Matrix Metalloproteinase-13 (MMP-13, Collagenase-3) is Highly Expressed in Human Tooth Pulp

Matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) participate into extracellular matrix degradation in physiological and pathological conditions. We hypothesized that MMP expression in pulp tissue changes in response to caries attack and investigated the gene expression profiles of MMPs and TIMPs in pulp tissue of sound and carious teeth with cDNA microarray. cDNA microarray demonstrated an extremely high MMP-13 (collagenase-3) mRNA expression in pooled pulp samples of sound and carious teeth, with less pronounced expression of MMP-16 (MT3-MMP) and TIMP-1. Real-time quantitative polymerase chain reaction of individual pulp samples revealed a wide range of the MMP-13 expression level between pulp samples with possible downregulation of MMP-13 expression during caries progression. Western blot and immunohistochemical staining confirmed the presence of MMP-13 with no observable differences between sound and carious teeth pulp tissues. The results reveal that MMP-13 is expressed and synthesized in pulp tissue, an interesting feature considering the very limited expression of MMP-13 in normal adult tissues. Further studies with a larger sample size are needed to clarify the changes in MMP-13 expression during caries progression.     

Ex vivo detection and characterization of early dental caries by optical coherence tomography and Raman spectroscopy

Early dental caries detection will facilitate implementation of nonsurgical methods for arresting caries progression and promoting tooth remineralization. We present a method that combines optical coherence tomography (OCT) and Raman spectroscopy to provide morphological information and biochemical specificity for detecting and characterizing incipient carious lesions found in extracted human teeth. OCT imaging of tooth samples demonstrated increased light backscattering intensity at sites of carious lesions as compared to the sound enamel. The observed lesion depth on an OCT image was approximately 290 microm matching those previously documented for incipient caries. Using Raman microspectroscopy and fiber-optic-based Raman spectroscopy to characterize the caries further, spectral changes were observed in PO4 (3-) vibrations arising from hydroxyapatite of mineralized tooth tissue. Examination of various ratios of PO4 (3-) nu2, nu3, nu4 vibrations against the nu1 vibration showed consistent increases in carious lesions compared to sound enamel. The changes were attributed to demineralization-induced alterations of enamel crystallite morphology and/or orientation. OCT imaging is useful for screening carious sites and determining lesion depth, with Raman spectroscopy providing biochemical confirmation of caries. The combination has potential for development into a new fiber-optic diagnostic tool enabling dentists to identify early caries lesions with greater sensitivity and specificity.     

Human decidua and invasive trophoblasts are rich sources of nearly all human matrix metalloproteinases

Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. As for all invasive cell types, the ability of CTB to infiltrate the uterine wall is facilitated by the activity of matrix metalloproteinases (MMPs), which is regulated by tissue inhibitors of MMPs (TIMPs). There is evidence for the expression of several MMPs and TIMPs in decidua. However, published data are limited. Therefore, to set a foundation for future research, we screened a panel of healthy human deciduas obtained during first, second and third trimester of pregnancy in addition to isolated decidual cell populations for the expression of all known human MMPs and TIMPs by RT-PCR, western blot and immunohistochemistry. In the decidual samples, we detected almost all MMPs and all four TIMPs at mRNA level. While the expression of proMMP-3 and active MMP-13 and -23 was down-regulated in the course of pregnancy, the pro forms of MMP-8, -19 and -23, active MMP-9, -10, -12, -15, -16, -26 and -28, and pro- and active MMP-14 increased towards the end of gestation. All MMPs and TIMPs were expressed in uterine natural killer cells, decidual fibroblasts and/or trophoblasts, with the exception of MMP-20 and -25. In summary, a remarkably broad spectrum of MMPs was expressed at the human feto-maternal interface, reflecting the highly invasive and remodelling effect on placenta formation. It can be speculated that expression of MMPs correlates with the invasive potential of CTBs together with a crucial role in activation of labour at term.     

Overexpression of tissue inhibitor of metalloproteinases-3 in intestinal and cutaneous lesions of graft-versus-host disease.

