Search PubMed⌕ Search

Biomedical subjects

A Z Eisen

Publications and source records attributed to A Z Eisen.

At least 19 recordsLinked to original sources
PubMed2000

Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity.

Epithelial cell adhesion, migration, and differentiation are controlled by interactions at the basement membrane zone (BMZ). Type VII collagen is the major collagenous component of anchoring fibrils that are essential for the attachment of the epidermis to the dermis. Gelatinase A (MMP-2) is believed to be necessary for the degradation of type VII collagen. In this study we have examined the in vivo distribution of type VII collagen and gelatinase A (Gel A) in the developing human epidermis and its appendages. At 13-15 wk of gestation a marked decrease in type VII collagen immunoreactivity was seen in the BMZ surrounding invading appendageal buds; however, type VII collagen mRNA was strongly expressed in the budding epidermal keratinocytes adjacent to the BMZ. At these stages, Gel A-positive mesenchymal-like cells were found scattered throughout the stroma with numerous Gel A-containing cells in direct contact with the developing appendageal buds. In situ zymography was used to show Gel A-activity in vivo. Gel A-mediated lysis was present at the interface between the appendageal buds and the underlying BMZ. By 20-25 wk of gestational age, immunostaining for type VII collagen protein was absent from the BMZ surrounding the distal portion of invading appendageal epithelial cords of both hair follicles and sweat glands. In contrast, type VII collagen mRNA was present in the basal keratinocytes adjacent to the BMZ surrounding the distal portion of these invading appendageal epithelial cords. At these stages Gel A-positive cells were present in the stroma directly adjacent to the distal portion of developing appendageal cords that lacked type VII collagen. In situ zymography showed zones of Gel A-mediated stromal lysis at the distal portion of developing appendageal cords. Interestingly, no differences were seen in the distribution of type IV collagen in the BMZ of both budding and resting fetal epidermis. These observations suggest that the absence of type VII collagen protein correlates directly with the presence of Gel A-activity at the BMZ. Gel A appears to play a major role in appendageal development and contributes to remodeling of the BMZ during fetal skin morphogenesis.

Basement Membrane↗
PubMed1998

Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate.

Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.

Base Sequence↗
PubMed1998

Matrix metalloproteinase (MMP)-9 type IV collagenase/gelatinase implicated in the pathogenesis of Sjögren's syndrome.

Type IV collagenases/gelatinases (matrix metalloproteinases MMP-2 and MMP-9) in labial salivary glands (LSG) and saliva in Sjögren's syndrome (SS) and healthy controls were studied. Zymograms and Western blots disclosed that SS saliva contained 92/82 kD MMP-9/type IV collagenase duplex. Specific activity measurement disclosed 53.1+/-9.8 U/mg protein MMP-9 in SS compared to 16.5+/-2.6 U/mg in healthy controls (p=0.01). MMP-2 did not differ between SS and controls. In SS salivary glands, MMP-2 and MMP-9 were also expressed, in addition to stromal fibroblasts and occasional infiltrating neutrophils, respectively, in acinar end piece cells. In addition, an effective proMMP-9 activator, human trypsin-2 (also known as tumor-associated trypsin-2 or TAT-2), was found in acinar end piece cells and in saliva. Interestingly, proteolytically processed MMP-9 was found in saliva (vide supra), and in vivo activated MMP-9 was significantly higher in SS than in controls (p=0.002). LSGs, particularly in SS, were characterized ultrastructurally by areas containing small cytoplasmic vesicles in the basal parts of the epithelial cells associated with areas of disordered and thickened basal lamina. Based on our results, we conclude here that SS saliva contains increased concentrations of MMP-9, which is of glandular origin in part. Pro MMP-9 is to a large extent proteolytically activated. This is probably mediated by the most potent pro MMP-9 activator found in vivo thus far, namely trypsin-2. Therefore, the MMP 9/trypsin-2 cascade may be responsible for the increased remodelling and/or structural destruction of the basement membrane scaffolding in salivary glands in SS. Due to the role of basal lamina as an important molecular sieve and extracellular matrix-cell signal, these pathological changes may contribute to the pathogenesis of the syndrome.

