Exposure of Cryptic Domains in the α1-chain of Laminin-1 by Elastase Stimulates Macrophages Urokinase and Matrix Metalloproteinase-9 Expression

Abstract

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that ␣1:Ser 2091 -Arg 2108 , a peptide derived from the ␣1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK erk1/2 , leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that ␣1:Ser 2091 -Arg 2108 is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparinbinding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the ␣1-chain including ␣1:Ser In earlier studies, we tested the hypothesis that the ECM regulates macrophage proteinase expression by culturing macrophages on ECM purified from Engelbreth Holm Swarm (EHS) sarcoma (Matrigel TM ) (8,9). Results demonstrated that the expression of urokinase type plasminogen activator (uPA) and MMP-9 by murine RAW264.7 macrophages, human THP-1 monocytes, and human bone marrow-derived macrophages were strongly up-regulated. This was the first demonstration that the engagement of ECM by macrophages stimulates their expression of both uPA and MMP-9. Since the uPA/plasmin system is a physiologic activator of MMPs (10, 11), the ECM emerges as a potent regulator of the macrophage-degradative phenotype.The ECM component responsible for stimulating macrophage proteinase expression was identified as laminin-1 (8).Laminins are large heterotrimeric molecules (ϳ500 -1000 kDa) with multiple domains that mediate their attachment to cells and other ECM components (12). Twelve laminin heterotrimers (assembled from five ␣, three ␤, and three ␥ chains) have been identified in mammals. Laminin-1 was the first of this family to be identified (13) and remains the best understood of the laminin isoforms (12,14). It consists of ␣1 (ϳ400 kDa), ␤1 (ϳ200 kDa), and ␥1 (ϳ200 kDa) chains. The NH 2 -terminal portions of the ␣1-, ␤1-, and ␥1-chains are free, whereas much of the rest of the chains are twisted in a coiled-coil. The COOH-terminal portion of the ␣1-chain extends past the coiled-coil region and forms a large oblong globule (G-domain) consisting of five homologous repeats. The G-domain is the principle heparin-binding region of laminin-1 (15, 16).In an effort to identify the domains of laminin-1 responsible for stimulating macrophage proteinase expression, we examined synthetic peptides, which were reported to support cell adhesion and stimulate a variety ...

Extracellular Matrix-induced Cyclooxygenase-2 Regulates Macrophage Proteinase Expression

“…In studies reported here, the activation of MAP-K erk1/2 was shown to be required for the up-regulation of the proteinase expression of macrophages induced by adhesion to SMC-ECM. These data confirm our earlier studies examining the role of MAPK erk1/2 in the induction of proteinase expression by synthetic ␣1-chain laminin peptides and laminin-1 fragments (42,43).…”

“…1B). As expected, conditioned media derived from thioglycollateelicited macrophages contained increased levels of uPA, MMP-9, and MMP-2 relative to resident cells (9,43,48). When elicited macrophages were cultured on ECM, their expression of uPA and MMP-9 activities was further increased.…”

“…In previous studies (42,43), we demonstrated that the induction of macrophage proteinase expression by synthetic laminin ␣1-chain peptides was mediated by protein kinase C-dependent activation of MAPK erk1/2 . Therefore, we determined whether the stimulation of uPA and MMP-9 expression by SMC-ECM was dependent on a similar pathway.…”

“…In addition, it has been reported that macrophage proteinase expression is either stimulated (33)(34)(35)(36)(37)(38)(39) or inhibited (40,41) by purified ECM components. Moreover, in some cases, the regions of ECM molecules that regulate macrophage proteinase expression are cryptic and require proteolytic processing to be exposed (39,42,43). Because the regulatory domains of individual matrix components may be masked by tertiary conformation or by their association with other matrix molecules in a complex ECM, it is difficult to predict the effect that intact cell-derived ECM would have on macrophage proteinase expression.…”

New insights into the role of extracellular matrix during tumor onset and progression

“…For example, cleavage of laminin-5, one of the ECM components that promotes stable cell attachment, by MMP-2 or MT1-MMP generates a proteolytic g2-chain fragment which promotes migration of human breast epithelial cells (Giannelli et al, 1997;Koshikawa et al, 2000). In addition, an a1-chain fragment generated by elastase proteolysis of laminin-1 has been shown to play a role in regulation of the macrophage degradative phenotype and in tissue remodeling (Khan et al, 2002). Note that the ECM fragments derived by MMP cleavage can also act as pro-angiogenic molecules, for example, the trimeric NC1 domain of collagen XVIII induces endothelial cell migration involved in angiogenesis (Kuo et al, 2001).…”