Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of β₁-integrin at threonine 788/789

Abstract

. Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of ␤1-integrin at threonine 788/789. Am J Physiol Cell Physiol 296: C193-C204, 2009. First published November 12, 2008 doi:10.1152/ajpcell.00355.2008.-Increased extracellular pressure stimulates ␤1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in ␤1-integrin binding affinity or avidity and whether these changes are phosphorylation dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(abЈ)2 multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of ␤1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-␤1-integrin-null murine fibroblasts stably transfected with either wild-type ␤1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of ␤1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(abЈ)2 multimeric complexes, but not soluble RGD-Fc ligands, was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting that pressure modulates ␤1-integrin affinity. Pressure stimulated both ␤1-integrin S785 and T788/9 phosphorylation. GD25-␤1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in ␤1-integrin-null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by ␤1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity. adhesion; mechanotransduction; metastasis INTEGRIN-MEDIATED TUMOR CELL adhesion to extracellular matrix components or endothelial cells is an important step in the development of metastatic lesions. Integrin binding affinity is regulated by distinct receptor activation states (23). Initial cell attachment requires integrin activation by cytoplasmic signals that convey large conformational changes to the extracellular domain and enhance ligand-binding affinity (56). The avidity and strength of the interaction is further increased through the redistribution of integrins in the cell membrane, often referred to as receptor clustering (65). The tendency of some malignancies to exhibit reduced integrin expression while maintaining adhesive properties underscores the significance of integrin activation states in mediating tumor cell adhesion (33, 47).Likewise, mutations conferring constitutive integrin activation have been shown to directly contribute to the metastatic potential of cancer cells (20,53). Mechanisms underlying integrin affinity and avidity modulation are therefore of significant interest in the control of tumor metastasis.It is increasingly clear that integrin modulation may...

Specific Phosphorylations Transmit Signals from Leukocyte β2 to β1 Integrins and Regulate Adhesion

“…The phosphorylation of ␤ 1 on Thr-788/ 789 corresponds to the phosphorylation triplet in ␤ 2 , including Thr-758. This phosphorylation reduces ␤ 1 association with the actin cytoskeleton (38) and increases pressure-induced cell adhesion in cancer cells (39). Importantly, cells expressing the activated form of LFA-1 showed reduced phosphorylation of ␤ 1 Thr-788/789.…”

Extracellular pressure stimulates adhesion of sarcoma cells via activation of focal adhesion kinase and Akt

“…0–60 mmHg p ressure above ambient was applied for 30 minutes using a prewarmed, airtight box with an inlet valve for gas and an outlet valve connected to a manometer as described 1–4. Control cells were incubated at 37°C under ambient pressure.…”

“…Adhesion of these shed tumor cells is crucial to metastasis. Previous studies 1–4,5 suggests that epithelial cancer cells can regulate their own adhesion to extracellular matrix proteins, endothelial cells, or surgical wounds via intracellular signals that ultimately regulate β 1 -integrin binding affinity.…”

β1-integrin mediates pressure-stimulated phagocytosis

“…In particular, β1-integrin has been linked to macrophage phagocytosis in regards to microbial pathogens. [19] Integrins are heterodimeric proteins that .may be activated after binding to an external target by integrin clustering, talin association and phosphorylation of the cytoplasmic tail [14, 16]. intracellular signals may influence integrin binding affinity for extracellular ligands prior to binding [15] Mechanical strain can stimulate conformational activation of integrins [17] as well as β1-integrin clustering [18].…”

“…Having previously shown that increased extracellular pressure promotes cancer cell adhesion by stimulating β1-integrin binding affinity [14], we now hypothesized that pressure stimulation of phagocytosis involves a similar effect, although cancer cells respond to pressure by different signals [9, 14]. We compared the effects of increased extracellular pressure on phagocytosis by β 1 -integrin-null GD-25 fibroblasts and GD-25 subclones expressing wild type β 1 -integrin subunit or various β 1 -integrin subunit point mutants that either mimic constitutive phosphorylation or constitutive dephosphorylation to determine whether β 1 -integrin-binding contributes to pressure stimulated phagocytosis, and to determine the specific phosphorylation sites on the β 1 -integrin subunit that might mediate this effect.…”