Blotting, Western↗
PubMed1998

Interstitial collagenase and the ED-B oncofetal domain of fibronectin are markers of angiogenesis in human skin tumors.

Collagenase-1 (C1) is the predominant matrix metalloproteinase present in newly formed microvessels and serves as a marker of neovascularization. The expression of the oncofetal fragment of fibronectin (Fn-f) was found to be increased during angiogenesis. In the present study, we investigated the relationship between the expression of collagenase-1 and the oncofetal fragment of fibronectin in newly formed microvessels as markers of tumor angiogenesis. In aggressive skin tumors (i.e., morpheaform and recurrent basal cell carcinomas) and squamous cell carcinomas, neovascularization was associated with a marked increase in the number of C1-positive and Fn-f-positive microvessels. At the beginning of elongation, microvessels begin to produce C1 but lose their ability to express type IV collagen and FVIII-related antigen. Later, this endothelium produces both Fn-f and C1. As maturation of microvessels occurs, C1-containing endothelium fails to express Fn-f but begins to produce a type IV collagen-containing basement membrane and FVIII-related antigen. These studies show that there is a selective expression of both Fn-f and collagenase by immature endothelial cells. C1 production begins at early stages of blood vessel formation and continues throughout angiogenesis. In contrast, Fn-f expression is limited to later stages of vasculogenesis, indicating that these proteins are reliable markers of angiogenesis.

Antigens, Neoplasm↗
PubMed1997

Activation of gelatinase A (72-kDa type IV collagenase) induced by monensin in normal human fibroblasts.

In monolayer culture, fibroblasts secrete all matrix metalloproteinases, including gelatinase A (72-kDa type IV collagenase), as inactive zymogens. Whereas limited proteolysis by plasmin or other matrix metalloproteinases (MMPs) can accomplish the extracellular activation of other proenzymes in this family, gelatinase A proenzyme is uniquely refractory to cleavage by such proteinases. Previously it has been shown that fibroblasts cultured in the presumably more physiologic culture milieu of a type I collagen lattice can be induced to secrete active gelatinase A. In monolayer culture, however, the plant lectin concanavalin A will induce gelatinase A activation. Here we show that in monolayer culture activation of gelatinase A by normal fibroblasts is also induced by the sodium ionophore monensin. The monensin response is dose-dependent, time-dependent, requires protein synthesis, and is specific to gelatinase A among the secreted matrix metalloproteinases. The activator appears to be associated with cell membranes and may be membrane-type matrix metalloproteinase 1(MT-MMP1). Both mRNA and immunodetectable protein of MT-MMP1 are increased with monensin treatment while message for the protein inhibitor of gelatinase A, TIMP-2, is unchanged. The monensin-induced signal transduction pathway leading to gelatinase activation in monolayer culture appears to be different from the integrin-mediated pathway operative in the collagen lattice system. The tyrosine kinase inhibitor genistein blocks monensin activation of gelatinase A in monolayer culture. In contrast, genistein has no effect on proenzyme activation in the collagen lattice. Likewise, the cyclooxygenase inhibitor indomethacin abrogates the monensin effect in monolayer culture and can be reversed by addition of exogenous prostaglandin E2 (PGE2). Neither indomethacin nor PGE2 affects activation of gelatinase A in the collagen lattice.

Cells, Cultured↗
PubMed1997

Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts.

