Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through α2β1 integrin

Abstract

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, α2β1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and α2β1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin.

BMP-1/Tolloid-like Metalloproteases Process Endorepellin, the Angiostatic C-terminal Fragment of Perlecan

“…3C). This provides a molecular explanation for the lack of LG3 affinity to heparin and for the lack of requirement for cell surface heparan sulfate for full biological activity (15). Moreover, electrostatic analysis indicated that the predicted Ca 2ϩ -coordinating region forms an acidic pocket on the surface, despite variations in nearby loop regions.…”

“…N-terminal sequences were determined by automated Edman degradation at the Harvard University Microchemistry Facility. Preparation of adenovirus-endorepellin (Ad-ER) and transduction experiments were performed as described previously (15).…”

“…To determine whether BMP-1/Tolloid-like proteases were capable of cleaving the LG3 domain from precursors, BMP-1 was incubated with recombinant endorepellin. As mentioned above, recombinant endorepellin contained small quantities of the 26-kDa LG3 that, due to a C-terminal His tag (11,15), co-purified with full-length endorepellin (Fig. 1A, lane 1).…”

“…We have recently discovered that the C-terminal lamininlike globular (LG3) domain of endorepellin harbors most of its anti-angiogenic activity (15). Notably, LG3 fragments cleaved at the amino acid residues identical to those generated in vitro by 293-EBNA cells in either human (11) or mouse (13,14) have been detected in the urine of patients with end-stage renal disease (16) and in the amniotic fluid of pregnant women with premature rupture of fetal membranes (17).…”

Caspase-3 Activation Triggers Extracellular Cathepsin L Release and Endorepellin Proteolysis

“…Recent work by our group and others demonstrated that EC apoptosis represents a previously unrecognized pathway contributing to ECM proteolysis (5,6,18,19,43,44). We showed that in EC, apoptosis triggers endorepellin proteolysis and LG3 release, which interacts with ␣2␤1 integrins on EC, platelets, and fibroblasts, and triggers angiostatic, thrombogenic, and anti-apoptotic responses (20,(45)(46)(47).…”