Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2

Matrix metalloproteinases (MMPs), a family of extracellular matrix-degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP-1, 2, 3, 7, 8, 9 and 13, and MT1-MMP) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non-neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP-1, MMP-2, MMP-13, MT1-MMP, and TIMP-1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP-2 zymogen (pro-MMP-2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro-MMP-2 and MT1-MMP were similar among these carcinoma groups, TIMP-2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro-MMP-2 activation ratio correlated directly with the MT1-MMP/TIMP-2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP-2, MT1-MMP, and TIMP-2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10-phenanthroline. These results suggest that enhanced activation of pro-MMP-2 mediated by MT1-MMP is implicated in the invasion and metastasis of MECs and that TIMP-2 may regulate pro-MMP-2 activation in salivary gland carcinomas.     

Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1 matrix metalloproteinase

Gelatin zymography is the common method for examining matrix metalloproteinase-2 (MMP-2) in cells and media samples. Activation of the latent MMP-2 zymogen involves its binding to the cell surface MT1-MMP*TIMP-2 (membrane type-1 matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase-2) complex with subsequent cleavage of proMMP-2 by TIMP-2-free adjacent MT1-MMP. This is followed by autolytic maturation of the activation intermediate and the release of the mature MMP-2 species from cell surfaces into the extracellular milieu. To observe the MMP-2 activation pathway in more detail, proMMP-2-deficient MCF7 breast carcinoma cells expressing MT1-MMP were incubated with excess proMMP-2 to saturate the available MT1-MMP*TIMP-2 surface receptors. After removal of the unbound material, the kinetics of proMMP-2 activation and MMP-2 release from cells into media was monitored by gelatin zymography and substrate cleavage. Our observations demonstrate that gelatin zymography is insufficient for providing meaningful information about the status of MMP-2. The proteolytically competent mature MMP-2 moiety alone, but not in its complex with TIMP-2, was released from the cells. In tissue culture conditions, the enzyme's proteolytic activity was suppressed in the next 30 to 60 minutes by tissue inhibitors of MMPs, especially by TIMP-1. The picture emerges that there is a likely temporal regulation of MMP-2 activity by TIMPs in tumor cells. These relatively rapid changes of the MMP-2 status cannot be detected by gelatin zymography. Additional studies are needed to examine the significance of this phenomenon in vivo.     

胸腺上皮肿瘤组织中基质金属蛋白酶-2活性与Ⅰ型膜型基质金属蛋白酶 mRNA表达的相关性

目的探讨胸腺瘤和胸腺癌中基质金属蛋白酶（MMP）-2、Ⅰ型膜型（MT1）-MMP、金属蛋白酶组织抑制剂（TIMP）-2mRNA的表达和MMP-2蛋白活性的关系。方法分别用real-time逆转录．聚合酶链反应（RT-PCR,Taqman法）、明胶酶谱法和Filmin situ gelatin-Zymography（FIZ）对正常的胸腺组织（2例）、胸腺瘤（12例）和胸腺癌（2例）患者的新鲜肿瘤组织中MMP-2、MT1-MMP、TIMP-2mRNA的表达,pro-MMP-2的活性率及活性蛋白的定位进行测定。结果MMP-2、MT1-MMP及TIMP-2mRNA在Ⅰ期与Ⅱ期和Ⅲ期与Ⅳ期中的表达差异均无统计学意义（P〉0．05）,在Ⅰ～Ⅱ期与Ⅲ～Ⅳ期和胸腺癌3组中差异均有统计学意义（P〈0．01）。在AB、B1型（混合型和淋巴细胞为主型）与B2、B3型（皮质型和多角细胞为主型）以及胸腺癌3组中差异均有统计学意义（P〈0．05）。MMP-2的蛋白活性率（MMP-2／pro—MMP-2＋MMP-2）在Ⅰ～Ⅱ期、Ⅲ～Ⅳ期和胸腺癌各组中差异有统计学意义（P〈0．05）,在AB、B1型与B2、B3型以及胸腺癌各组中的差异均具有统计学意义（P〈0．05）。胸腺瘤各期及各型中MT1-MMP、TIMP-2mRNA与MMP-2蛋白活性表达均呈正相关,且相关程度相似（r=0．7235、r=0．7647、P〈0．005）。MMP-9的蛋白表达在各组间差异均无统计学意义。结论MMP-2、MT1-MMP及TIMP-2的mRNA表达与胸腺瘤临床分期、病理分型相关,MMP-2的活性与MT1-MMP和TIMP-2的表达升高正相关。推测MT1-MMP通过TIMP-2对MMP-2的激活起促进作用。     