Normal fibroblasts cultured as monolayers secrete matrix metalloproteinases (MMP), including gelatinase A (72-kDa type IV collagenase) as inactive zymogens. Previously we found that normal fibroblasts cultured in a type I collagen lattice (dermal equivalent) secrete active gelatinase A. Here we show that the activation of progelatinase A occurs within the cell and that the activator copurifies with Golgi membranes. Cell extracts of fibroblasts cultured in collagen lattices contain active 62-kDa gelatinase A at least 4-6 h before active enzyme is detected in the culture medium. Pulse-chase experiments confirm these results. The activator is membrane-bound and localizes to the Golgi-enriched fraction. Highly purified plasma membranes from lattice cultures are unable to convert gelatinase A from the zymogen to its active form. The activator may be a metalloproteinase because EDTA prevents activation of exogenous proenzyme by membrane fractions. Membrane-type MMP1, the enzyme thought to be responsible for activation of gelatinase A on the plasma membrane of tumor cells, shows no significant change in either mRNA or protein levels during lattice culture. Intracellular levels of gelatinase A mRNA and protein increase during the culture period, and tissue inhibitor of metalloproteinases concentration does not change. Because of the greater availability of tissue inhibitor of metalloproteinases-free proenzyme as a substrate for the activator, it is possible that membrane-type MMP1 is the activating enzyme. In that case, malignant transformation may involve a change in the localization of the activator to the plasma membrane.

Biomarkers↗
PubMed1996

Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides.

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.

Amino Acid Sequence↗
PubMed1995

Matrix metalloproteinases in blood vessel development in human fetal skin and in cutaneous tumors.

In vitro angiogenesis models suggest that new blood vessel formation requires the induction and secretion by endothelial cells of matrix metalloproteinases. These enzymes assist in the controlled proteolytic degradation of the surrounding extracellular matrix during blood vessel formation. The results of in vitro studies cannot be extrapolated directly to the process of in vivo angiogenesis because the type of matrix employed and the repertoire of enzymes secreted by cells in vivo differ dramatically from in vivo conditions. To investigate the in vivo role of matrix metalloproteinases in blood vessel development, we looked for the presence of these proteinases in endothelial cells involved in fetal angiogenesis and in neovascularization of certain invasive skin tumors using immunofluorescent staining. In fetal tissue, interstitial collagenase was present in both early microvessels developing from undifferentiated mesoderm and in microvessels involved in elongation and sprout formation from preexisting blood vessels. In aggressive skin tumors, i.e., morpheaform and recurrent basal cell carcinomas and squamous cell carcinomas, there was a marked increase in the number of collagenase-containing blood vessels, often extending into the tumor nests. Immunofluorescent staining failed to detect stromelysin, matrilysin, or gelatinase A and B (72- and 92-kDa type IV collagenases, respectively) in fetal or tumor blood vessels. These findings are consistent with the hypothesis that proteolytic degradation of the extracellular matrix is required for the formation of new blood vessels. Interstitial collagenase appears to play an important role in this process.

Adult↗
PubMed1994

Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction.

The matrix metalloproteinase 72-kDa type IV collagenase (also known as gelatinase A) is thought to be involved in both normal connective tissue remodeling and invasive pathological processes. Like other matrix metalloproteinases, 72-kDa type IV collagenase is secreted by fibroblast monolayers as an inactive proenzyme, but is unique among this enzyme family in that it is not activated by serine proteinases such as plasmin. However, when fibroblasts are cultured in a collagen lattice, a situation thought to better approximate in vivo conditions, we have invariably found much of the secreted 72-kDa type IV collagenase in its enzymatically active 62-kDa form. Although collagen lattice contraction appeared to be required for the activation of 72-kDa type IV collagenase, we have found that the process of contraction can be dissociated from proenzyme activation. Both cytochalasin D and alpha-methylmannoside completely blocked lattice contraction, but not proenzyme activation. Furthermore, the monoclonal antibody M-13, which is directed against the beta 1 integrin chain, blocked collagen lattice contraction but not 72-kDa type IV procollagenase activation. At concentrations significantly higher than required to block lattice contraction or cell adhesion to collagen, M-13 was able to inhibit proenzyme activation. A second monoclonal antibody to the beta 1 integrin, P5D2, had little effect on collagen lattice contraction at low concentrations, but could significantly inhibit the activation of 72-kDa type IV procollagenase. Antibodies to the integrin alpha 2 chain also inhibited proenzyme activation. These data show that the activation of 72-kDa type IV collagenase proenzyme, like collagen lattice contraction, is mediated by beta 1 integrin receptors, possibly alpha 2 beta 1. Although both anti-beta 1 antibodies used are directed to the same site on the integrin chain, the fact that each antibody preferentially blocks a different event, either lattice contraction or activation of 72-kDa type IV collagenase, suggests the existence of branch points in the receptor-mediated signal transduction pathway.

