Abstract
Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel ‘RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.
Highlights
Isthmin (ISM), a 60 kDa secreted matricellular protein, was originally identified as a gene highly expressed in the midbrain-hindbrain organizer in Xenopus embryos.[3]
To decipher ISM’s mechanism of action, we addressed the following questions in this work: (1) Does avb[5] integrin mediate ISM’s antiangiogenic activities, such as inducing apoptosis? (2) How does ISM induce endothelial cell (EC) apoptosis? (3) Would ISM generate similar anti-endothelial effects in both soluble and anchored conditions? (4) Would ISM function as a cell adhesion molecule? If yes, how does it compare with other cell adhesion molecule in the extracellular matrix (ECM)? Using both neutralizing antibody as well as RNAi gene expression knockdown, we demonstrated that suppressing integrin avb[5] could stifle apoptosis induced by ISM and relieve ISM’s inhibition of EC proliferation
We have previously reported that soluble ISM produced from E. coli functions as an angiogenesis inhibitor, inhibiting EC tube formation, inducing EC apoptosis and suppressing vascular endothelial growth factor (VEGF)-stimulated EC proliferation in a dosedependent manner.[4]
Summary
Isthmin (ISM), a 60 kDa secreted matricellular protein, was originally identified as a gene highly expressed in the midbrain-hindbrain organizer in Xenopus embryos.[3]. We recently reported that ISM is a novel endogenous angiogenesis inhibitor with functions in both physiological and pathological angiogenesis.[4] It suppresses multiple aspects of in vitro angiogenesis including endothelial cell (EC) capillary network formation on Matrigel and EC proliferation. Soluble ISM potently induces apoptosis, immobilized ISM loses the proapoptotic activity and instead promotes EC adhesion, survival and haptotactic migration These results suggest that the matricellular protein ISM in different physical state can trigger different intracellular signaling pathways and generate different biological effects through the same integrin receptor. This is different from classical ECM integrin ligands such as vitronectin (VN) or fibronectin (FN)
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