Differential expression patterns of MMPs and their role in the invasion of epithelial premalignant tumors and invasive cutaneous squamous cell carcinoma

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumors. MMP-2, MMP-9, TIMP-2, and MT1-MMP are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. In this study, we investigated the expression and role of MMPs in cutaneous oncogenesis. Tissue microarray consisting of 62 squamous cell carcinomas (SCC), 32 Bowen's disease (BD) samples, 25 normal epidermis samples were obtained for the study. MMP-2,-9, MT1-MMP and TIMP-2 proteins were examined by immunohistochemical staining and mRNA level was detected by quantitative RT-PCR in fresh tissues consisting of 5 cutaneous SCCs and paired normal epidermis samples. Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatin zymography and protein levels of MT1-MMP and TIMP-2 were measured by western blot in 2 human SCC cell lines. The invasive property was evaluated with invasion assays using Transwell filters. SCC exhibited significantly increased MMP-2, MT1-MMP and decreased TIMP-2 mRNA and protein expression compared to that of the normal epithelium. Immunohistochemical staining revealed that MT1-MMP was strongly expressed on the invasive front of SCCs, whereas BD exhibited higher expression around the dyskeratotic cells in the epithelium. In comparison with the expression observed in BD, SCC exhibited significantly increased MMP-2 expression. In addition, high MMP-2 and MT1-MMP expression and low TIMP-2 expression had a significant positive correlation with the invasiveness of SCC cell lines in vitro. Our results revealed significantly increased MT1-MMP and MMP-2 expression and decreased TIMP-2 expression in cutaneous SCC, and the expression correlated with the invasiveness of SCC cell lines. Therefore, the expression of these factors in cutaneous tumors may serve as an indicator of tumor aggressiveness and invasion.     

Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma

Three different membrane-type matrix metalloproteinases (MT-MMPs) activate in vitro the latent form of matrix metalloproteinase-2 (MMP-2), which is one of the key proteinases in invasion and metastasis of various cancers. We examined the mRNA expression of MT1, 2, and 3-MMPs and MMP-2 in cell lines of head and neck squamous cell carcinoma (HNSCC) and quantitated the relative expression levels in human HNSCC tissues by Northern blotting. The tissue localization of MT1-MMP and MMP-2 was determined by immunohistochemistry and in situ hybridization. Their implications in clinicopathologic factors were statistically evaluated. All cell lines examined consistently expressed MT1-MMP and MMP-2, but not MT2, 3-MMP. In the clinical specimens, there was a significant correlation in coexpression of messenger of RNA (P = .0005) and colocalization by immunohistochemistry (P < .0001) for MT1-MMP and MMP-2. Relative mRNA expression levels of MT1-MMP and MMP-2 in the carcinoma tissues were significantly higher than those of the control tissues (P = .0045 and P = .0122, respectively). Both mRNA expression level and immunopositivity of MT1-MMP significantly correlated with lymph node metastasis (P = .0081 and P = .0193, respectively), which was confirmed by multivariate logistic regression analysis. Immunoreaction of MT1-MMP and its mRNA expression were observed in both carcinoma cells and stromal cells. The localization of MMP-2 closely corresponded to that of MT1-MMP. These observations suggest that MT1-MMP possesses a role as a determinant of lymph node metastasis in HNSCC, and that concurrent expression of MT1-MMP and MMP-2 are involved in progression of HNSCC.     