Antibodies, Monoclonal↗
PubMed1994

Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia.

Matrix-degrading metalloproteinases play a major role in tissue remodeling. Recent studies have shown that enzymes of this class are constitutively expressed primarily by stromal cells and not by epithelium. Here we present immunohistochemical evidence that matrilysin is localized within epidermal cells in developing skin and in tumor cells of cutaneous malignancies. The expression of matrilysin protein in developing fetal skin (6-15 weeks) is localized primarily to the germinative basal cell layer of fetal epidermis and early appendageal buds. The buds continue to express matrilysin during mesenchymal invasion. As development progresses (15-19 weeks) matrilysin is concentrated only in cells at the distal portion of the invading follicular and sweat gland appendageal cords. In adult skin, matrilysin was localized specifically to the outer root sheath of the hair follicles and the secretory cells of the eccrine glands but was absent in the epidermis. Nodulocystic, keratotic, adenoid basal cell carcinomas (BCCs) did not express matrilysin. In contrast, in the more aggressive morpheaform (infiltrative) BCCs and recurrent BCCs, matrilysin was localized at the tumor-stromal interface. In squamous cell carcinomas matrilysin was present in tumor cells at the stromal interface surrounding the tumor nests. The demonstration of matrilysin protein in germinal basal cells during fetal skin development and its presence in tumor cells at the stromal junction suggests that this enzyme may contribute to the proteolytic activity associated with cell-extracellular matrix interactions during appendageal development and tumor invasion.

Adult↗
PubMed1994

Collagenase in Sjögren's syndrome.

OBJECTIVE: To study collagenase production in labial salivary glands in patients with Sjögren's syndrome (SS). METHODS: Collagenases were localised in labial salivary glands by immunohistochemistry. Collagenase activity against triple helical type I collagen monomers in stimulated saliva was measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis and laser densitometry; tissue inhibitor metalloproteinase (TIMP) was measured by enzyme linked immunosorbent assay. RESULTS: Cells containing collagenase of matrix metalloproteinase (MMP)-1 type were more frequent and more intensely staining in SS than in healthy glands. Only SS saliva contained functional enzyme (11.7 (6.8) x 10(-6) IU/1). Cells containing MMP-8 type neutrophil collagenase were not found in situ, which was in accordance with sialochemical findings/doxycycline inhibition studies. TIMP was found in both SS and normal saliva. CONCLUSIONS: Fibroblast, but not neutrophil type, collagenase is synthesised, secreted, and subsequently activated, but is not inhibited by TIMP in labial salivary glands or saliva in SS. Collagenase may destroy glandular and salivary duct tissue and perturb factors influencing the morphogenetic extracellular matrix.

Collagenases↗
PubMed1994

Extracellular matrix metalloproteinases around loose total hip prostheses.

We have explored the tissue localization of extracellular matrix metalloproteinases MMP-1 (fibroblast collagenase), MMP-2 (72-kDa gelatinase/Type IV collagenase), MMP-3 (stromelysin), MMP-8 (polymorphonuclear leukocyte collagenase) and MMP-9 (92-kDa gelatinase/Type IV collagenase) in the tissues around loose hip prostheses. The findings were compared with those in synovial tissues obtained from patients with a fractured femoral neck. MMP-type specific antisera were applied in the sensitive avidin-biotin-peroxidase complex methods. MMP-1 was found in monocyte/macrophages, fibroblasts, and vascular endothelial cells in both interface tissues between bone and acetabular components and the pseudocapsular tissues obtained from loosening of hip prostheses. In these tissues, MMP-8 was occasionally found, but only in polymorphonuclear leukocytes. Cells showing immunoreactivity to 72- and 92-kDa gelatinase/Type IV collagenase, MMP-2 and MMP-9, respectively, and stromelysin, MMP-3, were abundant in both interface and pseudocapsular tissues in loose hip prostheses. In contrast, in hip fractures, immunoreactivity to MMP-1, 2, 3, and 9 was weak and only observed in synovial tissues. Immunoreactivity to MMP-8 was confined to polymorphonuclear leukocytes attached to the synovial membrane or in the infiltrate around blood vessels in the subsynovial connective tissues. The finding of MMP-1, 2, 3, and 9 in the tissues around loose hip prostheses suggests that they play a role in the weakening of connective tissues, and this leads to loosening.

