Matrix Metalloproteinases in Dog Brains Exhibiting Alzheimer-Like Characteristics

Abstract: We have previously reported that the amount of the neuronal matrix metalloproteinase (MMP) MMP-9, capable of cleaving β-amyloid_1–40 predominantly at Leu³⁴-Met³⁵, is increased in a latent form in hippocampal specimens from AD patients and have suggested that the lack of activation of this enzyme may contribute to the deposition of β-amyloid in plaques. The current study addresses whether similar matrix proteinases are detectable in amyloid-positive and -negative brain specimens of aged beagles. Using quantitative zymography, three major neutral proteinases with molecular masses of 60, 95, and 280 kDa were readily detected. These enzymes have the characteristics of MMPs because they were inhibited by EDTA and 1,10-phenanthroline, and their activities were restored by addition of both Ca²⁺ and Zn²⁺. The 95- and 280-kDa proteinases cross-reacted with specific monoclonal antibodies to human MMP-9 (gelatinase B; EC 3.4.24.35). Canine MMP-9 was latent because activation by organomercurial treatment resulted in a characteristic decrease in molecular mass. Statistical analysis revealed no difference in the 60-kDa proteinase activity in amyloid-positive and -negative brain specimens. However, significantly increased amounts of latent MMP-9 were observed in amyloid-positive brain specimens ( p ≤ 0.05) compared with amyloid-negative brain specimens. The observations document that changes in MMP-9 expression in amyloid-positive beagle brains are similar to those reported in the human Alzheimer's disease hippocampus and suggest the possibility that insufficient activation of MMP-9 may contribute to β-amyloid accumulation, a hypothesis that needs to be further investigated.     

‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC_A and TC_B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.     

A molecular analysis of matrix remodeling and angiogenesis during long bone development

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a ‘molecular map’ of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.     

Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade

In joint diseases of both the inflammatory (rheumatoid arthritis, or RA) or the degenerative variety (osteoarthritis, or OA), matrix metalloproteinases (MMPs) are essential mediators of irreversible tissue destruction. MMP-9 is secreted as a stable, inactive zymogen and is proteolytically converted to the active enzyme. To understand the activation mechanism of MMP-9 in joint diseases, the process was investigated in serum-free cocultures of human articular chondrocytes and macrophages. Macrophages extensively expressed and secreted pro-MMP-9 whereas chondrocytes failed to produce the enzyme. However, efficient activation of pro-MMP-9 required soluble and membrane-associated chondrocyte proteinases. Two alternative activation pathways mainly involved MMPs and, marginally, serine or cysteine proteinases. MT1-MMP (MMP-14), the only MT-MMP expressed in chondrocytes, converted pro-MMP-13 which, in turn, cleaved pro-MMP-9. Alternatively, pro-MMP-9 was activated less efficiently by MMP-3, which was converted by autocatalysis or by serine or cysteine proteinases. Both pathways were triggered by chondrocytes from OA, but not normal joints. Therefore, articular chondrocytes are not innocent bystanders in joint diseases. They not only produce destructive enzymes guided by environmental cues but also they can instruct inflammatory cells or cells from surrounding tissues to do so by converting in several ways zymogens produced but not activated by these cells themselves.     

Schistosoma mansoni: histological localization of gelatinase in the preacetabular glands of cercariae

Proteolytic activity was demonstrated in secretions from the preacetabular glands of cercariae of Schistosoma mansoni. Thin gelatin film substrates were lysed by living cercariae stimulated to penetrate by application on the films of skin surface lipid. Lysis was directly related to number of cercariae, time, and temperature of incubation and pH of the medium. Gelatinase activity in unfixed frozen sections of cercariae incubated on the gelatin films was in the preacetabular glands which are the source of the secretion emptied into skin during penetration. Protease activity, therefore, appears to be related to penetration. The schistosome larvae which made the penetration attempt satisfied the accepted criteria for schistosomules, and therefore appeared to have transformed into schistosomules even though they did not successfully penetrate anything.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP-1, TIMP-2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP-1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP-1 mRNA accumulation, the level of TIMP-2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP-1 was induced specifically on day 18. Interestingly, the normally high level of TIMP-1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP-2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP-2 in the decidua and ovary. Stromelysin-2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP-1 and TIMP-2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodelling of reproductive tissues in gestation. The peak of TIMP-1 mRNA levels in the uterus, decidua, and placenta at midgestation is associated with the most invasive period of embryo development. © 1993 Wiley-Liss, Inc.     

Presence of gelatinase A and etalloelastase type protease at the plasma membrane of human skin fibroblasts. Influence of cytokines and growth factors on cell-associated metalloendopeptidase levels

Gelatinase A and elastase type proteinase (Homsy, et al., 1988) present at plasma membranes of human skin fibroblasts (HSF) were separated by anion exchange chromatography on a DEAE Tris acryl M column. Elastase type proteinase (HSFE1) was able to convert 72 kDa progelatinase A to a lower 66 kDa M.W. active enzyme. Several cytokines (IL-1β, 1L4, IL6), interferon γ (IFN γ) and tumor growth factor β (TGFβ) were studied for their ability to modify the levels of those plasma membrane associated proteinases. Among these mediators, only IL-1β was found to enhance the amounts of HSF membrane-bound HSFE₁ and Gelatinase A.