Matrix metalloproteinases (MMPs) have been implicated in the pathobiology of various T-cell-mediated inflammatory disorders of the intestine and skin. Their synthetic inhibitor has been shown to prevent lethal acute graft-versus-host disease in animal models. We intended to determine the expression of MMPs 1, 3, 7, 9, 10, 12, and 19 and tissue inhibitors of metalloproteinases (TIMPs) 1 and 3 in intestinal and cutaneous lesions of patients suffering from graft-versus-host disease after bone marrow transplantation. In situ hybridizations for MMPs 1, 3, 7, 10, and 12 as well as TIMPs 1 and 3 were performed using (35)S-labeled cRNA probes on intestinal (n = 13) and cutaneous specimens (n = 9) from patients with graft-versus-host disease. Immunohistochemical stainings were carried out to localize MMP-9, MMP-19, TIMP-3, and TGF-beta1 proteins, and TUNEL staining, to detect apoptotic cells. TIMP-3 mRNA and protein were detected in cutaneous lesions in areas with vacuolar degeneration of the basal epidermal layer in all skin samples, and they colocalized with apoptotic keratinocytes and partly with staining for TGF-beta. None of the MMPs examined were overexpressed in skin lesions. Signals for MMP-1 and MMP-3 mRNA was found in 10/13 and 5/13 intestinal biopsies, respectively. In the gut, MMP-19-positive epithelial cells, particularly in the crypts, were found in 10/13 samples. Expression of MMPs 7, 9, 10, and 12 was absent or very low. TIMPs 1 and 3 were expressed by stromal cells in 12/13 and 10/13 gut samples, respectively. Whereas TIMP-1 was expressed particularly by subepithelial cells where epithelium had shed away, TIMP-3 was detected in deeper areas. We conclude that MMPs are differentially regulated in the skin and gut lesions of graft-versus-host disease. In agreement with previous data on cancer cells, TIMP-3, induced by TGF-beta1, may contribute to the apoptosis of keratinocytes in cutaneous graft-versus-host disease lesions, leading to typical histopathological changes. We also conclude that MMPs play a less important role as effector molecules in intestinal graft-versus-host disease than in celiac or inflammatory bowel disease.     

Matrix metalloproteinase expression is related to angiogenesis and histologic grade in spindle cell soft tissue neoplasms of the extremities

We defined the immunocytochemical expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in benign soft tissue neoplasms, fibromatoses, and sarcomas, together with the activity of gelatinase MMPs and TIMPs measured by zymography and reverse zymography in a subset of cases. The most strongly expressed MMP in all tumors was MMP-1, with weaker expression of MMP-10, MMP-11, and MMP-14 in most tumors. Nuclear expression of MMP-1, MMP-8, and MMP-13 was an unusual feature. TIMP-2 was expressed in all tumors, with stronger expression in fibromatoses than in sarcomas. Fibromatoses and high-grade sarcomas showed greater MMP-1 expression than other groups, and endothelial MMP-2 expression was more extensive in sarcomas. Differences in MMP and TIMP expression might be linked to the biologic behavior of soft tissue neoplasms. The activation of endothelial MMP-2 linked to widespread MMP-14 expression provides a mechanism for sarcomas to modulate their matrix and facilitate angiogenesis.     

Expression and Roles of MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2 in Allergic Nasal Mucosa

Purpose

Allergic rhinitis (AR) and asthma share many characteristics, but structural changes are observed far less often in AR. Matrix metalloproteinases (MMPs) constitute a family of Zn-dependent endopeptidases that can decompose the extracellular matrix and basement membrane, and regulate cell infiltration. We analyzed the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in allergic nasal mucosa after nasal allergen challenge (NAC) and determined their relationship to inflammatory cells.

Methods

Nasal mucosa specimens were obtained at surgery performed for hypertrophied turbinates. We performed NAC with house dust mite (HDM) allergen disks and control disks, and took biopsies at 30 minutes, 6 hours, and 12 hours after NAC. Cells expressing MMP-2, MMP-9, MMP-13, TIMP-1, and TIMP-2, as well as eosinophils and mast cells, were analyzed immunohistochemically. The MMPs and TIMPs in allergic nasal mucosa were quantified using enzyme-linked immunosorbent assays.

Results

At 30 minutes post-NAC, HDM-exposed nasal mucosa exhibited significantly more MMP-2+, MMP-9+, MMP-13+, TIMP-1+, and TIMP-2+ cells compared with control mucosa, and the numbers of MMP-9+ and TIMP-1+ cells correlated strongly with the number of mast cells. At 6 hours post-NAC, the numbers of MMP+ and TIMP+ cells did not differ significantly between HDM-exposed mucosa and control mucosa, but the ratios of MMP+ cells to TIMP+ cells were higher in HDM-exposed mucosa. At 12 hours post-NAC, the number of MMP-13+ cells tended to be higher in HDM-exposed mucosa and was strongly correlated with the number of eosinophils. Quantitatively, the levels of MMP-2 and MMP-13 were significantly higher than the MMP-9 level, and the TIMP-2 level was significantly higher than the TIMP-1 level in allergic nasal mucosa.