Aged↗
PubMed1993

Localization of 92-kDa type IV collagenase in human skin tumors: comparison with normal human fetal and adult skin.

To investigate the role of secreted metalloproteinases in the behavior of skin tumors we have studied immunoreactivity for 92-kDa type IV collagenase (92T4Cl) in benign tumors of sweat glands, basal cell carcinomas (BCC), baso-squamous cell carcinomas (BSCC), and squamous cell carcinomas (SCC). In all tumors, the enzyme was found in stromal cells, but not in tumor epithelium. 92T4Cl-positive cells contained the common leukocyte antigen HLe-1(CD45) and the polymorphonuclear leukocyte-specific antigen PMN-8C7. Only a few 92T4Cl-positive cells expressed either macrophage-specific Leu-M5 or eosinophil-specific cationic protein antigens. In benign sweat gland tumors, and in the majority of nodulocystic and adenoid BCCs, 92T4Cl-positive cells were relatively rare and no extracellular deposition of the enzyme was found. In the more aggressive tumors examined, SCCs, BSCC, recurrent, infiltrative, and morpheaform BCCs, 92T4Cl-positive cells were very abundant. In addition, a significant quantity of extracellular enzyme was deposited both within the extracellular matrix adjacent to the tumor nests and in their basement membrane zone. In normal adult skin only a few scattered 92T4Cl-containing cells were found in the dermis whereas in fetal skin, groups of 92T4Cl-positive, HLe-1-negative cells were present in the upper dermis. These observations suggest that in cutaneous tumors, extensive infiltration of 92T4Cl containing polymorphonuclear leukocytes and the extracellular deposition of the enzyme in the basement membrane zone are signs of more aggressive tumor behavior.

Adult↗
PubMed1993

Role of mesenchymal collagenase in the loosening of total hip prosthesis.

Fibroblast-type interstitial collagenase (E.C. 3.4.24.7) was associated with loosening of total hip prostheses in eight patients: there were four cemented stems and one cementless stem with the common type of loosening and two cemented stems and one cementless acetabular component with aggressive granulomatous lesions. The authors used a specific, well-characterized, heterologous, affinity-purified, polyclonal rabbit anti-human fibroblast collagenase antiserum applied in avidin-biotin-peroxidase-complex (ABC) staining. In the aggressive granulomatous type of loosening, collagenase was found in most of the fibroblast- and macrophagelike cells, including multinuclear giant cells and epithelioid cells in periprosthetic tissue. Collagenase-positive cells also were found in the periprosthetic tissue associated with common loosening. Collagenase was also found in capillary and postcapillary venule endothelial cells in the richly vascularized aggressive granulomatous tissue. Collagenase was extracted directly from the tissue samples and incubated with soluble Type I collagen. Collagen degradation products then were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the three-fourths length degradation product quantitated by gel scanning densitometry. In both aggressive granulomatosis and the common type of loosening, extractable collagenase was found in tissue. No significant differences between the sample groups were detected in respect to total measurable collagenase, however. The extractable collagenase was present in a latent form that could be activated by the organomercurial procollagenase activator, phenylmercuric chloride (PMC). It is likely that interstitial collagenase contributes to rapid growth of reactive infiltrative tissue, loosening of the prosthesis associated with aggressive granulomatosis, and the periprosthetic lytic process associated with the common type of hip prosthesis loosening.

Aged↗
PubMed1992

Expression of 92-kDa type IV collagenase mRNA by eosinophils associated with basal cell carcinoma.

