Overexpression of matrix metalloproteinases leads to lethality in transgenic Xenopus laevis: implications for tissue-dependent functions of matrix metalloproteinases during late embryonic development.

The extracellular matrix (ECM) functions as the structural support of cells and as a medium for cell-cell interactions. It is understood to play critical roles in development. ECM remodeling is mediated largely through the action of matrix metalloproteinases (MMPs), a family of Zn2+-dependent proteases capable of degrading various proteinaceous components of the ECM. MMPs are expressed in many developmental and pathologic processes. However, few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organogenesis. By using Xenopus development as a model system, we have previously shown that several MMP genes are expressed from neurulation to the completion of embryogenesis in distinct tissues/organs, suggesting that ECM remodeling during mid- to late embryogenesis occurs in an organ-specific manner. By using the recently developed transgenic technology for Xenopus laevis, we overexpressed Xenopus MMPs stromelysin-3 (ST3) and collagenase-4 (Col4) under the control of a ubiquitous promoter and observed that embryos with overexpressed ST3 or Col4, but not the control green fluorescent protein (GFP), died in a dose-dependent manner during late embryogenesis. The specificity of this embryonic lethal phenotype was confirmed by the failure of a catalytically inactive mutant of ST3 to affect development. Finally, overexpression of a mammalian membrane type-MMP also led to late embryonic lethality in Xenopus embryos, suggesting that membrane type-MMPs have functions in vivo for ECM remodeling, in addition to being activators of other pro-MMPs. These data together with the developmental expression of several MMPs during Xenopus development, suggest that MMPs play important roles during mid- to late embryogenesis and that proper regulation of MMP genes is critical for tissue morphogenesis and organogenesis.     

Expression of functional kappa-opioid receptors on murine dendritic cells

We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH(2) domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fabprime prime or minute)(2) from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflamed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.     

Proteolysis of AA amyloid fibril proteins by matrix metalloproteinases-1, -2, and -3

We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment.     

Elastolysis by proteinase 3 and its inhibition by alpha(1)-proteinase inhibitor: a mechanism for the incomplete inhibition of ongoing elastolysis

An excess of proteinase 3 (Pr3) is an assumed risk factor for elastin loss in chronic obstructive pulmonary disease. This study compared the degradation of [(14)C]elastin by Pr3 and its inhibition by alpha(1)-proteinase inhibitor (alpha(1)-PI) with the analogous reactions involving two other neutrophil serine proteases, human leukocyte elastase (HLE) and cathepsin G (CatG). The elastolytic rate catalyzed by Pr3 was estimated to be half of that of CatG and one-eighth of that of HLE. Evidence was obtained that indicated that absorption of Pr3 by the substrate was much less than that of HLE or CatG, and that the majority of absorbed Pr3 was highly mobile. These properties are consistent with the observation that elastolysis by Pr3 was almost completely and stoichiometrically inhibited by alpha(1)-PI even under conditions in which the protease had been preincubated with the substrate. In contrast, alpha(1)-PI in large molar excess was unable to inhibit completely ongoing elastolysis of the same substrate by HLE or CatG. An interfacial nonisotropic reaction mechanism has been proposed to address the incomplete inhibition of ongoing elastolysis. Pr3 was identified as being the most abundant neutrophil serine protease. However, two findings reported here, namely the low rate of elastolysis by Pr3 and the high efficacy of alpha(1)-PI against ongoing elastolysis by Pr3, imply that Pr3 might not necessarily be a major contributor to neutrophil-mediated elastin loss.     

1,25-Dihydroxy vitamin D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60-activated matrix vesicle matrix metalloproteinases

As growth plate chondrocytes mature and hypertrophy, they reorganize their proteoglycan-rich type II collagen extracellular matrix (ECM), involving 1,25(OH)(2)D(3)-dependent regulation of matrix metalloproteinases (MMPs). Stromelysin-1 (MMP-3) and 72-kD gelatinase (MMP-2) are found in extracellular matrix vesicles (MVs) and release and activate ECM-bound latent TGF-beta1 and TGF-beta2, respectively. 1,25(OH)(2)D(3) regulates incorporation of MMP-2 and MMP-3 into MVs and release of these enzymes in the ECM. Plasma membranes (PMs) and MVs contain the 1alpha,25(OH)(2)D(3) membrane receptor ERp60 (protein disulfide isomerase A3), phospholipase A(2) (PLA(2)), PLA(2)-activating protein, the nuclear vitamin D receptor and caveolin-1. 1,25(OH)(2)D(3) secreted by chondrocytes binds MV ERp60, activating PLA(2). Resulting lysophospholipids destabilize MV membranes, releasing active MMPs. We examined 1,25(OH)(2)D(3)-dependent activation of latent TGF-beta1 stored in cartilage ECM. Interestingly, TGF-beta1 regulates 1,25(OH)(2)D(3) production. 1alpha,25(OH)(2)D(3) activates PM protein kinase C (PKC)-alpha via ERp60-dependent PLA(2)-signaling, lysophospholipid production and phospholipase C-gamma. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching MVs in PKC-zeta. Direct activation of MV MMP-3 requires ERp60 based on blocking antibodies and PKC based on inhibitor studies. However, treatment of MVs with 1,25(OH)(2)D(3) decreases MV PKC-zeta activity, suggesting more complex feedback mechanisms, potentially involving MV lipid signaling. Our observations indicate that one role of MVs is to provide MMPs at sites distant from the cells. Chondrocytes secrete 1,25(OH)(2)D(3), which acts directly on MV-membranes via ERp60, releasing MMPs. MMP-specific ECM components are hydrolyzed, resulting in release and activation of growth factors that can act back on the cells.     

