Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin‐derived inhibition of angiogenesis in aortic ring cultures

Abstract

The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa protein, the laminin binding protein (LBP), is shed from malignant cells in significant amounts and binds to laminin-1 (Starkey et al., Cytometry 1999;35:37-47; Karpatova et al., J Cell Biochem 1996;60:226-34). However, the functions of shed LBP are not fully understood. We hypothesize that matrix-bound LBP could modulate local tumor angiogenesis. In support of this hypothesis, we demonstrate that shed LBP exhibits sulfhydryl oxidase-like activities, and modifies the production of angiostatins from plasmin in vitro. The molecular weights of the autocatalytic products of lys-plasmin incubated with LBP in vitro suggest that PMDs (plasmin A chains attached to degraded B chains) (Ohyama et al., Eur J Biochem 2004;271: 809-20) are preferentially generated. Using rat aortic ring assays, we also show that shed LBP reverses plasmin-dependent inhibition of vascular outgrowth. To elucidate which LBP region(s) are active in modulating angiogenesis, limited proteolysis experiments were conducted to determine stable rLBP domains likely to fold correctly, and these were cloned, expressed and purified. The stable LBP fragments were tested for binding to laminin-1 and for competition with shed LBP activity in the aortic ring assay. Results of these studies suggest that the active LBP domains lie within the 137-230 amino acid sequence, a region known to contain 2 bioactive sequences. Since this fragment binds to laminin-1 and modulates angiogenesis, it appears likely that binding of shed LBP to matrix laminin-1 is related to its functions in tumor angiogenesis. The findings presented in this manuscript suggest that LBP shedding could provide a useful therapeutic target. ' 2005 Wiley-Liss, Inc.Key words: laminin binding protein; angiogenesis; angiostatin; limited proteolysisInitially isolated from tumor cell membrane extracts, overexpression of the 67 kDa laminin binding protein (LBP) is strongly correlated with metastatic and aggressive tumor cell phenotypes. [1][2][3][4][5][6][7][8] Furthermore, recent microarray expression profiling and proteomic studies have confirmed this established correlation, for instance see Refs. 9 and 10. Tumor angiogenesis is an important component of malignant behavior, and high expression of the LBP is associated with both pathological and normal neovascularization processes. 11,12 The 67 kDa form of the LBP is shed from tumor cells and binds to laminin-1 in the substrate matrix. 13,14 Neither the mechanism of this shedding nor the function of the shed protein is known, although it is likely that this process promotes laminin degradation and cell migration. 15 Induced conformational changes in laminin-1 follow binding of the 67 kDa LBP to this disulphide rich macromolecule, and these changes facilitate laminin degradation and promote overall tumor cell aggression. 16,17 Pathological tumor associated angiogenesis is a key component allowing continued cancer growth and facilitating metastasis. Under nonpathol...

The Biochemical, Biological, and Pathological Kaleidoscope of Cell Surface Substrates Processed by Matrix Metalloproteinases

“…Full-length LR shed from malignant cells also induced conformational changes in laminin after binding. In addition, the shed LR modified production of anti-angiogenic angiostatins from plasmin in vitro, in this way promoting tumorassociated neoangiogenesis (Moss et al, 2006). As LRfragments released by MMP-11 contain the lamininbinding site, they might also modulate laminin conformation and enhance tumor cell invasiveness and angiogenesis.…”

Looking into laminin receptor: critical discussion regarding the non‐integrin 37/67‐kDalaminin receptor/RPSAprotein

“…). Similarly, LAMR 67‐Shed has been suggested to alter the profile of angiostatin fragments produced from plasmin, thereby reducing their anti‐angiogenic activity; LAMR would thus promote angiogenesis in this manner (Moss et al, ). Plasmin normally functions to degrade extracellular matrix components and activate collagenases.…”

Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells

“…Nuclear staining was detected in these strongly labeled tumor sections, which is an unexpected location for 67LR. Nevertheless, it has to be noted that shedding of 67LR by tumor cells has been reported (Berno et al, 2005;Karpatova et al, 1996;Moss et al, 2006), and it appears conceivable that artefactual location of shed 67LR molecules could occur during the fixation and inclusion procedures. In summary, over 71% of the assayed neuroblastoma samples showed expression of 67LR by immunocytochemistry, with a full range of expression levels.…”