Metalloproteinases are thought to be important for tumor invasion and metastasis. We used in situ hybridization with 35S-labeled cRNA probes to localize sites of expression for 92-kDa type IV collagenase mRNA in sections of nodular basal cell carcinoma. Positive signal for 92-kDa type IV collagenase mRNA was detected in eosinophilic granulocytes within inflammatory infiltrates surrounding the tumor nodules. Eosinophils, however, were not adjacent to tumor cells, suggesting that metalloenzyme production by these granulocytes in this disease may be targeted more to stromal components than to remodeling or destruction of the basement lamina. The identity of the eosinophils was confirmed by cell morphology and specific histochemical staining. No resident or other migratory cells were positive for enzyme mRNA in these samples. Signal specificity for in situ hybridization was shown by a duplication of the results with complementary oligomeric probes and by a lack of signal in sections hybridized with a sense RNA probe or nonspecific oligomer. No signal for 92-kDa type IV collagenase mRNA was detected in circulating eosinophils or in eosinophils associated with Hodgkin's lymphoma. These data suggest that eosinophils migrate into the dermis and express type IV collagenase in response to basal cell carcinoma and that this process may have a role in tumor growth.

Base Sequence↗
PubMed1992

Mosaic structure of the secreted ECM metalloproteases and interaction of the type IV collagenases with inhibitors.

SV-40 transformed human lung fibroblasts and HT 1080 fibrosarcoma cells secrete a 92-kDa type IV collagenase (in addition to 72-kDa type IV collagenase identical to that found in macrophages, phorbol ester differentiated U937 cells, and keratinocytes. The expression of this protease is induced by the tumor promoter TPA, and interleukin-1 and was not detected in the parental human lung fibroblast. The 92-kDa preproenzyme has a predicted Mr of 78,426, including a 19 amino acid long hydrophobic signal peptide. The apparent discrepancy between the predicted molecular weight and the molecular weight of the secreted protein is due to a post-translational modification of the enzyme through glycosylation. The 92-kDa type IV collagenase consists of five distinct domains, including a unique 54 amino acid long collagen--like domain, and is a member of the secreted ECM metalloprotease gene family. Both the 72 and 92-kDa type IV collagenase contain a fibronectin-like collagen binding domain. The mosaic structure of the secreted ECM metalloproteases is a result of a recruitment of the functional units from ECM structural macromolecules into an enzyme protein in the process of evolution. The 92-kDa and 72-kDa type IV collagenase proenzymes form a noncovalent complex with inhibitors, which is activatable by APMA, yielding an enzymes with similar if not identical substrate specificity profile. Our results demonstrate that while the 92-kDa type IV collagenase forms a stoichiometric complex with TIMP, the 72-kDa type IV collagenase, purified from the same starting material, contains a novel 24-kDa inhibitor-TIMP-2.

Amino Acid Sequence↗
PubMed1992

Activation of extracellular matrix metalloproteases by proteases and organomercurials.

Extracellular matrix metalloproteases are synthesized as proenzymes and are activated by certain physiological agents after secretion into the extracellular space. The identity of these agents and the stimulus that elicits their response in vivo is only recently becoming clear, but a variety of agents or stimuli are capable of activating these metalloproteases in vitro also. Of these, the most well studied and characterized are trypsin, plasmin and the organomercurials. These agents appear to have in common an ability to disrupt the structure of the stable latent enzyme in such a way as to allow the generation of a proteolytic active site. In the case of organomercurial activation, intramolecular proteolytic cleavage of the amino-terminus of the enzyme occurs subsequent to generation of activity. A similar intramolecular process is seen with trypsin and plasmin activation except that it is initiated by a single trypsin or plasmin catalyzed cleavage in the amino-terminus prior to the autocatalytic cleavages. A possible explanation for organomercurial activation is that the mercurial disrupts a cysteinyl residue coordination bond with the active site zinc that prevents interaction with substrate. Disruption of this complex would allow productive enzyme-substrate interaction via the newly available coordination site. In addition, activated stromelysin is capable of increasing the specific activity of active interstitial collagenase by approximately ten-fold through what appears to be proteolytic removal of a small peptide.

Amino Acid Sequence↗