Ryoichi Nemori

Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in rheumatoid synovium

In vitro, membrane-type matrix metalloproteinases (MT-MMP) are known to activate the zymogen of MMP-2 (proMMP-2, progelatinase A), which is one of the key MMP in joint destruction in rheumatoid arthritis. In the present study, we examined the production and activation of proMMP-2, and the expression of MT1-MMP, MT2-MMP, and MT3-MMP, their correlation with proMMP-2 activation, and their localization in rheumatoid synovial tissue. Using sandwich enzyme immunoassay and gelatin zymography techniques, proMMP-2 production levels and activation ratios were found to be significantly higher in rheumatoid synovium compared with normal synovium (p < 0.01). Quantitative RT-PCR analyses demonstrated that MT1-MMP and MT3-MMP were expressed in all rheumatoid synovial tissue (30 of 30 cases), but that the mean expression level of MT1-MMP was approximately 11-fold higher than MT3-MMP. Significant correlation was found between the mRNA expression level of MT1-MMP and the activation ratio of proMMP-2 (p < 0.01). In situ hybridization indicated that the hyperplastic lining cells of rheumatoid synovium expressed MT1-MMP. Immunohistochemistry demonstrated that MT1-MMP was co-localized with MMP-2 and with a tissue inhibitor of metalloproteinase-2, and was mainly located in the rheumatoid synovial lining cells. In situ zymography of rheumatoid synovium showed gelatinolytic activity, predominantly in the lining cell layer. This activity was blocked when incubated with BB94, a specific MMP inhibitor. These results demonstrate that MT1-MMP plays an important role in the activation of proMMP-2 in the rheumatoid synovial lining cell layer, and suggest that its activity may be involved in the cartilage destruction of rheumatoid arthritis.

Pericellular activation of proMMP-7 (promatrilysin-1) through interaction with CD151

Matrix metalloproteinase-7 (MMP-7) (also known as matrilysin-1) is secreted as a proenzyme (proMMP-7) and plays a key role in the degradation of various extracellular matrix (ECM) and non-ECM molecules after activation. To identify the binding proteins related to proMMP-7 activation, a human lung cDNA library was screened by yeast two-hybrid system using proMMP-7 as bait. We identified a candidate molecule CD151, which is a member of the transmembrane 4 superfamily. Complex formation of proMMP-7 with CD151 was demonstrated by immunoprecipitation of the molecules from CaR-1 cells, a human rectal carcinoma cell line, expressing both proMMP-7 and CD151, and CD151 stable transfectants incubated with proMMP-7. Yeast two-hybrid assays using deletion mutants of proMMP-7 and CD151 suggested an interaction between the propeptide of proMMP-7 and the COOH-terminal extracellular loop of CD151. The binding activity of 125I-labeled proMMP-7 to CD151 on the cell membranes was shown with CD151 stable transfectants. Laser-scanning confocal microscopy demonstrated that proMMP-7 colocalizes with CD151 on the cell membranes of CD151 stable transfectants and CaR-1 cells. In situ zymography using crosslinked carboxymethylated transferrin, a substrate of MMP-7, demonstrated proteinase activity on and around CD151 stable transfectants and CaR-1 cells, while the activity was abolished by their treatment with MMP inhibitors, anti-MMP-7 antibody or anti-CD151 antibody. In human lung adenocarcinoma tissues, colocalization of MMP-7 and CD151 was demonstrated on the carcinoma cells. Metalloproteinase activity was present in these tissues and could be inhibited by antibodies to MMP-7 or CD151. These data demonstrate for the first time that proMMP-7 is captured and activated on the cell membranes through interaction with CD151, and suggest the possibility that similar to the MT1-MMP/MMP-2 system, MMP-7 is involved in the pericellular activation mechanism mediated by CD151, a crucial step in proteolysis on the cell membranes under various pathophysiological conditions including cancer invasion and metastasis.

Impaired placental neovascularization in mice with pregnancy-associated hypertension.

