Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

Efeitos da azilsartana sobre a doença periodontal em um modelo experimental com ratos wistar

Periodontal diseases, highly prevalent disease in worldwide population, manifest primarily in two distinct entities: plaque-induced gingivitis and periodontitis. Periodontitis is a chronic inflammatory disease characterized of different levels of collagen, cementum, and alveolar bone destruction. Recent experimental studies demonstrated anti-inflammatory and antirreabsortive effect of antihypertensive agents of the angiotensin II receptor blockers class on periodontal disease. The aim of this study was to evaluate the effects of azilsartan (AZT), a potent inhibitor of the angiotensin II receptor which has minimal adverse effects on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs), receptor activator of nuclear factor kB ligand (RANKL), receptor activator of nuclear factor kB (RANK), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. Male Wistar albino rats were randomly divided into 5 groups of 20 rats each: (1) nonligated, water; (2) ligated, water; (3) ligated, 1 mg/kg AZT; (4) ligated, 5 mg/kg AZT; and (5) ligated, 10 mg/kg AZT. All groups were treated with water or AZT for 10 days. Periodontal tissues were analyzed by morphometric exam, histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1b, IL-10, TNF-a, myeloperoxidase (MPO), and glutathione (GSH) were determined by ELISA. Treatment with 5 mg/kg AZT resulted in reduced MPO (p˂0.05) and IL-1b (p˂0.05) levels and increased in Il-10 levels (p˂0.05). It was observed a reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and a increased expression of OPG in the animals subjected to experimental periodontitis and threated with AZT (5 mg/kg). Conclusions: These findings suggest an anti-inflammatory and anti-reabsortive effects of AZT on ligature-induced periodontitis in rats.

Konditionale Modellsysteme zur Untersuchung der ERBB2-induzierten Tumorgenese

Konditionale Modellsysteme zur Untersuchung der ERBB2-induzierten Tumorgenese Die Rezeptor-Tyrosinkinase ERBB2 ist in einer Vielzahl epithelialer Tumore, wie Mamma- und Ovarialkarzinomen, überexprimiert. Diese erhöhte Expression korreliert mit aggressivem Tumorwachstum, verstärkter Metastasierung und schlechter Prognose für den Patienten. Zur genaueren Untersuchung molekularer Mechanismen, die zur Tumorentstehung infolge der ERBB2-Überexpression führen, wurden im Rahmen dieser Arbeit mit Hilfe des Tet-Systems induzierbare MCF-7 Zelllinien generiert. Diese exprimieren bei Gabe von Doxyzyklin ERBB2 bzw. die zum humanen ERBB2 homologe und durch Punktmutation onkogen aktivierte Rattenvariante NeuT. Nachdem die stringente Regulierbarkeit durch Doxyzyklin für die untersuchten Zellklone gezeigt werden konnte, stellte sich bei der Charakterisierung der Zelllinien heraus, dass die Induktion von ERBB2 erstaunlicherweise nicht zur Proliferation der Zellen, sondern zum Wachstumsarrest führt. Bei der Untersuchung verschiedener Zellzyklusregulatoren konnte dieser Zellzyklusarrest dem CDK-Inhibitor P21 zugeordnet werden, dessen Expression durch ERBB2 induziert wird. In P21-Antisense-Experimenten konnte nachgewiesen werden, dass P21 eine Schlüsselrolle beim ERBB2-induzierten Zellzyklusarrest spielt. Neben der Induktion von P21 und der daraus resultierenden Wachstumsinhibition zeigten die Zellen starke morphologische Veränderungen und waren positiv beim Nachweis der Seneszenz-assoziierten -Galaktosidase. Erstmals konnte gezeigt werden, dass die Induktion des Onkogens ERBB2 nicht zur Proliferation, sondern zur Aktivierung eines verfrühten Seneszenz-Programms führt, welches der Zelle Schutz gegen die Onkogeneinwirkung bietet. Bei der Untersuchung verschiedener Signaltransduktionskaskaden mit Inhibitormolekülen konnte die Aktivierung dieses Seneszenz-Programms der Stress-aktivierten Proteinkinase P38 zugeordnet werden. Zur Identifizierung von Genen, die für die ERBB2-induzierte Tumorgenese relevant sind, wurde die differenzielle Genexpression eines NeuT-Klons nach 8- bzw. 48-stündiger Induktion mit Doxyzyklin in einem cDNA-Array untersucht. Dabei zeigte sich eine besonders starke Induktion von Integrin 5 und Integrin 1, die zusammen den Fibronektinrezeptor bilden. Der funktionale Nachweis des Rezeptors in einem Adhäsionsassay demonstrierte ein stark erhöhtes Adhäsionsverhalten ERBB2-überexprimierender Zellen an Fibronektin. Bei der Untersuchung von Mamma-, Ovarial- und Endometriumkarzinomen konnte die Expression von ERBB2 mit der von Integrin 5 korreliert werden. Diese Ergebnisse machen Integrin 5 zu einem potenziellen neuen Tumormarker und Therapieziel in ERBB2-überexprimierenden Tumoren. Ein weiteres interessantes Gen, das sich im Array durch ERBB2 überexprimiert zeigte, war die Matrix-Metalloproteinase MMP-9. In einem Zymografieassay konnte die erhöhte Gelatinaseaktivität von MMP-9 in Dox-induzierten Zellen nachgewiesen werden. Der Einsatz verschiedener Signaltransduktionsinhibitoren ergab, dass auch die ERBB2-induzierte Expression von MMP-9 über die Aktivierung von P38 läuft. Bei der Suche nach weiteren MMPs, die für die ERBB2-induzierte Tumorgenese relevant sein könnten, wurde MMP-13 untersucht. Erstmals konnte gezeigt werden, dass diese Matrix-Metalloproteinase von ERBB2 induziert wird. Dieser interessante Befund wurde auch in einem anderen Zellmodell in NIH3T3 Mausfibroblasten verifiziert. Durch ihre Matrix-degradierenden Eigenschaften sind MMPs potente „Werkzeuge“ für Tumorzellen und stellen ein wichtiges Ziel zur Unterbindung der Invasion und Metastasierung dieser Zellen dar. Neben den Zellkulturarbeiten wurden im Rahmen dieser Dissertation transgene Responder-Mäuse generiert, die NeuT unter Kontrolle eines Tet-responsiven Promotors exprimieren. Von vier transgenen Gründerlinien zeigten zwei eine unerwünschte, basale NeuT-Expression, für die beiden anderen Linien konnte sowohl in MEF-Assays, als auch nach Kreuzung mit rtTA- bzw. tTA-Effektor-Mäusen eine Dox-abhängige Regulation des Transgens gezeigt werden. Die Tiere dieser Linien sollen in Zukunft mit Effektor-Mäusen gepaart werden, die den rtTA bzw. tTA spezifisch in für die ERBB2-Tumorgenese relevanten Geweben, wie Ovarial- oder Lungenepithelzellen, exprimieren. So können individuelle Tumormodelle für die verschiedenen epithelialen Tumore, bei denen die Überexpression von ERBB2 von Bedeutung ist, entwickelt und untersucht werden.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Optical-limiting response of rare-earth metallo-phthalocyanine-doped copolymer matrix

