CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration.
(1/1501)
CD44 is a cell surface receptor for hyaluronate, a component of the extracellular matrix (ECM). Although CD44 has been implicated in tumor invasion and metastasis, the molecular mechanisms remain to be elucidated. Here we find that CD44 expressed in cancer cells is cleaved at the membrane-proximal region of the ectodomain and the membrane-bound cleavage product can be detected using an antibody against the cytoplasmic domain of CD44. Furthermore, we report that CD44 cleavage is mediated by a membrane-associated metalloprotease expressed in cancer cells. A tissue inhibitor of metalloproteases-1 (TIMP-1), as well as metalloprotease inhibitors, inhibit CD44 cleavage in the cell-free assay. Contrary, serine protease inhibitors enhance CD44 cleavage, and the enhancement can be prevented by pretreatment with a metalloprotease inhibitor. Thus, CD44 cleavage is regulated by an intricate balance between some proteases and their inhibitors. Interestingly, treatment with the metalloprotease blocker 1,10-phenanthroline, which strongly prevent the CD44 cleavage, suppressed RERF-LC-OK lung cancer cell migration on a hyaluronate substrate, but not on several other substrates. These results suggest that CD44 cleavage plays a critical role in an efficient cell-detachment from a hyaluronate substrate during the cell migration and consequently promotes CD44-mediated cancer cell migration. Our present data indicate that CD44, not only ECM per se, is one of the targets of pericellular proteolysis involved in tumor invasion and metastasis. (+info)
Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages.
(2/1501)
BACKGROUND: Macrophages in human atherosclerotic plaques produce a family of matrix metalloproteinases (MMPs), which may influence vascular remodeling and plaque disruption. Because oxidized LDL (ox-LDL) is implicated in many proatherogenic events, we hypothesized that ox-LDL would regulate expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in monocyte-derived macrophages. MWRHOSA AND RESULTS: Mononuclear cells were isolated from normal human subjects with Ficoll-Paque density gradient centrifugation, and adherent cells were allowed to differentiate into macrophages during 7 days of culture in plastic dishes. On day 7, by use of serum-free medium, the macrophages were incubated with various concentrations of native LDL (n-LDL) and copper-oxidized LDL. Exposure to ox-LDL (10 to 50 microg/mL) increased MMP-9 mRNA expression as analyzed by Northern blot, protein expression as measured by ELISA and Western blot, and gelatinolytic activity as determined by zymography. The increase in MMP-9 expression was associated with increased nuclear binding of transcription factor NF-kappaB and AP-1 complex on electromobility shift assay. In contrast, ox-LDL (10 to 50 microg/mL) decreased TIMP-1 expression. Ox-LDL-induced increase in MMP-9 expression was abrogated by HDL (100 microg/mL). n-LDL had no significant effect on MMP-9 or TIMP-1 expression. CONCLUSIONS: These data demonstrate that unlike n-LDL, ox-LDL upregulates MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages. Furthermore, HDL abrogates ox-LDL-induced MMP-9 expression. Thus, ox-LDL may contribute to macrophage-mediated matrix breakdown in the atherosclerotic plaques, thereby predisposing them to plaque disruption and/or vascular remodeling. (+info)
Luteinization and proteolysis in ovarian follicles of Meishan and Large White gilts during the preovulatory period.
(3/1501)
This experiment was conducted to determine why follicles luteinize faster in the Meishan breed than in the Large White breed of pig. Follicles were recovered during the late follicular phase from ovaries of both breeds before and after administration of hCG given to mimic the LH surge. First, the patterns of cholesterol transporters (high and low density lipoproteins: HDL and LDL) were compared. Cholesterol transporters detected in follicular fluid consisted of HDL only. Similar amounts of Apolipoprotein A-I were found in all samples. There was no obvious breed effect on minor lipoproteins found in the HDL-rich fraction, and this pattern was altered similarly by hCG in the two breeds. The LDL-rich samples of serum from both breeds contained similar amounts of protein. Second, three steroidogenic enzymes, adrenodoxin, 17 alpha-hydroxylase-lyase (P450(17) alpha) and 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) were detected by immunohistochemistry and quantified by image analysis on sections of the two largest follicles. Before hCG treatment, theca interna cells demonstrated immunoreactivities for adrenodoxin (strong), P450(17) alpha and 3 beta-HSD (very strong), whereas granulosa cells displayed immunoreactivities for adrenodoxin only. After hCG treatment, the localization of the enzymes was unchanged but the staining intensity of adrenodoxin on granulosa cells and 3 beta-HSD on theca cells increased (P < 0.01 and P < 0.05, respectively). Breed effects were detected for the amounts of adrenodoxin in theca cells (Meishan > Large White; P < 0.05) and of 17 alpha-hydroxylase (Large White > Meishan, P < 0.01). Breed x treatment interactions were never detected. Finally, gelatinases, plasminogen activator, plasminogen activator inhibitor, tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2) were visualized by direct or reverse zymography or western blotting. Whatever the stage relative to LH administration, follicular fluid from Large White gilts contained more TIMP-1, and TIMP-2 (P < 0.02 and P < 0.01, respectively). No breed effect was detected for the amounts of gelatinases and plasminogen activator inhibitor 1. However, for these parameters, a significant breed x time interaction was obvious, as the Meishan follicles had a greater response to hCG (P < 0.01). Since proteolysis plays a key role in the bioavailability of growth factors such as insulin-like growth factor 1, fibroblast growth factor and transforming growth factor beta, which have the ability to alter gonadotrophin-induced progesterone production in pigs, the differences observed in its control in the present study may explain, at least in part, the different patterns of luteinization observed in Meishan and Large White follicles. (+info)
Serum gelatinase B, TIMP-1 and TIMP-2 levels in multiple sclerosis. A longitudinal clinical and MRI study.