Conclusions

We demonstrated increased expression of MMP-2, MMP-9, and MMP-13 in allergic nasal mucosa, high MMPs-to-TIMP-1 ratios, and a strong correlation between MMP-9 and mast cells and between MMP-13 and eosinophils. The imbalance between MMPs and TIMPs may contribute to the migration of inflammatory cells such as eosinophils and mast cells to the nasal mucosa of AR patients, suggesting a possible active role of MMPs in AR.     

Expression and prognostic significance of matrix metalloproteinases and their tissue inhibitors in primary neuroendocrine carcinoma of the skin

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been implicated in the development and progression of many tumors, but data for primary neuroendocrine carcinoma (PNC) of the skin are lacking. The aim of the study was to assess the expression of MMPs and TIMPs in PNC and to evaluate their prognostic significance. Expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, MMP-13, and MMP-14 and TIMP-1, TIMP-2, and TIMP-3 was evaluated by immunohistochemistry on 23 samples of PNC of the skin. The results were matched with clinical features and patient survival. In the 23 specimens of PNC, high (>20% of positive neoplastic cells) MMP-1 expression was found in 13 (56.5%) cases. MMP-2 was evidenced in 12 (52.1%) cases, 8 (34.7%) of which showed high expression in neoplastic cells. MMP-3 was detected in 11 cases (47.8%), with high expression in 9 (39.1%) of them. High MMP-9 expression was observed in 3 (13%) cases, whereas high MMP-14 expression was detected in 11 (47.8%) specimens. Expression of TIMP-1 by neoplastic cells was found in 8 (34.7%) cases, with high expression in 3 cases, whereas high TIMP-3 expression was detected in 21 (91.3%) cases. No immunoreactivity for MMP-11, MMP-13, or TIMP-2 was found. Statistical analysis failed to identify a significant correlation between MMP/TIMP expression and clinical parameters. By univariate analysis, stage >I (P = 0.01), high expression of MMP-1 (P = 0.04) and MMP-3 (P = 0.01) resulted significant negative prognostic factors, whereas by multivariate analysis, stage was the only factor that affected survival (P = 0.02). Our results suggest that MMP-1 and MMP-3 may influence the invasive and metastatic potential of PNCs. It is conceivable that future attempts to specifically block MMP-1 and MMP-3 activity may provide a novel means to inhibit invasiveness and distant spread in selected patients with PNC.     

Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid

BACKGROUND: Matrix metalloproteinase (MMP)-2, MMP-9 and MMP-13 can degrade type IV collagen which is the major component of the basement membrane zone (BMZ). In bullous pemphigoid (BP), the separation occurs within the BMZ. OBJECTIVE: To evaluate the involvement of MMPs in the pathogenesis of BP, we examined the expression of MMP-2, MMP-9 and MMP-13 in the lesional skin of BP patients. METHODS: The expression of MMP-2, MMP-9, MMP-13, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 was analyzed by immunohistochemistry in the lesional skin of BP patients in comparison with that in normal human skin. Next, the cellular sources of MMP-2, MMP-9 and MMP-13 were analyzed by double immunohistochemistry. Finally, the levels of these MMPs in the serum and blister fluid of BP patients were measured by ELISA. RESULTS: The number of cells expressing MMP-2, MMP-9 and MMP-13 were significantly increased in the lesional skin of BP patients as compared to that in normal skin. Although the number of cells expressing TIMP-1 and TIMP-2 were also increased in the lesional skin of BP patients as compared to that in normal skin, the ratio of MMPs to TIMPs in the lesional skin of BP patients was high (2.4:1). T cells comprised the major source of MMP-2, MMP-9 and MMP-13, while a proportion of mast cells and eosinophils also expressed these MMPs. Furthermore, marked expression of MMP-2 was detected in the epidermal keratinocytes. The levels of these MMPs in the blister fluid were significantly greater than those in the serum. CONCLUSION: These results suggest that MMP-2, MMP-9 and MMP-13 may be involved in the mechanism of blister formation in BP and that besides infiltrating inflammatory cells, structural cells like epidermal keratinocytes may also participate in the induction of blister formation in BP.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP-13, MMP-14 and TIMP-2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP-1, MMP-7 and MMP-9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP-2, MMP-14 and TIMP-2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low-grade malignant chondrosarcoma and a benign growing enchondroma.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。     