宫颈癌中MMP-2及TIMP-2的表达及其临床意义

目的 :通过检测基质金属蛋白酶 - 2 (MMP - 2 )及基质金属蛋白酶组织抑制物 - 2 (TIMP - 2 )在宫颈癌中的表达 ,探讨其与宫颈癌侵袭转移的关系。方法 :采用免疫组化S -P法检测 5 1例宫颈癌和 16例正常宫颈组织中MMP - 2和TIMP - 2的表达情况。结果 :MMP - 2、TIMP - 2在正常宫颈上皮组织中均无表达 ,在宫颈癌组织中的阳性表达率分别为 74 .5 % (38/ 5 1)、4 7.1% (2 4 / 5 1) ,有显著性差异 (P <0 .0 1)。MMP - 2、TIMP - 2的表达与组织学类型无关 ,但与临床分期、细胞分化程度、淋巴结转移有关。结论 :MMP - 2、TIMP - 2的表达与宫颈癌的侵袭转移有关 ,MMP - 2、TIMP - 2可作为预测宫颈癌侵袭转移潜能和临床预后的指标。     

Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.     

Expression of matrix metalloproteinases in benign and malignant follicular thyroid lesions

AIMS: To examine expression of matrix metalloproteinases (MMPs) and related proteins in follicular thyroid lesions (FTLs) and to determine their usefulness for differential diagnosis of FTLs, particularly between minimally invasive carcinoma and adenoma. METHODS AND RESULTS: Six widely invasive follicular carcinomas (WIFCs), 15 minimally invasive follicular carcinomas (MIFCs), 19 follicular adenomas (FAs) and 10 adenomatous goitres (AGs) were analysed immunohistochemically for MMP-1, MMP-2, MMP-7, MMP-9, membrane-type 1-MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). MMP-1 was positive in all FTLs. MMP-2 and MMP-7 were positive in more than 80% of WIFC and MIFC cases, whereas they were negative in all FA and AG cases except one MMP-2+ FA (P < 0.001). MMP-9 stained positive significantly more in MIFC than FA or AG cases (P < 0.05, respectively). The positivity of MT1-MMP and TIMP-2 was different among some of the FTLs, but with no significant difference between MIFC and FA cases. In-situ hybridization of MMP-2 and MMP-7 mRNA in selected cases demonstrated the expression of these enzymes in the tumour cells as well as in some stromal cells. CONCLUSIONS: Our results confirm MMP expression mainly in malignant FTLs and suggest that MMP-2 and MMP-7 may be useful markers to distinguish MIFC from FA.     

Expression of MMP-2 and MMP-9, their inhibitors, and the activator MT1-MMP in primary breast carcinomas.

Enhanced expression of the type IV collagenases MMP-2 and MMP-9, or lack of their inhibitors TIMP-1 and TIMP-2, has been associated with tumour invasion and metastatic potential in several experimental models. Regulation of enzyme activity is clearly a key step in tumour invasion, and recently a potent activator of MMP-2, the membrane-associated MT1-MMP, has been described and characterized. Using an immunohistochemical approach, this study has examined the expression and distribution of the type IV collagenases, their inhibitors, and the activator MT1-MMP, in a series of 79 infiltrating ductal carcinomas (IDCs), 8 tubular carcinomas, and 27 infiltrating lobular carcinomas (ILCs). MMP-2 and MT1-MMP were expressed in more than 90 per cent of all carcinomas, with predominantly stromal and tumour cell cytoplasmic staining. However, reactivity localized on tumour cell membranes was recorded for MMP-2 in 34 per cent of cases with a monoclonal antibody and 55 per cent of cases with a polyclonal antibody, and for MT1-MMP in 68 per cent of tumours. In each case, this pattern of staining was significantly associated with the presence of lymph node metastasis (p=0.001, p=0. 008, and p=0.1, respectively). Both tumour cell and stromal staining was observed for TIMP-2, but there was no correlation with metastatic status. The 92 kD gelatinase MMP-9 was expressed by 68 per cent of carcinomas, either in the stromal compartment or by tumour cells. There was a highly significant correlation between the expression pattern of MMP-9 and tumour type, with ILCs displaying greater frequency and more homogeneous cytoplasmic staining than IDCs (p=0.0004). Staining for TIMP-1 was seen in the stroma and also in relation to small blood vessels, with more than 90 per cent of tumours showing this staining pattern using a polyclonal antibody. This study indicates distinct patterns of expression for different MMPs and demonstrates the potential importance of the MMP-2/MT1-MMP system in breast tumour progression. The association of MMP-9 with the infiltrating lobular phenotype may reveal novel mechanisms of control for this metalloproteinase.     

