Ok-Hee Jeon

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary.  Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin αIIbβ3 is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to αIIbβ3 was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with αIIbβ3 is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with αIIbβ3 to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.

MMP‐2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary.  Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active αIIbβ3. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin αIIbβ3 in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the αIIbβ3‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin αIIbβ3 was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active αIIbβ3, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin αIIbβ3. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to αIIbβ3.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg‐Gly‐Asp (RGD) motif in its disintegrin‐like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15‐mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12‐myristate 13‐acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane‐associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD‐dependent manner and suppress vitronectin‐ and fibronectin‐induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage‐derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome‐mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.—Lee, H. D., Koo, B.‐H., Kim, Y. H., Jeon, O.‐H., Kim, D.‐S. Exosome release of ADAM15 and the functional implications of human macrophage‐derived ADAM15 exosomes. FASEB J. 26, 3084–3095 (2012). www.fasebj.org

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg98 and Arg101 residues, whereas plasmin only cleaved the propeptide downstream of Arg101. Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn109-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

Integrin αvβ3-mediated transcriptional regulation of TIMP-1 in a human ovarian cancer cell line

We have previously reported that a disintegrin inhibits solid tumor growth and metastasis in mouse model [I.C. Kang, Y.D. Lee, D.S. Kim, A novel disintegrin salmosin inhibits tumor angiogenesis, Cancer Res. 59 (1999) 3754-3760; S.I. Kim, K.S. Kim, H.S. Kim, D.S. Kim, Y. Jang, K.H. Chung, Y.S. Park, Inhibitory effect of the salmosin gene transferred by cationic liposomes on the progression of B16BL6 tumors, Cancer Res. 63 (2003) 6458-462]. In this study, we have investigated the modulatory effect of a disintegrin, saxatilin, on the balance between MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human ovarian cancer cell line MDAH 2774. Functional mechanism of the disintegrin-mediated transcriptional regulation of MMP-9 and TIMP-1 was examined in the ovarian cancer cell line. Saxatilin strongly induced TIMP-1 expression in dose- and time-dependent manners, while the disintegrin suppressed MMP-9 expression. Further analyses clearly indicated that interaction of the disintegrin and integrin alphavbeta3 results in the TIMP-1 promoter activation via c-fos to suppress TNF-alpha-induced cancer cell invasion. These results demonstrate that integrin alphavbeta3-mediated transcriptional regulation of MMP-9 and TIMP-1 is critical for suppressing the ovarian cancer cell invasion.

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT‐c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT‐c is composed of a signal sequence, amino‐terminal propeptide, a central metalloprotease domain, and a Lys‐Gly‐Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coll. The expressed MT‐c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extraceUular matrix proteins including type I gelatin, type 1V and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving Aα chain of the protein.

Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases

Matrix metalloprotease‐2 is implicated in many biological processes and degrades extracellular and non‐extracellular matrix molecules. Matrix metalloprotease‐2 maintains a latent state through a cysteine–zinc ion pairing which, when disrupted, results in full enzyme activation. This pairing can be disrupted by a conformational change or cleavage within the propeptide. The best known activation mechanism for pro‐matrix metalloprotease‐2 occurs via cleavage of the propeptide by membrane type‐1 matrix metalloprotease. However, significant residual activation of pro‐matrix metalloprotease‐2 is seen in membrane type‐1 matrix metalloprotease knockout mice and in fibroblasts treated with metalloprotease inhibitors. These findings indicate the presence of a membrane type‐1 matrix metalloprotease‐independent activation mechanism for pro‐matrix metalloprotease‐2 in vivo, which prompted us to explore an alternative activation mechanism for pro‐matrix metalloprotese‐2. In this study, we demonstrate membrane type‐1 matrix metalloprotease‐independent propeptide processing of matrix metalloprotease‐2 in HEK293F and various tumor cell lines, and show that proprotein convertases can mediate the processing intracellularly as well as extracellularly. Furthermore, processed matrix metalloprotease‐2 exhibits enzymatic activity that is enhanced by intermolecular autolytic cleavage. Thus, our experimental data, taken together with the broad expression of proprotein convertases, suggest that the proprotein convertase‐mediated processing may be a general activation mechanism for pro‐matrix metalloprotease‐2 in vivo.

