Binding of Extracellular Maspin to β1 Integrins Inhibits Vascular Smooth Muscle Cell Migration

Bass, Rosemary; Wagstaff, Laura; Ravenhill, Lorna; Ellis, Vincent

doi:10.1074/jbc.m109.038919

Cited by 24 publications

(46 citation statements)

References 47 publications

(53 reference statements)

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“…Extracellular maspin interacts with ␤1 integrins to influence cell adhesion and migration directly (19,20). We identified ␣5␤1 as being critical for the effects of extracellular maspin on cell migration through a mechanism involving rapid modulation of the activation state of ␤1 (20).…”

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“…HT-29 cells were maintained in DMEM with 10% (v/v) FCS. Primary aortic smooth muscle cells (referred to as VSMCs) were cultured as detailed previously (20). All cell culture reagents including extracellular matrix (ECM) components were from Invitrogen.…”

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“…Cell Migration Assays-Cell migration was determined using time lapse video microscopy as described previously (20).…”

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“…Plasmids and Transfection-Generation and transfection of maspin plasmid referred to as pcDNA3.2-Maspin and the control empty vector pcDNA3.2 were described previously (20).…”

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G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Ravenhill¹,

Wagstaff²,

Edwards

et al. 2010

Journal of Biological Chemistry

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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...

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G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Ravenhill¹,

Wagstaff²,

Edwards

et al. 2010

Journal of Biological Chemistry

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“…[6] The literature contains several additional contradictory reports as to the effect of soluble RGD-containing proteins on adhesion and migration. [5,15] Mechanistic studies of the roles of ligand-receptor interactions in cell migration are challenging because it is difficult to control which interactions operate between cell and matrix. This limitation is particularly relevant to in vivo experiments, where a broad range of signals and matrix constituents are present and not always defined.…”

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An Inhibitor of a Cell Adhesion Receptor Stimulates Cell Migration

2010

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The integrins are a family of heterodimeric receptors that play a universal role in mediating the adhesion of cells to the extracellular matrix. As such, the differential interactions of the receptors at the front and back ends of the cell are important in regulating the migration of cells. [1] Approximately one-half of the integrins recognize the canonical RGD motif found in fibronectin, and a significant body of work has shown that model substrates presenting the RGD peptide support the adhesion and migration of cells. [2] Inhibitors of the integrin receptor have been explored as reagents that may block cell migration and recent work has advanced a class of soluble RGD peptides as possible therapeutics that display anti-metastatic activity. [3][4][5] But the molecular mechanisms by which these compounds block migration have not been elucidated and have recently been contradicted by work that demonstrates that the soluble antagonists can also promote the migration of tumor cells. [6][7][8] Herein, we use a well-defined model system to assess the influence of soluble inhibitors on cell migration and we show that the integrin antagonists are able to promote the migration of cells.The RGD-mimetic compounds cilengitide and S36578 were developed as integrin antagonists and are now in early phase clinical trials for cancer therapy. [9,10] These compounds, at micromolar concentrations, were shown to block cell migration, but recent work reported that lower concentrations of these drugs can actually enhance the growth of tumors in vivo by promoting migration and VEGF-mediated angiogenesis. [7] Other recent work has investigated the analogy between tumor angiogenesis and wound healing [3,11] with the finding that the fibronectin-derived Pro-His-SerArg-Asn (PHSRN) peptide can stimulate migration of human kerotinocytes and fibroblasts and accelerate wound healing in obese diabetic mice. [8] Our group [12,13] and others [14] have suggested that PHSRN antagonizes RGD binding, and therefore may accelerate migration by inhibiting the integrinmediated adhesion. Additionally, it has been reported that addition of insulin-like growth factor binding protein (IGFBP-1) which contains the RGD sequence in the cell culture medium increases the migration of CHO cells twofold compared to cells treated with Trp-Gly-Asp (WGD). [6] The literature contains several additional contradictory reports as to the effect of soluble RGD-containing proteins on adhesion and migration. [5,15] Mechanistic studies of the roles of ligand-receptor interactions in cell migration are challenging because it is difficult to control which interactions operate between cell and matrix. This limitation is particularly relevant to in vivo experiments, where a broad range of signals and matrix constituents are present and not always defined. [16] Even in the laboratory, it can be difficult to reproducibly present matrix ligands to a cell in order to obtain reproducible matrix formulations. [5] To address these limitations, we use selfassembled monolayers that presen...

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