Reverse Zymogram Research Articles

Abstract 3837: The physiological relevance of TIMP dimer formation

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are a family of four paralogous genes that modulate the activity of matrix metalloproteinases (MMPs) and are vital for tissue homeostasis. Additionally, TIMPs display activities that are independent of their MMP inhibitory activities. Dysregulation and perturbations within this system can lead to complex tissue-specific pathologies. TIMP2 is the most abundantly expressed member of the TIMP family and has been shown to display potential therapeutic uses in cancer and other diseases. We have noted the persistent presence of SDS-stable TIMP2 dimers/multimers in SDS-PAGE at a level of ~5% total protein. The goal of this study is to determine the stability and biological relevance of SDS-stable dimer formation of TIMP2 in in vitro and in vivo. We found that TIMP2 dimers display an enhanced ability to inhibit MMP2 activity in reverse zymography assays. Immunoblotting revealed gel extracted TIMP2 dimer entered an equilibrium with monomeric TIMP2, while the extracted monomer did not form new dimer. In addition, MMP2 activity assays do not corroborate the enhanced MMP2 inhibitory activity observed in reverse zymograms but are consistent with the observation that isolated TIMP2 dimers re-equilibrate with monomers. This implies that structural or sequence difference mediates the formation of dimers. Dimer/multimer formation is observed across the TIMP family. Mass spectrometry analysis of gel extracted TIMP monomers and dimers show that monomer and dimer forms of TIMPs display unique post-translation modification (PTM) profiles. We propose that understanding the interplay between TIMP multimers and PTMs will reveal new biological functions within this intriguing family of proteins. We are currently working to resolve the functional differences between monomer and dimer and reveal the underlying mechanisms at play. Citation Format: Carolyn Lazaroff, David Peeney, Sadeechya Gurung, William G. Stetler-Stevenson. The physiological relevance of TIMP dimer formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3837.

Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht

As the aim of this present study, a proteinaceous α-amylase inhibitor has been isolated from the rhizome of Cheilocostus specious (C. speciosus) and was purified using DEAE cellulose anion exchange chromatography followed by gel filtration using Sephacryl-S-200 column. The purity and molecular mass of the purified inhibitor was determined by SDS-PAGE and LC-MS respectively. The molecular mass of the purified inhibitor was determined to be 31.18kDa. Protein-protein docking was also carried out as molecular model. Model validation methods such as Ramachandran plot and Z-score plot were adopted to validate the structural description (sequence analysis) of proteins. The inhibitory activity was confirmed using spectrophotometric and reverse zymogram analyses. This 31.18kDa protein from C. speciosus inhibited the activity of fungal α-amylase by 71% at the level of ion exchange chromatography and 96% after gel filtration. The inhibition activity of the α-amylase inhibitor was stable and high at optimum pH6 (52.2%) and temperatures of 30–40°C (72.2%). Thus it was suggested that the main responsible for the versatile biological and pharmacological activities of C. speciosus is due to its primary metabolites (proteins) only.

Purification and characterization of a novel heterodimer protease inhibitor from Streptomyces spp. VL J2 with potential biopesticidal activity against H. armigera

A protease inhibitor (PI) was isolated and purified from halo-alkaliphilic Streptomyces spp. VL J2. SDS-PAGE of purified PI revealed it to be a heterodimer of two unidentical subunits of 27.5 and 11.08kDa, which corroborates well with intact molecular mass of 38.5kDa obtained by GPC and MALDI −TOF. Inhibitory activity was confirmed by activity staining and reverse zymogram studies and the inhibitor was found to retain activity at <50°C and pH 2–9.5. It showed presence of two isoforms with isoelectric point of 5.5 and 5.7. The inhibition of trypsin and chymotrypsin indicated it to be a serine protease type belonging to serpin family. The stoichiometry of trypsin-inhibitor interaction was 1:2. Modification of amino acid showed presence of arginine and free sulfhydryl group at active site. Kinetic studies revealed the non-competitive type of inhibition of trypsin with low Ki value (9.4×10−9M). Activity against H. armigera showed significant decline and delay in larval (51%), pupal weights and periods, respectively, prominent physical abnormalities and reduced nutritional indices as a function of treatment. The results suggest that the purified PI has promising pesticidal activity and could serve as a potential candidate gene for transgenic plant research.