The nanosecond optical-limiting characteristics (at 532 nm) of some rare-earth metallo-phthalocyanines (Sm(Pc)2, Eu(Pc)2, and LaPc) doped in a copolymer matrix of poly(methyl methacrylate) and methyl-2-cyanoacrylate have been studied for the first time to our knowledge. The optical-limiting response is attributed to reverse saturable absorption due to excited-state absorption. The performance of LaPc in a copolymer host is studied at different linear transmissions. The laser damage thresholds of all the samples are also reported.

Roles of the matrix metalloproteinases in mammary gland development and cancer

Tissue remodeling is a key process involved in normal development, wound healing, bone remodeling, and embryonic implantation, as well as pathological conditions such as tumor invasion and metastasis, and angiogenesis. The degradation of the extracellular matrix that is associated with those processes is mediated by a number of families of extracellular proteinases. These families include the serine proteinases, such as the plasminogen-urokinase plasminogen activator system and leukocyte elastases, the cysteine proteinases, like cathepsin D and L, and the zinc-dependent matrix metalloproteinases (MMPs). Accumulating evidence has highlighted the central role of MMP-driven extracellular matrix remodeling in mammary gland development and breast cancer.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Tb3+ sensitization in a deoxycholate organogel matrix, and selective quenching of luminescence by an aromatic nitro derivative

In this article, we present the discovery of a metallo-organogel derived from a Tb3+ salt and sodium deoxycholate (NaDCh) in methanol. The gel was made luminescent through sensitization of Tb3+ by doping with 2,3-dihydroxynaphthalene (DHN) in micromolar concentrations. Rheological measurements of the mechanical properties of the organogel confirmed the characteristics of a true gel. Significant quenching of Tb3+ luminescence was observed in the deoxycholate gel matrix by 2,4,7-trinitrofluorenone (TNF), but not by several other polynitro aromatics. Microscopic studies (AFM, TEM and SEM) revealed a highly entangled fibrous network. The xerogels retained luminescent properties suggesting the possibility for application in coatings, etc.

Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed `form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.

Matrix metalloproteinase gene polymorphisms and oral cancer

Since oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity, several researches have been performed to study the role of important enzymes in this disease. Among them, the matrix metalloproteinases (MMPs) are highlighted, due to the fact that they are proteinases responsible to degrade many extra-cellular matrix components, making possible the invasion of neoplasic cells. Important tools in cancer prognosis have been utilized aiming to correlate high levels of MMPs and OSCC, such as immunohistochemical, zymographic and mRNA detection methods. However, these techniques are usually applied after cancer detection, characterizing a curative but not a preventive medicine. Trying to make interventions before the development of the disease and making possible the identification of people at high risk and, analysis of modifications in MMP genes has been a chance for modern medicine. Recently, polymorphisms in MMP genes have been related to different neoplasias, including OSCC. Despite investigation is beginning, MMP gene polymorphisms seems to have a promising future in oral cancer research and some of the present results have shown that there are MMP polymorphisms related to an increased risk for developing oral cancer. Key words:Oral cancer, polymorphism, matrix metalloproteinase.

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Processing of procollagen III by meprins: new players in extracellular matrix assembly?

Meprins ? and ?, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins ? and ? release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin ? is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin ? increases and meprin ? begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.