Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the β1 integrin subunit, and subsequently both α3β1 and α5β1, but not αvβ3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to α5β1 was confirmed using a recombinant α5β1-Fc fusion protein. Using conformation-dependent anti-β1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of α5β1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human α5 integrin, but not those lacking α5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.Maspin is a member of the serpin family of serine protease inhibitors (SERPINB5).
2 It was originally identified as a gene down-regulated in invasive breast cancer and proposed as a class II tumor suppressor (
1), and has since been shown to have many effects on cellular behavior that are consistent with this activity. It has been shown to decrease the proliferation, migration, and metastasis of tumor cells
in vivo (
1,
2) and their invasion
in vitro (
3,
4), and to increase apoptosis of endothelial cells (
5) and inhibit angiogenesis (
6). However, the cellular effects of maspin are not restricted to tumor cells, and we have demonstrated that maspin can inhibit the migration of vascular smooth muscle cells (
7).VSMC migration is a key event in the development of atherosclerosis (
8), and contributes significantly to restenosis after angioplasty (
9) and transplant arteriosclerosis (
10). VSMC are not terminally differentiated and acquire migratory capacity as part of a phenotypic switch from a contractile, quiescent state to a dedifferentiated phenotype, characterized by proliferation and increased extracellular matrix synthesis, in addition to motility (
11). This allows VSMC to respond to environmental cues following vascular injury. The phenotypic plasticity of VSMC is regulated by an array of signals, among which integrin-mediated association with surrounding extracellular matrix and changes in the expression of matrix-degrading proteases are prominent (
12–
14).How maspin mediates its various cellular effects is unclear. Maspin has been reported to be an inhibitor of plasminogen activation (
3,
15,
16), but we have shown that maspin is unable to inhibit either uPA- or tPA-catalyzed plasminogen activation under conditions in which the serpin PAI-1 was completely inhibitory (
7). The anti-proteolytic inhibitory mechanism of serpins is dependent on characteristics of the reactive center loop (RCL) allowing it to adopt the necessary canonical conformation and rearrangements subsequent to protease binding (
17). The RCL of maspin does not have the required characteristics (
7,
18), and the conclusion that maspin is a non-inhibitory serpin is fully supported by its crystal structure (
19,
20).Another confounding factor in understanding the mechanisms underlying the cellular effects of maspin is that, in common with the serpin PAI-2, it lacks an authentic secretion signal sequence. Nevertheless it has been shown to enter secretory vesicles (
21) and is found extracellularly, in the cytoplasm and also in the nucleus (
21,
22). Cytoplasmic and nuclear binding proteins for maspin have been identified (
23–
25), and may be responsible for its effects on proliferation and apoptosis. How secreted, extracellular maspin exerts its effects is unclear, but a function as a cell signaling ligand has been proposed (
26–
28). However, the characteristics of the maspin inhibitory effect on VSMC migration point to a more direct effect of maspin.To determine the mechanism of the maspin effect on VSMC migration, we have now attempted to identify maspin-binding proteins on the surface of these cells. In this report we provide biochemical, cellular, and functional evidence that the effect of maspin on cell migration is mediated by specific binding to cell adhesion receptors of the integrin family. We find that maspin binds specifically to β1 integrins on the surface of dedifferentiated VSMC, which leads to a reduction in the activation status of the integrin, and that the binding of maspin to α5β1 is sufficient for its inhibitory effects on cell migration and may represent a more general mechanism underlying its diverse biological effects.
相似文献