A kidney bean trypsin inhibitor with an insecticidal potential against Helicoverpa armigera and Spodoptera litura

In the present study, trypsin inhibitor extracts of ten kidney bean seed (Phaseolus vulgaris) varieties exhibiting trypsin and gut trypsin-like protease inhibitor activity were tested on Helicoverpa armigera and Spodoptera litura. Trypsin inhibitor protein was isolated and purified using multi-step strategy with a recovery of ~15 % and purification fold by ~39.4. SDS-PAGE revealed a single band corresponding to molecular mass of ~15 kDa and inhibitory activity was confirmed by reverse zymogram analyses. The inhibitor retained its inhibitory activity over a broad range of pH (3–11), temperature (40–60 °C) and thermostability was promoted by casein, CaCl2, BSA and sucrose. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.85 μM. The purified trypsin inhibitor protein was further incorporated in the artificial diet and fed to second instar larvae. A maximum of 91.7 % inhibition was obtained in H. armigera, while it was moderate in S. litura (29 %) with slight varietal differences. The insect bioassay showed 40 and 22 % decrease in larval growth followed by 3 and 2 days delay in pupation of H. armigera and S. litura, respectively. Some of the adults emerged were deformed and not fully formed. Trypsin inhibitor protein was more effective against H. armigera as it showed 46.7 % mortality during larval growth period compared to S. litura (13.3 %).

Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization

Protease inhibitors have great demand in medicine and biotechnology. We report here the purification and characterization of a protease inhibitor isolated from mature leaf extract of Moringa oleifera that showed maximum inhibitor activity. The protease inhibitor was purified to 41.4-fold by Sephadex G75 and its molecular mass was calculated as 23,600Da. Inhibitory activity was confirmed by dot-blot and reverse zymogram analyses. Glycine, glutamic acid, alanine, proline and aspartic acid were found as the major amino acids of the inhibitor protein. Maximal activity was recorded at pH 7 and at 40°C. The inhibitor was stable over pH 5–10; and at 50°C for 2h. Thermostability was promoted by CaCl2, BSA and sucrose. Addition of Zn2+ and Mg2+, SDS, dithiothreitol and β-mercaptoethanol enhanced inhibitory activity, while DMSO and H2O2 affected inhibitory activity. Modification of amino acids at the catalytic site by PMSF and DEPC led to an enhancement in the inhibitory activity. Stoichiometry of trypsin–protease inhibitor interaction was 1:1.5 and 0.6nM of inhibitor effected 50% inhibition. The low Ki value (1.5nM) obtained indicated scope for utilization of M. oliefera protease inhibitor against serine proteases.

The defensive role of latex in plants: detrimental effects on insects

The defensive role of the latex of Calotropis procera has recently been reported. In this study, latex proteins involved in detrimental effects on insects were evaluated on another important crop pest. The latex was fractionated to obtain its major protein fraction, which was then used to evaluate its insecticidal properties against Callosobruchus maculatus (Coleoptera: Bruchidae) in artificial bioassays. Laticifer proteins (LP) were investigated to characterize their action in such an activity. LP was highly insecticidal at doses as low as 0.1% (W/W). This effect was slightly augmented in F1 generation reared in artificial seeds containing LP at similar proportions of F0, but was fully reversed when F1 developed in LP-free seeds. The insecticidal proteins were not retained in a chitin column, and did not lose their insecticidal activity, even after heat treatment or pronase digestion. However, these samples inhibited papain (EC 3.4.22.2) activity and gut proteases of C. maculatus larvae, and a reverse zymogram showed the presence of protein bands resistant to papain digestion. These activities were not observed in unheated LP as they were probably masked by abundant endogenous cysteine protease (EC 3.4.22.16) activity present in unheated LP. LP was resistant to proteolysis when assayed with C. maculatus gut extract. However, gut proteins of C. maculatus were digested when incubated with LP. These observations and the deleterious effects of LP upon C. maculatus, reinforce the hypothesis that laticifer fluids are involved in plant defense against insects and indicate C. procera latex to be a source of promising insecticidal proteins. The inhibitor of proteolysis present in the latex seems to be resistant to heat and proteolysis and is certainly involved in the detrimental effects observed.

