Opposite effect of stable transfection of bioactive transforming growth factor-beta 1 (TGF beta 1) versus exogenous TGF beta 1 treatment on expression of 92-kDa type IV collagenase in mouse skin squamous cell carcinoma CH72 cells

We have previously shown that transforming growth factor-beta 1 (TGF beta 1) mRNA is consistently overexpressed in squamous cell carcinomas relative to normal mouse skin. Here we show that 92-kDa type IV collagenase (matrix metalloproteinase) (MMP-9) mRNA was likewise progressively overexpressed during mouse skin carcinogenesis. To determine if overexpression of MMP-9 and TGF beta 1 are linked, we stably transfected a bioactive TGF beta 1 into a mouse skin squamous cell carcinoma cell line (CH72), which resulted in about twofold to three-fold higher levels of secreted active TGF beta 1. Active TGF beta 1-transfected cells grew only slightly, but not significantly, more slowly in vitro and in vivo than vector-only transfectants. Two clones overexpressing active TGF beta 1 secreted much reduced levels of MMP-9 activity, as determined by zymogram analyses. However, treatment of these clones with 40 pM exogenous TGF beta 1 for 48 h enhanced secretion of MMP-9 activity. Constitutive mRNA expression of MMP-9 was reduced twofold to 70-fold in five untreated active TGF beta 1-transfected clones relative to the other transfectants. In contrast, treatment with 40 pM exogenous TGF beta 1 induced MMP-9 mRNA expression in a time-dependent fashion, from twofold to fourfold after 4 h to a maximum of 12- to 19-fold after 24-48 h. Induction of MMP-9 mRNA was dose dependent at TGF beta 1 concentrations of 4-400 pM. Thus, stable transfection of bioactive TGF beta 1 downregulated whereas exogenous TGF beta 1 treatment upregulated MMP-9 activity and expression. Treatment of transfectants with a neutralizing TGF beta 1 antibody slightly downregulated constitutive MMP-9 mRNA (20-30%) but completely blocked induction by exogenous TGF beta 1. Thus, the effect of TGF beta 1 transfection was not due to secreted TGF beta 1 but may have been a secondary effect.     

TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages

Macrophages are present in inflammatory tissue sites where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of macrophages to participate in such matrix destruction, we studied the effects of three cytokines present in inflammatory tissue sites, TNF-alpha, IL-1beta, and IFN-gamma, on the production of three matrix-degrading metalloproteinases, interstitial collagenase, stromelysin, and 92-kDa gelatinase, as well as their natural inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases number 1), by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of interstitial collagenase and stromelysin by these cells was minimal, and was not influenced by the cytokines. In contrast, the cells secreted substantial basal amounts of 92-kDa gelatinase, the secretion of which was stimulated (2- to 15-fold; on average 5-fold) by both TNF-alpha and IL-1beta, while the production of TIMP-1 was unaffected. IFN-gamma suppressed the production of the 92-kDa gelatinase induced by TNF-alpha- and IL-1beta. TNF-alpha and IL-1beta regulated the expression of 92-kDa gelatinase by monocyte-derived macrophages at the pretranslational level. The results show that expression of 92-kDa gelatinase, but not its natural inhibitor TIMP-1, by human tissue-type macrophages is selectively up-regulated by proinflammatory cytokines; which suggests that these cells, when actually present in an inflammatory environment, will actively participate in the destruction of the extracellular matrix.     

Ovarian steroid-modulated stromelysin-1 expression in human endometrial stromal and decidual cells