Abstract B42: Modulation of integrin‐mediated cell adhesion by ADAM15 disintegrin‐like domain

Integrin‐mediated cell adhesion on extracellular matrix works essentially on numerous physiological processes such as cell adhesion, migration and angiogenesis. As integrin ανβ3 and integrin α5β1 are the representative receptors for cell adhesion and due to their important function in cancer biology, the antagonists targeting the integrins has been sought for long. Lately, the interactions between ADAM (A Disintegrin And Metalloprotenase) 15, the only member in ADAM family with Arg‐Gly‐Asp (RGD) motif, and the ανβ3 and α5β1 intergins was observed. For that, we experimented on the cellular function of recombinant ADAM15‐derived disintegrin‐like domain containing Asp‐Typ‐Lys‐Arg‐Gly‐Asp (rNWKRGD) in integrin‐mediated cell adhesion. The binding affinity of HUVEC, COS‐1, MCF‐7 and MDAH 2274 cells to various extracellular matrix proteins were increased by the disintegrin‐like domain in a dose‐dependent manner, and the presence of inhibitory integrin α5β1 antibody thoroughly abrogated the rNWKRGD‐stimulated binding. By mutagenic analysis, it was founded that RGD motif is essential for the binding. In comparison, saxatilin, RGD disintegrin from snake venom, inhibited binding of cells to ECM proteins. Through flow cytometric analysis, we confirmed that β1 integrin on the cell surface was activated by rNWKGRD. All these results indicated that the activation of integrin α5β1 mediates the rNWKRGD‐stimulated cell adhesion. Therefore rNWKRGD could function as the potential activator of cell migration and proliferation. Although disintegrin is known as inhibitor of cell migration and proliferation, it is possible that ADAM15 disintegrin domain could have a regulatory role in integrin function such as angiogenesis and invasion of cancer cells. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. 2009‐0081759), KIST grant, and the Brain Korea 21 (BK21).) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B42.

Abstract B56: Regulation of immune‐related genes by integrin signaling affects the cancer cell proliferation and invasion

Cytokines have been implicated in tumor proliferation and metastasis. Saxatillin, snake venom‐derived disintegrin, is known to suppress tumor progression in vivo and in vitro but its correlation with cytokine9s functions has not been known yet. We have investigated the role of immune‐related genes in cancer cell proliferation and metastasis in human ovarian cancer cell (MDAH 2774). We demonstrate that saxatilin, an integrin antagonist, mitigated MDAH 2774 proliferation and invasion by reducing the level of TNF‐ induced matrix metalloproteinase‐9 (MMP‐9) and IL‐8 expression in a dose‐dependent manner. And Immunoblot assay of nuclear extracts of the cancer cells implicated that signal transducer and activator of transcription (STAT) is related in integrin signaling pathway. We also observed the activity of human glioma cell‐invasion mediated by IL‐8, as examined by a Boyden chamber assay, involved the mechanism of enhancing the actin stress fiber formation. IL‐8 increased the phosphorylation of focal adhesion kinase (FAK), known to be the site of integrin clustering and nuclear translocation of STAT. We have shown here that interleukin‐8 promotes the cancer proliferation and that mRNA levels of immune‐related genes are regulated by disintegrin in human ovarian cancer cell line. These results demonstrate immune‐related genes as a mediator of metastasis and proliferation and disintegrin would contribute in slowing of cancer development by regulating integrin signaling. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (No.2009‐0081759), KIST grant, and the Brain Korea 21 (BK21) program.) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B56.