Detection of a Lysozyme Inhibitor in Proteus mirabilis by a New Reverse Zymogram Method

A reverse zymogram method for the detection of bacterial lysozyme inhibitors was developed. This method was validated by using a periplasmic protein extract of Escherichia coli containing a known inhibitor and subsequently led to the detection of a new proteinaceous hen egg white lysozyme inhibitor in Proteus mirabilis.

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1-10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β-2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose-dependent manner. Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1-10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissueinhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions(4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β-2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose-dependent manner. Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN.

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy

Canine TIMP-2: Purification, Characterization and Molecular Detection

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.

Canine TIMP-2: Purification, Characterization and Molecular Detection

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.

Vitronectin deficiency is associated with increased wound fibrinolysis and decreased microvascular angiogenesis in mice

Background: Vitronectin has several putative functions including regulating hemostasis, cell adhesion, and cell migration. However, the targeted deletion of vitronectin in mice results in normal development and normal coagulation parameters. To determine whether vitronectin may be necessary for nondevelopmental processes, we examined the response to tissue injury in vitronectin-null mice. Methods: We examined wound healing in control and vitronectin-null mice by healing rate, zymography, reverse zymography, and Western blots. Results: We found that dermal wound healing was slightly delayed in mice lacking vitronectin. More importantly, we found extensive areas of delayed hemorrhage near the sprouting tips of microvessels between days 7 and 14, which temporally coincided with increased urokinase-type plasminogen activator and tissue-type plasminogen activator activity by zymography. Though Western blots confirmed the presence of plasminogen activator inhibitor-1 protein throughout wound repair and reverse zymograms showed decreased plasminogen activator inhibitor-1 activity between days 7 and 14. Conclusions: Loss of vitronectin in mice was associated with changes in the fibrinolytic balance, and this may have led to focal sites of delayed hemorrhage. The mechanism that resulted in decreased angiogenesis and the formation of larger blood vessels in response to tissue injury remains unknown. This study suggests that vitronectin may have several distinct functions that are not required for normal development but are manifested in response to tissue injury. (Surgery 2000;127:696-704.)

Suppression of cell invasion on human malignant glioma cell lines by a novel matrix-metalloproteinase inhibitor SI-27: in vitro study.

Matrix metalloproteinase (MMP) has come to be highlighted by its close relation to the cell invasion of gliomas. Suppression of MMP activity in malignant glioma cells would be meriting to local delivery of genes or chemotherapeutic agents. In this study, we employed a novel MMP inhibitor, SI-27 to investigate inhibition of cell invasiveness in human malignant glioma cell lines, U87MG, U251MG, and U373MG. We evaluated with zymogram, reverse zymogram, and cell invasion assay after exposure of SI-27 for 24 h followed by preliminary MTT assay to find non-cytotoxic dose range, 5, 10, 50, 100 microg/ml compared with non-treatment group as the control. Common to three glioma cell lines, zymogram disclosed that expressions of MMP-2 and -9 were suppressed in a dose-dependent fashion, meanwhile those of tissue inhibitor of MMP (TIMMP) in reverse zymogram were not. The numbers of invading cells through Boyden chamber were significantly reduced in a dose-dependent manner, while those with 5 microg/ml were not diminished common to those three lines. In conclusion, dose concentration ranging 10-100 microg/ml of SI-27 inhibited MMP-2 and -9 mediated cell invasiveness in malignant glioma cell lines. This is the first report for chemotherapeutic effect of SI-27 on glioma cells.

Localization of the Functional Domains of Human Tissue Inhibitor of Metalloproteinases-3 and the Effects of a Sorsby's Fundus Dystrophy Mutation

A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino- and carboxyl-terminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non-ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH2-terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH2-terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby's fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby's fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3.

Imbalance Between Matrix Metalloproteinase (MMPs) and their Inhibitors in BALs of Mechanically Ventilated Neonates † 1704

Background: Neutrophil-derived proteinases (mainly serine proteinases) play an important role in neonatal lung tissue damage. However the exent to which MMPs (92 kD Type IV and 72 kD (Interstitial collagenases) participate in the remodelling of the extracellutar matrix (ECM) is unknown. Intrapulmonary leucocytes (neutrophils and alveolar macrophages) are the main secretors of MMPs gelatinases, induced through a prostaglandin-E2 (PG-E2) dependent pathway involving cyclooxygenase-2 (COX-2) and balanced by the natural tissue inhibitors of MMPs (TIMPs). Aims: 1) To evaluate the gelatinolytic activity of MMPs isoforms and their inhibitors in BAL fluids from mechanically ventilated infants; 2) To study the effect of bacterial lipopolysaccaride (LPS) on MMPs activity and inhibition in conditioned media (CM) of cultured BAL cells, in the presence and absence of Ibuprofen (Ibu) or Dexamethasone (Dex). Methods: BAL samples were obtained from 13 intubated and mechanically ventilated neonates(GA>27 629<4039 gr) age 1-30 days with RDS, perinatal asphyxia, diaphragmatic hernia, BPD) and from 3 pediatric patients with chronic pulmonary disease (dysmotile cilia syndrome, cystic fibrosis). After separation of cellular components from fluid phase (FP) by centrifugation, washed pellets were resuspended in RPMI medium, (supplemented with 0,1% FCS and antibiotics), allowed to adhere to Petri dishes at 37°C and 5% CO2 for 1 hour (to select the monocytic-macrophagic population) and then tested for induction of MMPs and production of PG-E2, under basal condition and after 24 hours of treatment with diluent (PBS) versus LPS (10 mg/ml). +/- 50mM Ibu or 1mM Dex. Radioimmunoassays for PG-E2 quantitation, zymograms and reversezymograms for MMPs gelatinolytic activity and inhibition, were of CM of cultured adherent cells and FPs from BALs. Results: Zymograms performed on samples from FPs and CM of cultured BAL cells, in basal conditions, show an appreciable gelatinolytic activity (mainly type IV collagenase MMP-9), whereas reverse zymograms show less evident bands of inhibition (TIMPs). A marked induction of MMP-9 (more evident in the neonates than in the pediatric patients) and a consistent reduction of TIMPs activity, associated with a 10-100 fold increase of PG-E2 production, were observed after 24 hours of LPS stimulus. The patterns of gelatinolytic activity and inhibition were almost totally reversed in the presence of Ibu, with only minimal changes induced by Dex. Conclusions An imbalance between ECM degradative activity (particularly of MMP-9) and inhibition is observed in FPs and cells from BALs, under basal conditions and after LPS induction. Sub-epithelial basal membrane ECM components (particular type IV collagen) are more susceptible to damage since MMP-9 specifically degrades type IV collagen and can significantly contribute to the sustained increase in pulmonary permeability characteristic of the flogistic reaction that follows acute neonatal lung injury. The role of the MMPs/TIMPs imbalance in the evolution of neonatal lung disease and its possible modulation by acting on the COX-2-mediated PG-E2 inducible synthesis, warrants further studies.

Quantitative Reverse Zymography: Analysis of Picogram Amounts of Metalloproteinase Inhibitors Using Gelatinase A and B Reverse Zymograms

Matrix metalloproteinases are a growing family of neutral pH optima, zinc atom-dependent endopeptidases that collectively degrade all components of the extracellular matrix. This family of related proteases is further defined by their inhibition of protease activity by a class of low-molecular-weight endogenous inhibitors known as tissue inhibitors of metalloproteinases or TIMPs. Reverse zymography is an electrophoretic technique used to identify TIMP inhibitory activity within acrylamide gels. Previous methods have generally used biochemically complex sources of proteolytic activity (such as cell culture conditioned media) copolymerized with a proteinase substrate in the gel to identify the zones of inhibited proteolysis. We describe a novel system for reverse zymography using purified recombinant human gelatinase A or gelatinase B in place of conditioned media. These reverse zymograms using recombinant gelatinase have sensitivities for TIMPs that are favorable in comparison to immunoblotting techniques but have the benefit of visualizing multiple inhibitors simultaneously. We have developed and characterized these methods for the evaluation of inhibitors and have shown them to be highly sensitive, convenient, and reproducible. Both systems detect TIMPs 1, 2, and 3 simultaneously, but with differential sensitivities for TIMPs 1 and 2. Using gelatinase A the system can detect as little as 1 pg of rTIMP-2, but the limit of detection for rTIMP-1 is 40 pg. Gelatinase B shows less differential activity in that the limits of detection are 60 and 40 pg for TIMP-2 and TIMP-1, respectively. We demonstrate how these varied sensitivities of the gelatinases for the TIMPs can contribute to potential pitfalls in systems using uncharacterized reagents (i.e., conditioned media).

Fibronectin and fibronectin fragments modulate the expression of proteinases and proteinase inhibitors in human periodontal ligament cells.

Fragments of the matrix molecule fibronectin (FN) have been shown to modulate tissue remodeling activity by inducing matrix metalloproteinases (MMPs) in synovial fibroblasts. These molecules could contribute to the tissue degradation that occurs during periodontal disease if they also modulate the expression of proteinases in cells of the periodontal ligament (PDL). We tested the hypothesis that FN and specific FN fragments induce the expression of specific proteinases in PDL cells. Using substrate zymograms, reverse zymograms and Western immunoblots, we found that PDL cells constitutively express 72 kDa gelatinase, urokinase-type plasminogen activator (uPA) and at least three inhibitors whose molecular masses correspond to those of the tissue inhibitors of metalloproteinases (TIMPs). A fourth, previously uncharacterized, proteinase inhibitor of approximately 22 kDa was also observed in some cell isolates. PDL cells, when exposed to a 120 kDa proteolytic FN fragment containing the cell-binding domain, were induced to express collagenase and stromelysin and also demonstrated an increased secretion of the serine proteinase uPA. Expression of collagenase increased with increasing concentrations (0.001 microM-1 microM) of the 120 kDa FN fragment. This fragment also induced the expression of a 20 kDa inhibitor, but not of the higher-molecular-mass inhibitors, in PDL cells. The observed alterations in proteinases were associated specifically with the 120 kDa FN fragment, since similar responses were not seen when PDL cells were exposed to either a 60 kDa heparin-binding FN fragment or a 45 kDa collagen/gelatin-binding FN fragment. PDL cells exposed to intact FN did not express the proteinases induced by the 120 kDa fragment but did express 92 kDa gelatinase and the 20 kDa proteinase inhibitor. These data suggest that FN and specific FN fragments can differentially induce the expression of proteinases in PDL cells. Thus, functional regions of FN may modulate many of the functions of PDL cells that contribute to periodontal disease, wound healing and maintenance of extracellular matrix in periodontal tissues.

Characterization and Identification of Proteinases and Proteinase Inhibitors Synthesized by Temporomandibular Joint Disc Cells

The adult mammalian temporomandibular joint (TM) disc is a fibrocartilaginous tissue that undergoes normal developmental remodeling, requiring removal of the existing extracellular matrix and its replacement by new matrix macromolecules. This remodeling is probably mediated by matrix-degrading enzymes, but to date none has been demonstrated in association with the TMJ disc. We characterized, identified, and determined the regulation of proteinases and proteinase inhibitor (PIs) synthesized by TMJ disc cells in organ and cell cultures. TMJ discs were retrieved from 14-week-old male NZW rabbits and both tissue- and disc-derived cells were cultured in serum-free medium. The conditioned media were retrieved at 12-hour intervals and assayed for proteinases and PIs in gelatin- and casein-impregnated polyacrylamide gels. Three proteinases with gelatinolytic activities at 92 kDa, 72 kDa, and 42/57 kDa and one caseinolytic activity at 51/54 kDa were detected. All were inhibited by 1,10-1 phenanthroline, thus characterizing these enzymes as matrix metalloproteinases (MMPs), most likely 92-kDa gelatinase (proMMP-9), 72-kDa gelatinase (proMMP-2), procollagenase (proMMP-1), and prostromelysin (proMMP-3). The identity of the latter two MMPs was confirmed by Western blots. Two PIs and 30 kDa and 20 kDa, probably tissue inhibitors of metalloproteinase (TIMP) and TIMP-2, were observed on reverse zymograms. TPA, a protein kinase-C agonist, increased the expression of 92-kDa gelatinase and 30-kDa PI by both explanted discs and isolated disc cells. The profile of MMPs constitutively expressed by disc cells is similar to that of synovial fibroblasts but different from that of chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)