). Additionally, the EphB2 receptor is cleaved by MMP-2 and MMP-9, and this release is ephrinB2 induced (42
). EphrinA2 is also cleaved by a member of the MMP family (22
), and stimulation of EphA2 by the soluble form of ephrinA2 leads to a functional ligand-receptor interaction and osteoclast differentiation (31
). On the other hand, ADAM13 cleavage of ephrinB1 and ephrinB2, as demonstrated by Wei et al., is not dependent on ligand-receptor interaction. In fact, they postulate that cleavage of the ligand prevents ligand-induced receptor activation and forward signaling into the Eph-expressing cell (64
EphrinA5 and ephrinA2 are cleaved by the metalloprotease ADAM10 (a disintegrin and metalloprotease 10) (22
). Analysis of ADAM10 substrates revealed a conserved motif that is also present in ephrinA1 (17
). However, this motif lies within the G-H loop of ephrinA1, the region known to interact with and bind the EphA2 receptor. We have never detected an immunoreactive band of ephrinA1 present in conditioned medium that would correspond to the size of ephrinA1 cleaved toward the middle of the protein. Additionally, we have demonstrated the functionality of released ephrinA1, which would not be the case if cleavage of the ligand occurred within the receptor binding domain. Furthermore, cleavage studies implicating ADAMs in ephrin proteolysis indicate the requirement of receptor-ligand interaction in order for cleavage to occur (17
). Conversely, we demonstrated that ephrinA1 release is not Eph receptor dependent. In fact, ephrinA1 is present in conditioned medium of cancer cells even when plated at a density at which cell-cell contact is less likely to occur (70
). Incubation of ephrinA1-Fc with GBM cell-conditioned medium in an acellular assay caused cleavage, as did incubation with nonconditioned, serum-containing medium, implicating a secreted protease in ephrinA1 release. In a physiologically relevant scenario, ephrinA1-Fc is also cleaved by human serum. Long-lasting effects of exogenous ephrinA1-Fc treatment in cell culture in previous studies may be due, in part, to monomeric ephrinA1 formed from cleavage of the homodimeric protein by secreted MMPs. While MMPs cleaved ephrinA1-Fc and also ephrinA5 in an acellular cleavage assay, incubation of ephrinA1-Fc with ADAM10 did not produce smaller ephrinA1 immunoreactive fragments (data not shown).
Similar to ephrinA1, ephrinA5 is also released from cells and has been demonstrated to be present in medium from cells endogenously expressing ephrinA5 as well as in cells in which ephrinA5 has been overexpressed (3
). While previous studies have reported the cleavage of ephrinA5 from cells by ADAM10 (32
), our acellular cleavage assay suggests that it may be susceptible to cleavage by various other MMPs as well. Of note, while MMP cleavage patterns between ephrinA1 and ephrinA5 were different, both were cleaved readily by members of the collagenase class of MMPs (MMP-1, -8, and -13). Unlike ephrinA1, ephrinA5 was also readily cleaved by MMP-7.
When residues 174 to 181 or 175 to 181 of ephrinA1 are deleted, ephrinA1 is no longer released from membranes, thus confirming that proteolysis of ephrinA1 occurs within the region encompassing these amino acids. Comparison of proteolytic-site-deficient and wild-type ephrinA1-transfected cells did not show significant differences in the migratory capacity of the cells or in EphA2 protein levels. This is to be expected since extensive cell-cell contact occurs within these assays and, although ephrinA1 is not released from proteolytic-site-deficient mutants, it remains on the membrane and could still activate EphA2 upon contact or be a continuous partner for proteins involved in reverse signaling. Although the protein levels of EphA2 are comparable, the wild-type and proteolytic-site-deficient mutants display various levels of ephA2. Therefore, in this system, EphA2 may not be subjected solely to genetic regulation. Thus, this represents a complex system that needs to be explored in a systematic manner.
In addition, the longest proteolysis-resistant form of ephrinA1, ending at amino acid 175, retains the ability to activate the EphA2 receptor. This form of ephrinA1 or similarly truncated or mutated forms can be exploited for pharmaceutical targeting.
EphA2, the primary receptor for ephrinA1, is overexpressed in multiple human malignancies, making it a promising target for new cancer therapeutics (66
). In GBM, a disease of dismal prognosis, EphA2 is highly overexpressed while the ligand ephrinA1 is nearly absent. On the other hand, EphA2 is not expressed in the normal adult brain (68
). Therefore, ephrinA1 conjugated to an agent such as a toxin could be delivered specifically to GBM cells while sparing normal brain tissue (67
). In order for the ephrinA1-based therapy to successfully reach its target, however, it is critical that proteolysis of the protein not occur within the tumor microenvironment, which would release the cytotoxic or imaging agent from its targeting ligand before encountering cancer cells. Our study has demonstrated that a form of ephrinA1 ending in amino acid 175 would represent a prototype cleavage-resistant functional binding unit serving such a purpose.