MMP-7、MMP-10和TIMP-4在心力衰竭心室重构中的表达

目的:应用细胞因子抗体芯片技术筛选与心力衰竭心室重构关系密切的基质蛋白酶。方法:从本院的心脏病组织库中挑选6例病理诊断明确和各方面资料比较齐全的致心律失常性右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行细胞因子抗体芯片分析,筛选在致心律失常性右室心肌病引起的心力衰竭中差异表达的基质蛋白酶,并应用酶联免疫分析和免疫组织化学的方法加以验证。结果:在所筛选的17种基质金属蛋白酶中,只有MMP-7和MMP-10在致心律失常性右室心肌病引起心力衰竭中高表达,而在4种基质金属蛋白酶内源性组织抑制剂中,只有TIMP-4 低表达。经酶联免疫分析和免疫组织化学的方法证实,不仅在致心律失常性右室心肌病引起心力衰竭的心肌,在缺血性心肌病和扩张性心肌病引起的心力衰竭心肌中也发现MMP-7和MMP-10 的高表达及TIMP-4的低表达。结论:心肌组织中MMP-7和MMP-10的高表达及TIMP-4的低表达在不同心肌病引起的心力衰竭心室重构分子机制中可能发挥重要的作用。     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in joint fluid of the patients with loose artificial hip joints.

The pseudojoint cavity formed in patients undergoing total hip arthroplasty (THA) is later remodeled to synovial membrane-like tissue, which produces pseudosynovial fluid. This pseudosynovium also is an important source of matrix metalloproteinases (MMPs). As it is widely speculated that synovial fluid MMPs may contribute to local tissue degradation in rheumatoid arthritis (RA) and osteoarthritis (OA), we hypothesize that locally produced MMPs are found in the pseudosynovial fluid, via which they have access to the implant-host interface, and that if they retain their proteolytic potential, they might contribute to aseptic loosening. Enzyme-linked immunosorbent assay (ELISA), immunoblotting, and zymography were used to analyze MMPs and tissue inhibitors of metalloproteinases (TIMPs) in synovial fluid in aseptic loosening, which was compared to RA and OA. Pseudosynovial THA fluid was characterized using low levels of MMP-1 but moderate levels of MMP-13 and MT1-MMP (MMP-14). Due to the lack of an appropriate assay, MMP-13 and MT1-MMP were not similarly assessed, but the immunoblotting indicated that they were in the 56 kD intermediate proteolytically processed forms. The MMP-9 level was intermediate between RA and OA. MMP-2 was on a significant level, but there were no differences among study groups. The THA group also was characterized using relatively high levels of TIMP-1 and TIMP-2. Accordingly, MMP-9 and MMP-2 were found to occur in the 92 kD and 72 kD proenzyme form, respectively, with full activity retained in all study groups. The data suggest that proMMP-2-TIMP-2 and proMMP-9-TIMP-1 complexes are formed in the pseudosynovial fluid due to the excess of TIMPs over MMPs in aseptic loosening of THA. TIMP-complexed MMPs are resistant to MMP-mediated proteolytic activation, which may explain their latency and proenzyme zymogen form. Thus, formation of stabilizing proMMP-TIMP complexes enable transportation of proMMPs far from their original site of production. Due to motion-associated cyclic changes of the intra-articular pressure, fluid-phase MMPs stabilized by TIMPs might be absorbed to implant surfaces and interface tissues and help to dissect the implant/cement-to-bone interface in situ. Consequently, they may contribute to local proteolytic/tissue destructive events and aseptic loosening.     

Matrix metalloproteinases and their tissue inhibitors in the lesions of cardiac and pulmonary sarcoidosis: an immunohistochemical study

The pathogenesis of the tissue damage and fibrosis in sarcoidosis is poorly understood. The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) must be considered in this regard, because they control the lysis of connective tissue components. Immunohistochemical studies (peroxidase and dual labeling for confocal microscopy) of reactivity for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and the 4 membrane-type-MMPs were made on tissues from patients with cardiac (n = 4) and pulmonary (n = 5) sarcoidosis. The granulomas were histochemically similar in both organs. The multinucleated giant cells (MGCs) showed moderate reactivity for MMP-1 and MMP-9 and variable reactivity for MMP-2 and MMP-3; in addition, they showed colocalization of MT-1-MMP, which activates MMP-2. The reactivity of epithelioid cells (ECs) was moderate for MMP-2 and mild for other MMPs. Macrophages showed weaker reactivity for MMPs than did MGCs and ECs. All 3 types of cells showed very low reactivity for TIMPs. Staining for type IV collagen showed focal damage to the basement membranes of cardiac myocytes and pulmonary alveoli near the granulomas. The cells in sarcoid granulomas contain an abundance of MMPs and a paucity of TIMPs. The MGCs also contain MT-1-MMP and thus can activate MMP-2 in the granulomas. The MMPs can cause damage to adjacent cardiac myocytes and pulmonary alveoli, leading to the interstitial fibrosis produced by sarcoidosis.     

Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinase, collagens, and Ki67 antigen in pleural malignant mesothelioma: an immunohistochemical and electron microscopic study

Because matrix metalloproteinases (MMPs) degrade extracellular matrix, including basement membrane, and because tissue inhibitors of MMP (TIMPs) suppress MMP activities, MMPs and TIMPs are considered to play important roles in invasion and metastasis in many malignancies. We examined immunohistochemically the expression of MMPs (MMP-1, -2, -3, -7, and -9), TIMPs (TIMP-1 and -2), and collagens (types I, III, and IV) in 16 patients with pleural malignant mesothelioma (PMM; 8 with the epithelial, 4 with the sarcomatous, and 4 with the biphasic type). Electron microscopy revealed that the tumor cells in all types possessed the characteristics of malignant mesotheliomas, including numerous microvilli and moderate amounts of intermediate filaments. Basement lamina was present only focally. The proliferative Ki67 index was at a high level, compared with values reported in various other malignancies. Positive staining for MMP-1 was observed in most tumor cells in all 16 patients (100%). MMP-2 was expressed in most tumor cells in 2 patients (13%). In contrast, MMP-3, -7, and -9 were not detected in any PMM. TIMP-1 and TIMP-2 were expressed in 3 patients (19%) and 2 patients (13%), respectively. The stromal cells were simultaneously positive for MMPs or TIMPs in the patients whose tumor parenchymal cells were positive for each enzyme. These results indicate that the expression of MMP-1 and MMP-2 may be related to PMM invasion and spread. In particular, as MMP-1 was overexpressed in contrast to the lower expression of TIMP-1, MMP-1 is strongly suggested to play an important role in PMM invasion by degrading the tumor stroma. In spite of general agreement that epithelial-type PMM has a better prognosis than other types, there was no significant difference in the Ki67 index among the histological types of PMM.     

Increased matrix metalloproteinases as possible cause of osseoarticular tissue destruction in long-term haemodialysis and β2-microglobulin amyloidosis

Immunolocalization of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in periarticular tissues of ₂-microglobulin amyloidosis patients was investigated. MMP-1 (interstitial collagenase) the most strongly expressed of the MMPs, was localized in the synovial lining cells, mesenchymal cells in granulation tissue and nodular amyloid deposits, and chondrocytes within areas of cartilage erosion. Expression of MMP-1 was correlated with the degree of macrophage infiltration and synovial cell hyperplasia, but it was not correlated with the degree of amyloid deposition or haemodialysis period. Expression of MMP-1 appeared more intense than that of TIMP-1 and TIMP-2 in highly inflammatory cases. MMP-2 was mildly expressed in the interstitial fibroblasts and MMP-3 was faintly stained in the extracellular matrix of the synovial membrane. MMP-9 (gelatinase B) was found to be strongly positive in the osteoclasts which increased in the progressing osteolytic lesion from the destructive arthropathy. These results suggest involvement of MMPs in inflammation with an imbalance between expression of MMPs and TIMPs being closely related to pathogenesis of the destructive arthropathy.     

Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways.Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting.Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3.Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.     

Monocyte-derived dendritic cell subpopulations use different types of matrix metalloproteinases inhibited by GM6001

Matrix metalloproteinases (MMPs) are endopeptidases with the potential to cleave extracellular matrix, support tissue renewal and regulate cell migration. Functional activities of MMPs are regulated by tissue inhibitors of MMPs (TIMPs) and disruption of the MMP–TIMP balance has pathological consequences. Here we studied the expression and secretion of MMPs and TIMPs in CD1a⁻ and CD1a⁺ monocyte-derived dendritic cell (DC) subpopulations. Our results showed that monocytes express TIMPs but lack MMPs, whereas upon differentiation to moDCs and in response to activation signals the expression of MMPs is increased and that of TIMPs is decreased. MMP-9 is expressed dominantly in the CD1a⁻ subpopulation, while MMP-12 is preferentially expressed in CD1a⁺ cells. Experiments performed with the synthetic MMP inhibitor GM6001 revealed that this drug efficiently inhibits the migration of moDCs through inactivation of MMPs. We conclude that modulation of MMP activity by GM6001 emerges as a novel approach to manipulate DC migration under inflammatory conditions.     

Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.