Elizabeth Hahn-Dantona

Activation of Matrix Metalloproteinase-9 (MMP-9) via a Converging Plasmin/Stromelysin-1 Cascade Enhances Tumor Cell Invasion

Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03–8.1 nm). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1–4 nm. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50–75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.

Activation of ProMMP‐9 by a Plasmin/MMP‐3 Cascade in a Tumor Cell Model: Regulation by Tissue Inhibitors of Metalloproteinases

ABSTRACT: To examine MMP‐9 activation in a cellular setting we employed cultures of human tumor cells that were induced to produce MMP‐9 over a 200‐fold concentration range (0.03 to 8.1 nM). The secreted levels of TIMPs in all the induced cultures remain relatively constant at 1‐4 nM. Quantitation of the zymogen/active enzyme status of MMP‐9 in the cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP‐9 to TIMP dictates whether proMMP‐9 activation can progress. When the MMP‐9/TIMP ratio exceeds 1.0, MMP‐9 activation progresses, but only via an interacting protease cascade involving plasmin and stromelysin 1 (MMP‐3). Plasmin, generated by the endogenous plasminogen activator (uPA), is not an efficient activator of proMMP‐9. Plasmin, however, is very efficient at generating active MMP‐3 from exogenously added proMMP‐3. The activated MMP‐3, when its concentration exceeds that of TIMP, becomes a potent activator of proMMP‐9. Addition to the cultures of already‐activated MMP‐3 relinquishes the requirement for plasminogen and proMMP‐3 additions and results in direct activation of the endogenous proMMP‐9. The activated MMP‐9 enhances the invasive phenotype of the cultured cells as their ability to transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodeling capability is due to the activation of MMP‐9 is demonstrated through the use of a specific anti‐MMP‐9‐blocking monoclonal antibody.

MMP-2 expression during early avian cardiac and neural crest morphogenesis

Matrix metalloproteinase‐type 2 (MMP‐2) degrades extracellular matrix, mediates cell migration and tissue remodeling, and is implicated in mediating neural crest (NC) and cardiac development. However, there is little information regarding the expression and distribution of MMP‐2 during cardiogenesis and NC morphogenesis. To elucidate the role of MMP‐2, we performed a comprehensive study on the temporal and spatial distribution of MMP‐2 mRNA and protein during critical stages of early avian NC and cardiac development. We found that ectodermally derived NC cells did not express MMP‐2 mRNA during their initial formation and early emigration but encountered MMP‐2 protein in basement membranes deposited by mesodermal cells. While NC cells did not synthesize MMP‐2 mRNA early in migration, MMP‐2 expression was seen in NC cells within the cranial paraxial and pharyngeal arch mesenchyme at later stages but was never detected in NC‐derived neural structures. This suggested NC MMP‐2 expression was temporally and spatially dependent on tissue interactions or differed within the various NC subpopulations. MMP‐2 was first expressed within cardiogenic splanchnic mesoderm before and during the formation of the early heart tube, at sites of active pharyngeal arch and cardiac remodeling, and during cardiac cushion cell migration. Collectively, these results support the postulate that MMP‐2 has an important functional role in early cardiogenesis, NC cell and cardiac cushion migration, and remodeling of the pharyngeal arches and cardiac heart tube. Anat Rec 259:168–179, 2000.

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.