Integrin αvβ3-Mediated Activation of Apoptosis

doi:10.1006/excr.1999.4559

Experimental Cell Research

Volume 251, Issue 1, 25 August 1999, Pages 33-45

https://doi.org/10.1006/excr.1999.4559 Get rights and content

Abstract

The α_vβ₃ integrin mediates endothelial cell binding to the extracellular matrix and transduces an intracellular signal promoting survival of endothelial cells and various tumor cells. While the α_vβ₃ integrin-mediated survival signal has been shown to be adhesion dependent, a thorough analysis has not been performed comparing the biochemical effects of antagonist binding to α_vβ₃ integrin with the effects induced by the growth of cells in suspension. In this study we demonstrate that expression of α_vβ₃ integrin in human embryonic kidney 293 cells transfers the α_vβ₃ integrin survival pathway to an epithelial cell line. Furthermore, we show that α_vβ₃ integrin-expressing cells respond differently to α_vβ₃ integrin-specific antagonist treatment and growth in suspension conditions. Treatment with the α_vβ₃ antagonist echistatin resulted in an apoptotic response occurring prior to cell detachment and was not observed in either suspended cells or antagonist-treated suspended cells. These data suggest that the death induced by antagonist binding to α_vβ₃ integrin results in an apoptotic signal with different kinetics than the apoptotic signal induced by matrix detachment (anoikis). Since aberrant α_vβ₃ integrin expression in tumor models is thought to play a role in tumor cell survival, these data have implications for the use of α_vβ₃ antagonists as anti-tumor agents.

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SPRY domain-containing SOCS box protein 2 (SPSB2) is a negative regulator of inducible nitric oxide synthase (iNOS) that modulates the lifetime of iNOS and thus the levels of nitric oxide (NO) production. Inhibitors that can disrupt the endogenous SPSB2-iNOS interaction and augment NO production have potential as novel antimicrobial and anticancer drugs. In this study, we have designed a cyclic peptide (cR8), containing an RGD motif and the SPSB2 binding motif (DINNNV). ITC and chemical shift perturbation showed that cR8 binds to the iNOS binding site on SPSB2 with a K_d of 671 nM, and saturation transfer difference NMR showed that cR8 binds to α_vβ₃ integrin-expressing cells. Moreover, we determined the crystal structure of SPSB2 in complex with cR8, at a resolution of 1.34 Å. cR8 forms extensive hydrogen bonding with SPSB2 residues, but loss of an intramolecular hydrogen bond that is present in SPSB2-bound iNOS peptide may destabilize the bound conformation of cR8 and lead to a gentle reduction in SPSB2 binding affinity. These results serve as a useful basis for designing site-directed SPSB2 inhibitors in the future.
Functional analysis of four single (RGDWL, RGDWM, RGDWP, RGDMN) and two double (RGDNM, RGDMP) mutants: The importance of methionine (M) in the functional potency of recombinant mojastin (r-Moj)
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We have demonstrated in previous studies that a single amino acid change can alter the activity of the recombinant disintegrin r-Moj. In this study, four r-Moj recombinants containing single mutations (r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-MN) and two containing double mutations (r-Moj-MP and r-Moj-NM) at the binding loop were produced, purified, and tested. All r-Moj-W_, r-Moj-M_, and r-Moj-NM mutant peptides inhibited platelet aggregation at higher potency than r-Moj-D_ mutants. Five of the seven r-Moj peptides inhibited angiogenesis at different levels. Two of the mutant peptides with a methionine at the second position carboxyl of the RGD (r-Moj-WM and r-Moj-NM) were the strongest angiogenesis inhibitors, with r-Moj-WM being the most potent. Recombinant r-Moj-MP and r-Moj-WN failed to inhibit angiogenesis. Only the r-Moj-MP mutant peptide induced apoptosis of SK-Mel-28 cells significantly (p = 0.001). This was confirmed by chromatin condensation. Proliferation of SK-Mel-28 cells was inhibited at high levels (>70%) by all r-Moj mutant peptides. Recombinant r-Moj-MN and r-Moj-WN failed to inhibit cell migration significantly (p > 0.5). Recombinant r-Moj-NM was the strongest cell migration inhibitor (98% ± 0.69), followed by r-Moj-MP (80% ± 2.87), and r-Moj-WM (61.8% ± 5.45). The lowest inhibitor was r-Moj-WL (50% ± 12.16). Our functional data suggest that the most potent r-Moj disintegrins contain a methionine in the first or the second position carboxyl to the RGD.
RNA sequence analyses of r-Moj-DM treated cells: TXNIP is required to induce apoptosis of SK-Mel-28
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RNA sequencing of untreated and r-Moj-DM treated SK-Mel-28 cells was performed after 6 h, to begin unraveling the apoptotic pathway induced by r-Moj-DM. Bioinformatic analyses of RNA sequencing data yielded 40 genes that were differentially expressed. Nine genes were upregulated and 31 were downregulated. qRT-PCR was used to validate differential expression of 13 genes with known survival or apoptotic-inducing activities. Expression of BNiP3, IGFBP3, PTPSF, Prune 2, TGF-ß, and TXNIP were compared from cells treated with r-Moj-DN (a strong apoptotic inducer) or r-Moj-DA (a non-apoptotic inducer) for 1 h, 2 h, 4 h, and 6 h after treatment. Our results demonstrate that significant differences in expression are only detected after 4 h of treatment. In addition, expression of TXNIP (an apoptotic inducer) remains elevated at 4 h and 6 h only in r-Moj-DN treated cells. Based on the consistency of elevated TXNIP expression, we further studied TXNIP as a novel target of disintegrin activation. Confocal microscopy of anti-TXNIP stained SK-Mel-28 cells suggests nuclear localization of TXNIP after r-Moj-DM treatment. A stable TXNIP knockdown SK-Mel-28 cell line was produced to test TXNIP' role in the apoptotic induction by r-Moj-DM. High cell viability (74.3% ±9.1) was obtained after r-Moj-DM treatment of TXNIP knocked down SK-Mel-28 cells, compared to 34% ±0.187 for untransduced cells. These results suggest that TXNIP is required early in the apoptotic-inducing pathway resulting from r-Moj-DM binding to the αv integrin subunit.
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Background. The regenerative capacity of the liver is critical for proper responses to injury. Fibrin extracellular matrix (ECM) deposition is a common response to insult and contributes to inflammatory liver injury. However, the role of this matrix in hepatic regeneration has not been determined. Objective. The purpose of this study was first to determine the role of fibrin ECM in hepatic regeneration followed by the role of the fibrin-binding α_vβ₃ integrin in mediating this effect.
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Regular ArticleIntegrin αvβ3-Mediated Activation of Apoptosis