(4/1501)
Metalloproteinases have been implicated in the pathogenesis of multiple sclerosis. We report longitudinal serum levels of gelatinase B and of the tissue inhibitors of matrix metalloproteinases (TIMP), TIMP-1 and TIMP-2, in 21 patients with relapsing multiple sclerosis. Patients had monthly clinical and gadolinium-enhanced MRI follow-up for 10 months. Longitudinal samples in nine healthy controls and cross-sectional samples from 12 patients with inflammatory CNS disease and 15 patients with other neurological diseases were used for comparison. Average serum gelatinase B, TIMP-1 and TIMP-2 levels were significantly higher in multiple sclerosis patients and those with other neurological diseases than in healthy controls. In the patients with multiple sclerosis, gelatinase B levels were significantly higher during clinical relapse compared with periods of clinical stability. Multiple sclerosis patients with high mean serum gelatinase B levels had significantly more T1-weighted gadolinium-enhancing MRI lesions than those with mean levels within the control range. TIMP-1 levels were not different during relapse and between relapses. There was a trend for TIMP-2 levels to be lower during relapse compared with non-relapse periods. For similar levels of serum gelatinase B, associated TIMP-1 levels were significantly lower and TIMP-2 levels significantly higher in multiple sclerosis patients compared with the inflammatory CNS control group. We propose that an abnormality in the inhibitory response to metalloproteinases may play an aetiological role in the chronicity of multiple sclerosis. (+info)
Expression of matrix metalloproteinases during murine chorioallantoic placenta maturation.
(5/1501)
A large body of experimental evidence supports the participation of two groups of extracellular proteases, matrix metalloproteinases (MMPs), and plasminogen activators/plasmin, in tissue remodeling in physiological and pathological invasion. In the late mouse placenta, several tissue remodeling and cell invasion processes take place. Spongiotrophoblast migration into maternal decidua, as well as decidual extracellular matrix remodeling require the coordinated action of extracellular proteolytic enzymes. Via Northern and in situ hybridization, we have analyzed the spatio-temporal expression patterns of members of the MMP family (stromelysin-3, gelatinases A and B), as well as their inhibitors TIMP-1, -2 and -3 in late murine placenta (days 10.5 to 18.5 of gestation). Gelatinase activity in placental extracts was assessed by substrate zymography. Gelatinase A and stromelysin-3 were found to be prominently expressed in decidual tissue; shortly after midpregnancy, the decidual expression patterns of gelatinase A and stromelysin-3 became overlapping with each other, as well as with the expression domain of TIMP-2. On the other hand, gelatinase B transcripts were expressed only by trophoblast giant cells at day 10.5, and were downregulated at later stages. TIMP-1 and TIMP-3 transcripts were detected in decidual periphery at day 10.5, while later the expression was restricted to the endometrial stroma and spongiotrophoblasts, respectively. The areas of stromelysin-3 expression were the same (giant trophoblasts) or adjacent (decidua) to those where urokinase (uPA) transcripts were detected, suggesting a possible cooperation between these proteinases in placental remodeling. We generated mice doubly deficient for stromelysin-3 and uPA, and report here that these mice are viable and fertile. Furthermore, these animals do not manifest obvious placental abnormalities, thereby suggesting the existence of compensatory/redundant mechanisms involving other proteolytic enzymes. Our findings document the participation of MMPs and their inhibitors in the process of late murine placenta maturation, and warrant the characterization of other members of the MMP family, like membrane type-MMPs, in this process. (+info)
Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases-1 in breast cancer cells.
(6/1501)
HER2/neu, a Mr 185,000 tyrosine kinase receptor that is overexpressed in breast cancer, undergoes proteolytic cleavage of its extracellular domain (ECD). In contrast with other membrane-bound proteins, including growth factor receptors, that are cleaved by a common machinery system, we show that HER2 cleavage is a slow process and is not activated by protein kinase C. Pervanadate, a general inhibitor of protein-tyrosine phosphatases, induces a rapid and potent shedding of HER2 ECD. The shedding of HER2 ECD is inhibited by the broad-spectrum metalloprotease inhibitors EDTA, TAPI-2, and batimastat. The tissue inhibitor of metalloproteases-1; an inhibitor of matrix metalloproteases that does not inhibit cleavage by the general protein kinase C-dependent shedding machinery, also inhibited HER2 ECD shedding, whereas tissue inhibitor of metalloproteases-2 did not. These data suggest that HER2 cleavage is a process regulated by an as-yet-unidentified distinct protease. (+info)
Tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA is elevated in advanced stages of thyroid carcinoma.
(7/1501)
Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor. (+info)
Role of interleukin 10 and transforming growth factor beta1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice.
(8/1501)
Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis. (+info)