Ronald T. Aimes

Localization of Matrix Metalloproteinase MMP-2 to the Surface of Invasive Cells by Interaction with Integrin αvβ3

Abstract Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin αvβ3. MMP-2 and αvβ3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of αvβ3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind αvβ3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M+HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M+HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M+HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

The Isolation, Characterization, and Molecular Cloning of a 75-kDa Gelatinase B-like Enzyme, a Member of the Matrix Metalloproteinase (MMP) Family AN AVIAN ENZYME THAT IS MMP-9-LIKE IN ITS CELL EXPRESSION PATTERN BUT DIVERGES FROM MAMMALIAN GELATINASE B IN SEQUENCE AND BIOCHEMICAL PROPERTIES

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90–110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75–90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.

Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts

Rous sarcoma virus‐transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase‐2 (MMP‐2) that exists in a form free of complexed tissue inhibitor of metalloproteinase‐2 (TIMP‐2). In order to ascertain whether the increased levels of TIMP‐free MMP‐2 in RSVCEF cultures are due to diminished expression of TIMP‐2 or alterations in TIMP‐2 that diminish its MMP‐2 binding ability, it was necessary to clone, characterize, and express chicken TIMP‐2 cDNA. The TIMP‐2 cDNA was cloned from a chick embryo λgt11 library by RT‐PCR using primers based on amino‐acid sequences determined from isolated TIMP‐2. The deduced amino acid sequence for chicken TIMP‐2 is 81% identical to human TIMP‐2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP‐2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP‐2 mRNA levels are increased in RSVCEF. However, TIMP‐2 protein levels, relative to proMMP‐2 levels, appear to decrease upon transformation and suggest additional control of TIMP‐2 at the post‐transcriptional level. Addition of recombinantly expressed TIMP‐2 to RSVCEF cultures causes a disappearance of TIMP‐free (TF) proMMP‐2 with a corresponding increase in the TIMP‐complexed (TC) proMMP‐2 levels, demonstrating that TF proMMP‐2 is capable of converting to TC pro‐MMP‐2 when free TIMP‐2 is available. Surprisingly, RSVCEF cultures manifest a TIMP‐2 population that is not complexed to MMP‐2, despite the coexistence of TIMP‐free proMMP‐2. Gel‐filtration analysis indicates that this uncomplexed TIMP‐2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP‐2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP‐2/MMP‐2 balance by transcriptional and post‐translational modifications, yielding a population of inhibitor‐free, proteolytically active MMP2. J. Cell. Physiol. 174:342–352, 1998.

Serine protease and metallo protease cascade systems involved in pericellular proteolysis

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.

What structure and function of avian plasminogen activator and matrix metalloproteinase-2 reveal about their counterpart mammalian enzymes, their regulation and their role in tumor invasion.

Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF) constitute a well-characterized model system for oncogenic transformation, matrix degradation, and cancer invasion. As RSVCEF cultures employ both serine protease and metalloprotease cascades in the process of matrix degradation, they have contributed significantly to understanding the nature and regulation of these molecules involved in invasive cell behavior. RSVCEF produce elevated levels of a matrix metalloprotease-2 (MMP-2) whose hemopexin domain differs from mammalian MMP-2. The majority of MMP-2 produced by RSVCEF is present in a TIMP-free form which enhances its activation, catalytic activity and substrate specificity and therefore its matrix-degrading ability. RSVCEFs also exhibit high levels of urokinase-type plasminogen activator (uPA), which is found in active form in their conditioned medium in complete absence of plasminogen. Recombinantly expressed avian uPA is also in active form, while an active-site mutant of the same maintains its zymogen form, indicating the mechanism of activation of chicken uPA is autocatalytic. A domain and sequence comparison between chicken and human uPA attempts to identify motifs potentially responsible for the zymogen instability of avian uPA and its capability to autoactivate.