Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.     

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.     

基质金属蛋白酶及其抑制剂在乳腺癌中的表达

目的:研究基质金属蛋白酶2(Matrix Metalloproteinase-2,MMP-2),基质金属蛋白酶7(MMP-7),基质金属蛋白酶9(MMP-9),膜型基质金属蛋白酶(Membrane Type-1 Matrix Metalloproteinase,MT1-MMP),金属蛋白酶组织抑制剂1(Tissue Inhibitor of Metalloproteinase,TIMP-1),金属蛋白酶组织抑制剂2(TIMP-2)在乳腺癌组织中mRNA的表达,及与临床病理变量之间的关联。方法:采用150例乳腺癌患者的组织样本。使用半定量逆转录-聚合酶链反应(RT-PCR)法来测定肿瘤组织和正常乳腺组织中MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2的mRNA表达。结果:MMP-2,MMP-7,MMP-9,MT1-MMP,TIMP-1和TIMP-2在乳腺癌中的mRNA表达显著高于正常组织。结论:MMP-2,MMP-7,MMP-9,和MTI-MMP的表达增加和临床病理参数之间的关联,可以用来预测乳腺癌的侵害行为。     

Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The goal of this study was to elucidate peculiarities of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF). The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papillomavirus (HPV-16). Papillomaviruses type16 and 18 are the etiological factor for cervical cancer. A primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF included determination of MMP-2 and MMP-9 activity by hydrolysis of the specific substrate, radioactive collagen type IV; analysis of MMP spectra by a zymographic assay, and estimation of the mRNA expression by RT-PCR. It was found that: (1) collagenolytic activity of MMP was increased only in TF and it depended on the degree of cell tumorigenicity; (2) the study of MMP spectra revealed the presence of MMP-9 only in TF, whereas MMP-2 was found in IF as well; (3) the mRNA expression of MMP-9, MT1-MMP and TIMP-2 increased in all TF while the MMP-2 expression increased in TF only after TF cell selection on rats; (4) the collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 and endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to the PF culture. The data obtained indicate changes in the ratio enzyme/activator/inhibitor and also suggest a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV16 gene in a cell culture.     

Normal variation in thermal radiated temperature in cattle: implications for foot-and-mouth disease detection

Background

Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth.

Results

MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs.

Conclusions

Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors.     

Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2

Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts.     

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

Spatial analysis of RECK,MT1-MMP,and TIMP-2 proteins during early Xenopus laevis development

Proper cell-cell and cell-ECM interactions are vital for cell migration and patterning of the vertebrate embryo. MMPs and their inhibitors, RECK and TIMPs, are all differentially expressed during embryogenesis to regulate such ECM remodeling and cell interactions. MT1-MMP, RECK, and TIMP-2 are unique amongst other ECM-regulating proteins as they act in the pericellular space. Past studies have shown that RECK and TIMP-2 interact with MT1-MMP on the cell surface, thereby influencing cell behaviour as well as the microenvironment immediately surrounding the cells. We investigated the localization of RECK, TIMP-2, and MT1-MMP proteins throughout early X. laevis development using immunohistochemistry. We found that during neural tube formation, axis elongation, and organogenesis, RECK, TIMP-2, and MT1-MMP proteins show highly similar localization patterns, particularly in the ectoderm and in the dorsal-ventral differentiation of the neural tube. Our data suggests they function together during patterning events in early Xenopus development.     

Ras modifies proliferation and invasiveness of cells expressing human papillomavirus oncoproteins

Infection by human papillomavirus (HPV) is a major risk factor for human cervical carcinoma. However, the HPV infection alone is not sufficient for cancer formation. Cervical carcinogenesis is considered a multistep process accompanied by genetic alterations of the cell. Ras is activated in approximately 20% of human cancers, and it is related to the metastatic conversion of tumor cells. We investigated how Ras activation was involved in the malignant conversion of HPV-infected lesions. The active form of H-ras was introduced into human primary keratinocytes expressing the HPV type 18 (HPV18) oncoproteins E6 and/or E7. We analyzed the keratinocytes' growth potentials and found that the activation of the Ras pathway induced senescence-like growth arrest. Senescence could be eliminated by high-risk E7 expression, suggesting that the pRb pathway was important for Ras-induced senescence. Then we analyzed the effect of Ras activation on epidermis development by using an organotypic "raft" culture and found that the E7 and H-ras coexpressions conferred invasive potential on the epidermis. This invasiveness resulted from the upregulation of MT1-MMP and MMP9 by H-ras and E7, respectively, in which the activation of the MEK/extracellular signal-regulated kinase pathway was involved. These results indicated that the activation of Ras or the related signal pathways promoted the malignant conversion of HPV-infected cells.     

Expression of interstitial collagenase and its endogenous regulators in immortalized and transformed by HPV-16 E7 gene fibroblasts

Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study was to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators during oncogenic transformation of fibroblasts by HPV-16 E7 gene. Papilloma virus types 16 and 18 are etiological factor of cervical cancer. We have studied expression of MT1-MMP, MMP-1, tissue inhibitor of these proteases, TIMP-1, and urokinase-like plasminogen activator (uPA), activating MMP-1 via plasmin. The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression, and enzymatic activity were studied by RT-PCR and by hydrolysis of fluorogenic type I collagen, respectively. Cell transformation was accompanied by: (a) 2–3 fold induction of MT1-MMP mRNA expression vs PF; (b) the decrease in mRNA level of TIMP-1 (1,5–2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: (a) the increase of MT1-MMP expression (1,5–2 fold); (b) unchanged TIMP-1 expression; (c) the increase of uPA expression (2–4 fold) vs PF and TF. MMP secreted activity and activity in lysates of TF increased but level of free endogenous MMP inhibitors decreased vs IF. Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF.     

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.     

Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.