The metalloproteinase inhibitor TIMP-2 is down-regulated by androgens in LNCaP prostate carcinoma cells

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to perform several biological functions in tumor promotion, principally by their action of inhibiting matrix metalloproteinases (MMPs) at different steps of the metastatic process. In particular, TIMP-2 is involved in a functional complex with the membrane-type 1 (MT1) MMP to convert the secreted MMP-2 progelatinase into the fully active proteolytic enzyme. We used the human, androgen-sensitive prostate carcinoma cell line LNCaP in coculture with the human osteosarcoma cell line OHS to experimentally address the possibility of androgen-dependent regulatory effects on the functional MT1-MMP/TIMP-2/MMP-2 complex upon interaction between prostate carcinoma and osteoblastic cells in metastasis of prostate cancer to bone. In the LNCaP cells a gradual, time-dependent decline in TIMP-2 mRNA expression was observed in the presence of the synthetic androgen analogue R1881 (100 nM), reaching ∼25% of the control level after 48 h of incubation. Consistent with this, the accumulation of secreted TIMP-2 in media from R1881-treated cells was significantly inhibited already after 3 h. Neither MMP-2 gelatinolytic activity nor expression of MT1-MMP was detected in LNCaP cells. In contrast, the OHS cells showed membrane-associated MT1-MMP expression as well as MMP-2 secretion. However, R1881 treatment of the LNCaP/OHS coculture model did not seem to change the overall proteolytic activity of the MT1-MMP/TIMP-2/MMP-2 complex. Hormonal control of TIMP-2 expression in prostate carcinoma cells has not been previously reported, but whether such regulation has any functional role in the development of osteoblastic metastases in prostate cancer is still unclear. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neutrophil activator of matrix metalloproteinase-2 (NAM)

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.     

Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.     

Ezrin、MMP-2及TIMP-2在子宫颈癌中的表达与侵袭转移的关系

目的 通过观察埃兹蛋白(Ezrin)、金属蛋白酶-2(MMP-2)及金属蛋白细胞组织抑制因子-2(TIMP-2)蛋白水平在子宫颈癌组织中的表达情况,探讨它们与子宫颈癌侵袭转移的关系. 方法 采用免疫组织化学方法对40例子宫颈鳞癌,20例腺癌,29例子宫颈上皮内瘤样病变和16例正常子宫颈组织进行标记及分析. 结果 Ezrin、MMP-2及TIMP-2蛋白主要表达在子宫颈鳞癌、腺癌、上皮内瘤变的异型细胞质内,各实验组中的表达量与正常组比,差异均有统计学意义(P<0.01).Ezrin和MMP-2蛋白在有淋巴结转移组中的表达量较无淋巴结转移组明显高(P<0.01), 而TIMP-2蛋白水平的表达结果却相反.同时发现Ezrin、MMP-2及TIMP-2蛋白水平在宫颈病变组织表达有正相关性. 结论 Ezrin、MMP-2及TIMP-2 蛋白在子宫颈鳞癌、腺癌的表达量较对照组高. Ezrin和MMP-2在有淋巴结转移的子宫颈癌中高表达,而TIMP-2为低表达,提示它们在子宫颈癌的侵袭转移中起重要的作用.Ezrin、MMP-2及TIMP-2蛋白水平在宫颈病变组织表达有正相关性,这三者在子宫颈癌的侵袭转移中可能起协同作用.     

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。     

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels. This revised version was published online in July 2006 with corrections to the Cover Date.