This study examined steroid-regulated expression of the metalloproteinase stromelysin-1 in primary human endometrial stromal and decidual cells. Immunoblot analysis using a specific polyclonal antibody against stromelysin-1 revealed that the progestin medroxyprogesterone acetate (MPA) produced a time-dependent reduction in a band at 50,000 mol wt. Although the cells were refractory to estradiol (E2) alone, E2 plus MPA further reduced the intensity of this stromelysin-1 zone. By 6 days of incubation, MPA inhibited levels of secreted stromelysin-1 by one third, and E2 plus MPA inhibited stromelysin-1 levels by two thirds compared with the control values. This differential responsiveness of the stromal cells to the two steroids is reported for several biochemical end points of decidualization. Northern analysis indicated pronounced inhibition of stromelysin-1 messenger ribonucleic acid (mRNA) by E2 plus MPA over a concentration range that simulated circulating progesterone levels of the luteal phase (10(-8) mol/L) through pregnancy (10(-6) mol/L). After suppression of stromelysin-1 expression in the stromal cell monolayers by E2 plus MPA, steroid withdrawal led to a several-fold enhancement of stromelysin-1 mRNA by 4 days and of the stromelysin-1 protein by 7 days. Given its actions in degrading several extracellular matrix components and activating other MMP zymogens, steroid withdrawal-enhanced stromelysin-1 activity could mediate a proteolytic cascade that promotes the rapid tissue destruction and vascular disruption associated with menstruation. Stromelysin-1 expression by cultured decidual cells isolated from first trimester endometrium was also reduced by MPA and synergistically reduced by E2 plus MPA. As activation of the 92-kilodalton gelatinase/type IV collagenase, a crucial mediator of trophoblast invasiveness, is stromelysin-1 dependent, reduced decidual stromelysin-1 production could help to limit trophoblast invasion.     

Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

BACKGROUND: Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. METHODS: Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. RESULTS: Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.3, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). CONCLUSIONS: Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.     

Expression of the tissue metalloproteinase inhibitors TIMP-1 and TIMP-2 in malignant fibrous histiocytomas and dermatofibromas as studied by in situ hybridization and immunohistochemistry

The expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was studied in eight malignant fibrous histiocytomas (MFH) and in eight dermatofibromas (DF) using in situ hybridization methods (ISH). Immunohistochemical stainings were also performed using corresponding antibodies to TIMP-1 and TIMP-2. In ISH the neoplastic cells of MFHs showed a high level of expression for both TIMP-1 and TIMP-2 mRNAs. The cells usually expressed similarly both TIMPs, except for osteoclast-like giant cells, which showed a distinct signal for TIMP-2 but not for TIMP-1. A distinctly lower level of both TIMP-1 and TIMP-2 mRNAs was seen in DFs. Immunohistochemical stainings were concordant with the results obtained by ISH. The findings suggest that the behavior of MFHs and DFs is not directly or solely dependent on the quantity of type IV collagenase inhibitors. The increased TIMP synthesis in MFHs might represent a chaotic response of malignant cells to increased matrix degradation. Alternatively, it may reflect a deranged communication between type IV collagenases and TIMPs in malignant tissues.     

Clinical and experimental studies of JPYS in reducing side-effects of chemotherapy in late-stage gastric cancer

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective-tissue-matrix remodelling and the accelerated breakdown associated with tumor development. These MMPs and tissue inhibitor of MMPs (TIMP1) could be expressed by either the cancer or the stromal cells. Expression of mRNAs encoding interstitial collagenase (MMP1), 72-kD type IV collagenase (MMP2) and stromelysin (MMP3), which are probably involved in tumor invasion and metastasis, and of TIMP1 were studied in human mammary pathology by in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells. These data suggest that there is a cooperation between tumor and stromal cells, in particular for the production of 72-kD type IV collagenase, involved in the disruption of basement membranes. A lack of TIMP1 expression from invasive cancer cells would also contribute to matrix destruction.     

Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes

PURPOSE: Matrix metalloproteinases (MMP) are a family of extracellular matrix degrading enzymes associated with the development of neovascularization. To investigate the possible role of these enzymes in choroidal neovascularization, the mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) were analyzed in subfoveal fibrovascular membranes from patients with age-related macular degeneration (AMD). METHODS: Surgically removed subfoveal fibrovascular membranes from five eyes were analyzed for the expression of MMP and TIMP mRNA. In situ hybridization anti-sense and sense riboprobes were generated using DNA complementary to human collagenase (MMP-1), 72 kDa gelatinase (MMP-2), stromelysin (MMP-3), 92-kDa gelatinase (MMP-9), TIMP-1, TIMP-2, and TIMP-3. Vascular endothelial cells were detected using immunostaining for von Willebrand factor. RESULTS: MMP-2 and MMP-9 mRNA were detected in all specimens. Most of the membranes also expressed TIMP-1 and TIMP-3 mRNA, and two of the membranes expressed TIMP-2 mRNA. MMP-2, TIMP-1, and TIMP-2 mRNA had a similar overall distribution that was relatively uniform within the vascularized membrane stroma. MMP-2 expression appeared to be localized mainly to the vascular endothelial cells, whereas TIMP-1 and TIMP-3 were detected in other cell types such as fibroblastlike cells. MMP-9 expression was distinctly expressed by cells at the margins of the membranes and often in proximity to a thickened Bruch's membrane-like layer under the retinal pigment epithelial cells. TIMP-3 mRNA was strongly expressed within the retinal pigment epithelial cell layer and also in the stroma of one membrane. None of the membranes showed detectable MMP-1 or MMP-3 expression. CONCLUSIONS: The results support a role for MMPs in the development of choroidal neovascularization in AMD. The localization of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and MMP-9 may be cooperatively involved in the progressive growth of choroidal neovascular membranes in AMD.     

Effects of TIMP-2 gene transfection on biological behaviors of a metastatic human lung carcinoma cell line

OBJECTIVES: To explore the suppressive effects of tissue inhibitor of metalloproteinase 2 (TIMP-2) on malignant phenotype of human carcinoma cells and to evaluate its potential application in cancer gene therapy. METHODS: A man malian expression vector containing TIMP-2 cDNA was constructed and transfected into a metastatic human lung carcinoma cell line PG. In vitro and in vivo tests such as Northern blotting, immunohistochemistry as well as x enografting in nude mice experiment were used to analyse expression levels of TIMPs and MMPs, in vitro and in vivo behaviors of the tumor cells before and after the gene transfection. RESULTS: After transfection, the TIMP-2 mRNA expression was upregulated significantly. Changes, in some malignant phenotypes of the transfectants were seen. For instance, the abilities of in vitro invasion through Matrigel, colony formation on soft agar, tumorigenecity as well as spontaneous metastasis in nude mice were remarkably decreased. Immunohistochemical staining and in situ hybrydization showed that MMP2, MMP9, TIMP-1 and TIMP-2 were expressed by both tumor cells and stromal cells, with stronger staining at the site of tumor invasion. CONCLUSION: Up-regulation of TIMP-2 in tumor cells could suppress their expression of malignant phenotype and could be used for cancer therapy.     

Frequency of grandparent contact with grandchild sets: six factors that make a difference

PURPOSE: We hypothesize that regulated trabecular extracellular matrix (ECM) turnover, initiated by the matrix metalloproteinases, is critical for the maintenance of normal aqueous humor outflow rates. However, very little is known about the regulation of trabecular ECM turnover. To identify candidate trabecular regulators, we evaluated the effects of several growth factors and cytokines on trabecular matrix metalloproteinase and TIMP expression. METHODS: Porcine trabecular meshwork cells were treated with several doses of a variety of growth factors and cytokines and culture media was analyzed after 24, 48, and 72 h. Zymograms were used to evaluate stromelysin, gelatinase A and B activity levels, while immunoblots of Western transfers were used to evaluate stromelysin, collagenase, TIMP-1 and TIMP-2 protein levels. RESULTS: A phorbol mitogen (TPA), and TNF alpha and beta, interleukin-1 alpha and PDGF BB stimulate gelatinase B, stromelysin, interstitial collagenase and TIMP-1 expression, while having negligible effects on gelatinase A expression; TIMP-2 levels are reduced by TNF but not affected by the other treatments. Acidic and basic FGF, IL-1 beta, TGF beta and PDGF AB produce similar but smaller effects, while HGF, VEGF, EGF, KGF, and LIF produce small to moderate elevations in stromelysin with minimal other responses. PDGF AA, gamma INF, oncostatin-M and endothelin-1 produce negligible changes in these proteinases and inhibitors. CONCLUSIONS: In addition to providing potential ways to modulate trabecular metalloproteinase and TIMP levels, the responsiveness of these cells to some of these growth factors and cytokines suggests possible roles in normal or pathogenic trabecular cell regulation and some may affect aqueous humor outflow.     