Spatio-temporal regulation and cleavage by matrix metalloproteinase stromelysin-3 implicate a role for laminin receptor in intestinal remodeling during Xenopus laevis metamorphosis.

The 37-kd laminin receptor precursor (LR) was first identified as a 67-kd protein that binds laminin with high affinity. We have recently isolated the Xenopus laevis LR as an in vitro substrate of matrix metalloproteinase stromelysin-3 (ST3), which is highly upregulated during intestinal metamorphosis in Xenopus laevis. Here, we show that LR is expressed in the intestinal epithelium of premetamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in the connective tissue but with little expression in the developing adult epithelium. Toward the end of metamorphosis, as adult epithelial cells differentiate, they begin to express LR. Furthermore, LR is cleaved during intestinal remodeling when ST3 is highly expressed or in premetamorphic intestine of transgenic tadpoles overexpressing ST3. These results suggest that LR plays a role in cell fate determination and tissue morphogenesis, in part through its cleavage by ST3. Interestingly, high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3, suggesting that LR cleavage by ST3 plays a role in both physiological and pathological processes.     

Subunit-dependent block by isoflurane of wild-type and mutant alpha(1)S270H GABA(A) receptor currents in Xenopus oocytes

The volatile anesthetic isoflurane both prolongs and reduces the amplitude of GABA-mediated inhibitory postsynaptic currents (IPSCs) recorded in neurons. To explore the latter effect, we investigated isoflurane-induced inhibition of steady-state desensitized GABA currents in Xenopus oocytes expressing wild-type alpha(1)beta(2), alpha(1)beta(2)gamma(2s), mutant alpha(1)(S270H)beta(2) (serine to histidine at residue 270) or alpha(1)(S270H)beta(2)gamma(2s) receptors. The alpha(1) serine 270 site in TM2 (second transmembrane domain of the subunit) is postulated as a binding site for some volatile agents and is critical for positive modulation of sub-maximal GABA responses by isoflurane. For all receptor combinations, at < or =0.6 mM isoflurane (< or =2 minimum alveolar concentration (MAC)) current inhibitions were not pronounced ( approximately 10%) with block reaching half-maximal levels at supraclinical concentrations ( approximately 2 mM isoflurane, 6 MAC). Comparisons with other GABA(A) receptor blockers indicated that isoflurane blocks in a similar manner to picrotoxin, possibly via the pore of the receptor. The extent of isoflurane-induced inhibition was significantly attenuated by inclusion of the gamma(2s)-subunit but was unaffected by introduction of the S270H mutation in the alpha(1)-subunit. In conclusion, isoflurane binds with low affinity and with subunit-specificity to an inhibitory site on the GABA(A) receptor that is distinct from the site that facilitates positive modulation at the extracellular end of the pore.     

Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis A virus (HAV) and implications for the polyprotein processing in HAV

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.     

Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid

A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab')(2), and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab')(2). Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab')(2) or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab')(2)s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab')(2) and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.     

Reporter gene construct containing 1.4-kB alpha 1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice.

A 1.4-kb human alpha1-proteinase inhibitor (alpha1-PI) 5'-flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The 1.4-kb alpha 1-PI fragment was found to target LacZ expression preferentially in the epithelium and stroma of the mouse cornea, and moderately or weakly in white blood cells and a few other tissues, such as the skin and brain. This finding implies that the alpha 1-PI promoter may offer an option for targeting foreign genes in both the epithelial and stromal layers of the cornea in future transgenic experiments.     

Regulation of alpha1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.     

Inhibition of polymorphonuclear leukocyte-mediated endothelial cell detachment by antileukoprotease: a comparison with other proteinase inhibitors

The role of elastase and proteinase inhibitors in polymorphonuclear leukocyte(PMN)-mediated injury to human umbilical cord venous endothelial cells (HUVEC) was investigated. Both purified human neutrophil elastase and PMN that were stimulated with serum-treated zymosan (STZ) induced detachment, but not lysis of HUVEC. PMN-, but not purified elastase-mediated detachment was enhanced by the presence of methionine, which indicates a role for reactive oxygen metabolites in PMN-mediated HUVEC detachment. Detachment of HUVEC could be inhibited by secretory leukocyte proteinase inhibitor or antileukoprotease (ALP), alpha 1-proteinase inhibitor (alpha 1-PI) and N-methoxy-succinyl-ala-ala-pro-val-chloromethyl ketone (CMK). At concentrations at which elastase-mediated detachment was maximally inhibited, ALP and CMK, but not alpha 1-PI, were also able to inhibit maximally PMN-mediated detachment. An explanation for this difference could be that the larger size of alpha 1-PI reduces the access of alpha 1-PI to the interface between the PMN and the HUVEC.     