Abstract

J. Biol. Chem.

Cell

J. Biol. Chem.

Curr. Opin. Cell Biol.

J. Biol. Chem.

Curr. Opin. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Virology

Cell

Blood

Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model

Science

Apoptosis induced by inhibition of intercellular contact

J. Cell Biol.

Requirement of vascular integrin αvβ3 for angiogenesis

Science

The vitronectin receptor αvβ3 binds fibronectin and acts in concert with α5β1 in promoting cellular attachment and spreading on fibronectin

J. Cell Biol.

Geometric control of cell life and death

Science

Human endothelial cells synthesize and express an Arg-Gly-Asp directed adhesion receptor involved in attachment to fibrinogen and von willebrand factor

Proc. Natl. Acad. Sci. USA

Integrins and signal transduction pathways: The road taken

Science

An antagonist of integrin αvβ3 prevents maturation of blood vessels during embryonic neovascularization

J. Cell Sci.

Integrin αvβ3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis

J. Cell Biol.

Dependence of cyclin E-cdk2 kinase activity on cell anchorage

Science

Requirement of the NPXY motif in the integrin β3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo

J. Cell Biol.

Disruption of epithelial cell-matrix interactions induces apoptosis

J. Cell Biol.

Regular Article
Integrin α_vβ₃-Mediated Activation of Apoptosis

Requirement of vascular integrin α_vβ₃ for angiogenesis

The vitronectin receptor α_vβ₃ binds fibronectin and acts in concert with α₅β₁ in promoting cellular attachment and spreading on fibronectin

An antagonist of integrin α_vβ₃ prevents maturation of blood vessels during embryonic neovascularization

Integrin α_vβ₃ requirement for sustained mitogen-activated protein kinase activity during angiogenesis

Requirement of the NPXY motif in the integrin β₃ subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo