Jun-Ichi Kishi

Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland

A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium.

Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid-potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment

In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM‐102 had erythroid‐potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role ofTIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.

Monoclonal Antibodies to Bovine Collagenase Inhibitor

Hybridoma antibodies against bovine collagenase inhibitor were produced by fusion of myeloma cells NS-1 (P3-NS1-1) with spleen cells from mice hyperimmunized with collagenase inhibitor purified from the explant medium of bovine dental pulps. Hybridomas positive by an enzyme-linked immunosorbent assay (ELISA) for bovine collagenase inhibitor were cloned by the dilution method. Seventeen hybridomas producing antibodies were isolated, four of which also recognized purified human collagenase inhibitor in the ELISA. Using a monoclonal antibody-Sepharose affinity column, we easily purified both bovine and human collagenase inhibitors to homogeneity. They showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 32,000 daltons.

A Sandwich Enzyme Immunoassay for Collagenase Inhibitor Using Monoclonal Antibodies

A sandwich enzyme immunoassay for collagenase inhibitor was set up with a pair of monoclonal antibodies prepared against bovine dental pulp collagenase inhibitor. Two different combinations of the antibodies were found to be applicable to the immunoassay; one was for the determination of both bovine and human collagenase inhibitors, but the other was only for the bovine one. Minimum sensitivity was 1 pg/tube (3 pg/ml) for bovine collagenase inhibitor and 1.5 pg/tube (5 pg/ml) for human inhibitor. The levels of collagenase inhibitor in several normal human body fluids were measured by the immunoassay, and the values obtained were as follows: serum, 183 +/- 30 ng/ml (mean +/- SD); platelet-poor plasma, 68 +/- 13; cerebrospinal fluid, 60 +/- 13; amniotic fluid, 1,780 +/- 969; tear fluid, 418 +/- 145; mixed saliva, 316 +/- 166; urine, 7 +/- 8.

Synthesis of latent collagenase and collagenase inhibitor by bovine aortic medial explants and cultured medial smooth muscle cells.

Bovine aortic medial tissue and medial smooth muscle cells were demonstrated for the first time to synthesize a latent collagenase together with collagenase inhibitor in culture. Molecular weights of the latent collagenase and its inhibitor derived from aortic medial tissue explant were estimated to be about 52 K by gel filtration and 26.5 K by electrophoresis, respectively. Activated aortic collagenases cleaved type I collagen in solution into 3/4 (alpha A) and 1/4 (alpha B) length cleavage fragments and were inhibited by EDTA, o-phenanthroline, dithiothreitol, bovine serum, and highly purified dental pulp and aortic collagenase inhibitors. The aortic inhibitors showed inhibitory activity against all the animal collagenases tested, except for bacterial collagenase. Double-immunodiffusion analysis using a monospecific antiserum prepared against dental pulp inhibitor showed that the aortic inhibitors are immunologically identical to the pulp inhibitor. Using the same antiserum, we found immunoreactive collagenase inhibitor protein to be localized along the collagen fibers between elastic membranes in aortic medial tissue.

The Activation of Matrix Metalloproteinases by a Whole-cell Extract from Prevotella nigrescens

Periradicular lesions are primarily evoked as a response to a bacterial challenge emanating from an infected root canal. Many bacteria such as those of the genera Porphyromonas, Prevotella, and others have been isolated from infected root canals. The cause of periradicular lesions is related to the destruction of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), and so on are products of inflammatory cells and, once activated, are intimately involved in the degradation of the ECM. However, there are no reports regarding the destruction of the ECM by bacterial extracts from Prevotella nigrescens (P. nigrescens). The present study was conducted to evaluate the activating effect of a whole-cell extract (WCE) of P. nigrescens on proMMP-2 and proMMP-9. P. nigrescens WCE was mixed with proMMP-2 or proMMP-9 under many conditions, and the activation of these MMPs was determined by gelatin zymography. A band indicating a lower molecular weight of 66 kd or 84 kd, which migrated faster than the band of proMMP-2 (72 kd) or proMMP-9 (92 kd) respectively, was detected, which could be the active form of either MMP. The present study suggests that P. nigrescens might be able to activate proMMP-2 and proMMP-9 in vivo and that this activation might be related to the destruction of periapical tissues.

Immunoelectron microscopic localization of collagenase inhibitor in bovine dentin.

The localization of collagenase inhibitor in bovine dentin was investigated by immunohistochemistry and immunoelectron microscopy using a specific rabbit antiserum raised against the inhibitor purified from culture medium of bovine unerupted third molar pulps. Immunoreactive collagenase inhibitor was mainly localized as an amorphous or granular accumulation along the wall of dentinal tubules in decalcified bovine dentin. A part of the immunoreactive inhibitor appeared as two periodic bands on the collagen fibrils surrounding the dentinal tubules. Those two, a major and a minor band, corresponded to D-periodic fibril bands V-VII and I, respectively. As those two bands are located on both borders between overlap and hole zones of the collagen fibril, C- and N-terminal non-helical peptides and eight regions which are nD (n = 1-4) apart from each non-helical peptide along the triple helix are all equally possible binding sites of the collagenase inhibitor on the collagen molecule. It is interesting that the major band is located not exactly at, but fairly close to, the locus of collagenolytic cleavage, which lies between D-periodic fibril bands IV and V.

Dental pulp collagenase: initial demonstration and characterization.

Abstract A collagenase, active against native helical collagen, was initially found in the explant medium of bovine dental pulp. In contrast to the collagenases from other oral tissues, all the pulp enzyme released was in a latent form which was activated by trypsin treatment, 4-aminophenylmercuric acetate, and some chaotropic agents. The activated enzyme was inhibited by low concentrations of EDTA and calf serum. The molecular weight of activated enzyme was tentatively estimated at 45,000 daltons by gel filtration. The enzyme attacked undenatured collagen in solution at 20°C producing characteristic products α A ( 3 4 ) and α B ( 1 4 ) .

Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP‐1 and TIMP‐2

AIM To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria (Porphyromonas gingivalis, P. endodontalis and F. nucleatum) on the activation of matrix metalloproteinase (MMP-2) and the inactivation of tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2). METHODOLOGY Each SBE was added to cultures of human periodontal ligament (PL) cells or HT1080 cells and their supernatants were analysed by zymography for MMP-2. Each SBE was added to PL cell cultures, and the amount of TIMP-1 was determined by ELISA. P. gingivalis SBE was incubated with HT1080 cell culture supernatants, and the amounts of TIMP-1 and TIMP-2 were determined by ELISA. Statistical analysis was performed with the paired Students t-test. RESULTS In extracts of PL cells that had been incubated in the presence of P. gingivalis SBE, one representing pro-MMP-2 (72 kDa) and a band corresponding to the active MMP-2 (66 kDa) were observed; but in the other extracts it was not detected. When HT1080 cells were treated with P. gingivalis SBE, the pro-MMPs was processed into 86- and 66-kDa fragments, but in the other extracts, the processing did not occur when the other SBEs were used. When PL cells were incubated with the same SBEs, the amount of TIMP-1 was markedly decreased (P < 0.01), but in the other extracts, it was not. The amounts of both TIMP-1 and TIMP-2 were decreased in a dose-dependent manner when HT1080 cell culture supernatant was incubated with P. gingivalis SBE. CONCLUSIONS These findings suggest that P. gingivalis SBE may cause connective tissue to be destroyed, contributing to the process of periapical disease, by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2.

Immunohistochemical localization of collagenase inhibitor in bovine dental pulp, pulp cells in monolayer culture, and in some oral connective tissues

The development of an antiserum, monospecific to the collagenase inhibitor, from bovine dental pulps permitted localization of immunoreactive inhibitor protein, by means of both immunofluorescence and immunoperoxidase-staining techniques in sections of bovine dental pulps. The immunoreactive inhibitor protein in bovine dental pulps is present both in cells and extracellular matrices. When cultured in Eagle minimal essential medium, coronal pulps from bovine-unerupted teeth were shown, by assay of the medium, to produce only about 1/10 of the amount of inhibitor produced by the root pulps. When compared by immunohistochemical observation, however, essentially no differences in fluorescent activity was found between coronal and root pulps. Specific cytoplasmic staining was seen both in explanted root-pulp tissues and in immature fibroblast-like pulp cells from monolayer cell cultures of bovine root pulps, which indicate that the pulp cells are responsible for inhibitor production. Sections of dental follicle and gingiva from the same animal, showed a distribution of immunoreactive inhibitor protein similar to that in dental pulps.