Conservation in rotaviruses of the protein region containing the two sites associated with trypsin enhancement of infectivity

The primary structure of the region in the outer layer protein VP3, containing the two sites associated with trypsin enhancement of infectivity of rotavirus was found to be greatly conserved in cultivable human rotavirus serotypes 1 (Wa), 2 (DS1), and 3 (P) and in four human rotaviruses directly purified from feces. Significant differences with this conserved sequence were found in human rotavirus serotype 4 (ST3), isolated from an asymptomatic neonate, and in seven animal rotaviruses. However, the two trypsin cleavage sites were conserved in every rotavirus VP3 sequence analyzed.     

Inhibition of cysteine proteinases by autolytic digestion is mediated by CBP2/Hsp47

CBP2/Hsp47 is a glycoprotein normally limited to the ER-Golgi where it is first associated with procollagen chains at a very early point during translation of nascent chains and later with properly folded procollagen. Although CBP2/Hsp47 is regarded as a molecular chaperone belonging to the serpin superfamily, this protein does not appear to inhibit serine proteinases. Here we demonstrate that CBP2/Hsp47 functions in a manner similar to other serpin superfamily members by cross class inhibiting cysteine proteinases. A CBP2/Hsp47 to cathepsin L inactivation stoichiometery of approximately 1.5 revealed concurrent cleavage of CBP2/Hsp47 with proteinase inactivation. Cleavage of the CBP2/Hsp47 was shown to occur outside the P1-P1' at the P16-P15 and P2'-P3' bonds. In addition, the proteinase bands in SDS/PAGE diminished on reaction of the enzyme with CBP2/Hsp47. These results sustain a mechanism advocated by Bjork et al. (1998), in which cysteine proteinases assault a peptide bond in the reactive site loop of serpins, (CBP2/Hsp47) adjacent to the P1-P1' bonds involved in serine proteinase inhibition. The reaction proceeds with the substrate pathway dominating in the cysteine proteinase reaction. In these complexes the cysteine proteinases, papain and cathepsin L, are rendered more susceptible to proteolysis and are degraded by active enzyme. These properties help explain the mechanism by which CBP2/Hsp47 increases the fidelity of collagen production. Moreover, if CBP2/Hsp47 is shown to involve the multiplexin subclass of collagens, it may further provide a mechanism by which the motogen and angiogenic properties during development and/or neoplasia are regulated.     

Prostaglandin F(2alpha), cytokines and cyclic mechanical stretch augment matrix metalloproteinase-1 secretion from cultured human uterine cervical fibroblast cells

Human uterine cervical tissue is composed mainly of fibroblast cells and the extracellular matrix in which collagen types I and III predominate. It is hypothesized that these collagens are degraded by matrix metalloproteinases (MMPs) in the initial step of uterine cervical ripening during parturition. Among the MMPs, MMP-1, -8 and -13 have substrate selectivity for collagen types I and III. In the present study, we examined the regulation of MMP-1 secretion from the human uterine cervix. Immunohistochemistry detected strong staining of MMP-1, but not of MMP-8 or -13, in stromal cells of the pregnant uterine cervix. The MMP-1 expression in the pregnant uterine cervix was further confirmed by Western blot analysis and RT-PCR. To clarify the regulation of MMP-1 production, we subsequently investigated the effects of prostaglandins, inflammatory cytokines and cyclic mechanical stretch on the secretion of MMP-1 from cultured human uterine cervical fibroblast cells. Treatment with prostaglandin (PG)F(2alpha) (10(-7) to 10(-5) mol/l) or interleukin (IL)-1alpha (0.01-1.0 ng/ml) or stimulation with cyclic mechanical stretch increased MMP-1 secretion from cultured human uterine cervical fibroblast cells, with maximal increases of 3.4-, 4.5- and 1.9-fold respectively (24 h of treatment, P < 0.05 for all comparisons). These data suggest that MMP-1 may play a significant role in the degradation of extracellular collagen types I and III in the pregnant uterine cervix during the process of cervical ripening, in response to various stimulations such as PGF(2alpha), IL-1alpha and mechanical stretch.     

Structural and functional consequences of cleavage of human secretory and human serum immunoglobulin A1 by proteinases from Proteus mirabilis and Neisseria meningitidis

The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseria meningitidis and cleavage by the proteinase from Proteus mirabilis have been compared. For serum IgA1, both proteinases cleaved only the alpha chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the alpha1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fcalpha receptors, whereas the (Fcalpha1)(2)-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcalpha receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fcalpha receptors. The results are consistent with the Fcalpha receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcalpha receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.