Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G ␣-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on ␤1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.Maspin (SERPINB5) is a member of the serpin family of serine protease inhibitors that acts as a type II tumor metastasis suppressor, decreasing tumor growth and metastasis in vivo (1, 2) and invasion in vitro (3, 4). It is down-regulated in cancers including those of the breast (1) and prostate (5). Exogenous maspin decreases proliferation and increases cell adhesion in vitro (6). It inhibits angiogenesis in vivo (7) and causes apoptosis when expressed in endothelial cells (8). In addition, we have shown that maspin can inhibit the migration of vascular smooth muscle cells (VSMCs) 3 (9), which has potential ramifications for conditions resulting from vascular injury such as atherosclerosis.Maspin is expressed by epithelial cells and is essential for normal development because maspin-null mice die at the periimplantation stage due to a failure of early differentiation events, resulting from aberrant adhesion and cell migration (10). However, the mechanism of action of maspin remains largely unresolved. Although early evidence suggested that maspin was an inhibitory serpin able to block plasminogen activation by urokinase plasminogen activator and tissue-type plasminogen activator (11-13), we demonstrated that this was not the case in a number of conditions where the serpin PAI-1 was inhibitory (9). That maspin is a noninhibitory serpin is supported by crystal structure data revealing that its RCL does not correspond with those found in inhibitory serpins (14,15). It remains possible that maspin influences protease activity indirectly by noninhibitory interactions with the plasminogen activators (16, 17) and protection of matrix from degradation by cathepsin D (18).In common with the serpin PAI-2, maspin lacks an authentic signal sequence, but is found outside the cell as well as in t...

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 ␤1 integrin subunit, and subsequently both ␣3␤1 and ␣5␤1, but not ␣v␤3, 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 ␣5␤1 was confirmed using a recombinant ␣5␤1-Fc fusion protein. Using conformation-dependent anti-␤1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of ␣5␤1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human ␣5 integrin, but not those lacking ␣5 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 integr...

Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively.

Maspin (SERPINB5) is a type II metastasis suppressor that influences multiple cellular functions. To date, maspin has been shown to increase adhesion and apoptosis and to decrease cell migration, proliferation, invasion and metastases in tumour malignancy. At the subcellular level, maspin influences morphological changes in the cell cytoskeleton which regulates complex biological processes including cell migration, cell adhesion and EMT (epithelial to mesenchymal transition). Here non-Euclidian fractal and image analyses have been applied to measure changes in the actin cytoskeleton using confocal microscopy images to confirm the effects of maspin. Results show that maspin contributes to maintaining the regular epithelial like shape, increases cell-cell adhesion and restricts tumour cells from showing the premigration and EMT characteristics. Characterization of these changes in the actin cytoskeleton using microscopic image analysis will establish maspin as a potential prognostic marker in future.

The cellular cytoskeleton is a dynamic subcellular structure that can be a marker of key biological phenomena including cell division, organelle movement, shape changes and locomotion during the avascular tumor phase. Little attention is paid to quantify changes in the cytoskeleton while nuclei and