Cytogenetic analysis of sister chromosome sets in the second polar body and in pronuclei of unicellular mouse embryos. I. Frequency and origin of aneuploidy in embryos heterozygous for the reciprocal chromosome translocation T[14;15]6Ca]

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.     

Influence of heparin(s) on the interleukin-1-beta-induced expression of collagenase, stromelysin-1, and tissue inhibitor of metalloproteinase-1 in human gingival fibroblasts

Here, we describe the influence of heparin(s) on the interleukin-1-beta (IL-1beta)-induced expression of collagenase (matrix metalloproteinase-1, MMP-1), stromelysin-1 (matrix metalloproteinase-3, MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in human gingival fibroblasts (HGF). Amounts of secreted enzymes and inhibitors as well as their mRNA steady-state levels increased significantly following supplementation of HGF culture medium with 2 ng/mL of IL-1 beta1. Addition of heparin to cell culture medium 1 hour following IL-1beta decreased MMP and TIMP-1 expression in a dose-dependent manner. The inhibitory effect of heparin was significant at a concentration as low as 1 microg/mL. These findings could be reproduced with a low Mr heparin fragment devoid of anticoagulant activity. Heparin and fragments might therefore reduce the excessive proteolytic capacity of the gingival fibroblast during inflammation and could be useful as pharmacological agent(s) in gingivitis and periodontitis.     

Cytokine (IL-10, IL-6) induction of tissue inhibitor of metalloproteinase 1 in primary human prostate tumor cell lines

Northern blots and scintillation counting showed that tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA was expressed by low passage, primary epithelial cultures (n = 5) of low-grade human prostatic carcinoma. TIMP-1 mRNA levels were normally low in the primary cell lines, but were inducible by interleukin (IL) 10 and 6. Dose and time-course studies indicated that IL-10 was the most potent stimulator of TIMP-1 expression. Cycloheximide blocked the effects of IL-10 in a reversible manner. In situ hybridization assays with TIMP-1 oligonucleotide antisense probes confirmed the northern blot results and indicated that IL-10 preferentially stimulated TIMP-1 mRNA synthesis. We suggest that IL-10, and to a lesser extent IL-6, may normally influence TIMP-1 expression by human prostatic epithelial cells.     

A search for uniparental disomy in carriers of supernumerary marker chromosomes

Dietary supplementation with 4% eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid, suppressed the development of lung metastases in nude mice from MDA-MB-435 human breast cancer cell mammary fat pad solid tumors. Zymography of primary tumor homogenates showed that this inhibition of metastasis was associated with reduced levels of 92-kDa type IV collagenase gelatinolytic activity; this isoform has been previously associated with the metastatic phenotype. The 72-kDa isoform, which was also present, may have arisen from the cancer cells, or tumor-associated host cells. In vitro, the incorporation of EPA, 0.25-1.0 micrograms/ml into the medium caused a concentration-related suppression of cultured MDA-MB-435 cell 92-kDa type IV collagenase mRNA expression.     

Type IV collagenase (M(r) 72,000) expression in human prostate: benign and malignant tissue

The expression of type IV collagenase (M(r) 72,000) has been examined in tissues from patients with benign prostatic hyperplasia (6 patients) and varying Gleason grades of malignant prostate cancer (18 patients). Immunoperoxidase labeling indicated that expression of the type IV collagenase was weak or nonexistent in benign tissue but consistently strong in the glandular and ductal epithelial cells of prostate tumors diagnosed at Gleason grades 1-8. In moderate to advanced cancer (i.e., Gleason grades 2 to 8), invasive tumor foci in the stromal tissue produced relatively modest amounts of type IV collagenase. The normal stromal tissue (i.e., fibroblasts) uniformly failed to produce detectable levels of type IV collagenase in the 24 patients examined. Northern and quantitative slot blot hybridization assays demonstrated that collagenase type IV mRNA levels were low in benign tissue and high in malignant tumors. In contrast, the stromal cells did not express significant amounts of type IV collagenase mRNA. Enzyme-linked immunosorbent assays demonstrated that the amounts of type IV collagenase protein correlated directly with the mRNA levels in the tumor tissue. The studies suggest that type IV collagenase may be selectively overexpressed by malignant, preinvasive prostatic epithelial cells.     

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.