Preeclampsia is a serious disorder that may result in severe morbidity and mortality for mother and fetus, and it is thought that the placental dysfunction is important in the pathogenesis of preeclampsia. As the model of preeclampsia, we previously generated a transgenic mouse model that developed pregnancy-associated hypertension (PAH) by mating females expressing human angiotensinogen with males expressing human renin. In PAH mice, maternal blood pressure started to rise from days 12 to 13 of gestation (E12–13) to term (E19–20), which is accompanied by the fetal intrauterine growth retardation and systemic maternal disorders including proteinuria and convulsion. To understand the pathology of the complications in PAH mice that overlap with those in human preeclampsia, we analyzed the PAH placenta sequentially from the onset of hypertension to the term of delivery. In PAH placenta, histological analysis revealed that the microvessel densities of fetal vasculature at term were significantly lower than those of normal placenta, and the majority of terminal vessels of PAH placenta were lacking for pericytes and basement membrane. The interaction between fetal vasculature and maternal blood canal at labyrinth of PAH placenta was morphologically distorted, and the expression patterns of key molecules in neovascularization of PAH placenta were distinct from those of normal placenta during pregnancy. In addition, maternal plasma level of soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) was significantly increased in PAH at E19. Furthermore, in uteroplacental site, in situ proteolytic activity of PAH mice was suppressed from E16 to term compared to that of normal pregnancy, and the expression of matrix metalloproteinase-2 mRNA was strikingly downregulated at E16 in PAH mice. Collective data suggest that the impairments of fetoplacental neovascularization and uteroplacental remodeling contribute to the development of complications in PAH.

Development of In Situ Zymography to Localize Active Matrix Metalloproteinase-7 (Matrilysin-1)

Matrix metalloproteinase-7 (MMP-7) is upregulated during carcinogenesis and its expression correlates with metastasis of human endometrial and gastrointestinal carcinomas. In the present study, we have developed a new method to localize the activity of MMP-7 within tissues. Polyethylene terephthalate films were uniformly coated with crosslinked carboxymethylated transferrin (CCm-Tf) as a substrate and incubated with frozen tissue sections mounted on the films. CCm-Tf on the films was degraded selectively by MMP-7, but showed little or no susceptibility to MMP-1, -2, -3, -9, or -13; MT1-MMP; MT3-MMP; or ADAMTS4. Although some serine proteinases such as elastase also digested CCm-Tf, CCm-Tf films impregnated with serine proteinase inhibitors prevented the digestion. When frozen sections of human endometrial carcinoma and lung carcinoma tissues were incubated on CCm-Tf films or those treated with proteinase inhibitors, the activity was detected in the carcinoma cell nests, where MMP-7 was immunolocalized. The present in situ zymography using CCm-Tf may be a useful method to analyze the functions of MMP-7 in pathophysiological conditions.

Vascular tissue fragility assessed by a new double stain method.

Although matrix metalloproteinases (MMPs) are known to be involved in the development of atherosclerosis and the instability of atheromatous plaques, much remains to be learned about their roles at the tissue level. To help clarify this area, we established a new double staining method using film in situ zymography and immunohistochemistry. Using this technique, a comprehensive analysis of the gelatinolytic activity in human vessel tissue demonstrated that gelatinolytic activity is enhanced in the shoulder region and fibrous cap at superficial areas of the atheromatous plaque in the presence of thrombolysis. Enzyme assay clarified high activity in the superficial area (7.50 ± 5.04 U/mg weight;P < 0.001). Gelatin zymography also indicated that addition of the antiplatelet agent, trapidil, alters the amount of secretion of MMP-2 and MMP-9 and their activation ratio. This novel approach to detect the activity of gelatinases in resected tissues may aid in the selection of optimal treatment of individual patients.

Erratum: Pericellular activation of proMMP-7 (promatrilysin-1) through interaction with CD151 (Laboratory Investigation (2005) 85 (1489-1506) DOI: 10.1038/labinvest.3700351)

Correction to: Laboratory Investigation (2005) 85, 1489–1506. doi:10.1038/labinvest.3700351 Following the publication of the above paper, the author has identified errors in the text on pp 1494, 1495, and 1501. On p 1494, Figure 2 legend, line 7, incorrectly states: 125I-labeled anti-MMP-7 antibody (125-20H1; lanes 1 and 2) or 125I-labeled anti-CD151 antibody (14A2.-CD151 antibody (14A2.H1; lanes 3 and 4).