Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An imbalance between the activity of matrix metalloproteinases (MMPs) (proteolytic enzymes that degrade protein components of the extracellular matrix) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), may be one of the mechanisms responsible for tumor cell invasion. We have investigated the regulation of MMP-1 and TIMP-1 gene expression in benign and malignant (follicular, anaplastic, and papillary) human thyroid cells. As expected of cells with invasive potential, detectable MMP-1 messenger RNA (mRNA) levels were observed in malignant cells under basal conditions, in contrast to undetectable levels in benign cells. Exposure of these cells, for 1 h, to the active phorbol ester, phorbol 12-myristate 13-acetate (TPA, 100 nmol/L), acting via protein kinase C (PKC), elicited an increase in MMP-1 mRNA, with a peak stimulation after a 3- to 4-h culture period. Epidermal growth factor (EGF, 25 ng/mL), however, acting via protein tyrosine kinase (PTK), stimulated such gene expression in malignant cells but failed to do so in benign cells. TIMP-1 mRNA was not significantly altered by the TPA-PKC, EGF-PTK, or TSH-protein kinase A (PKA) pathways in malignant cells. In benign cells, however, TPA induced a small, though significant, increase in TIMP-1. The MMP-1 stimulation by EGF and lack of TPA-induced rise in TIMP-1 in malignant cells, in sharp contrast to the effects obtained in benign thyrocytes, seems to indicate that the MMP: TIMP balance favors a more extensive extracellular matrix protein breakdown by malignant thyrocytes, as expected of cells exhibiting invasive capacity. TSH (10-500 microU/mL) failed to significantly influence basal MMP-1 or TIMP-1 mRNA levels, but it caused a dose-dependent inhibition in TPA- and EGF-induced MMP-1 mRNA in malignant cells, and TPA-stimulated MMP-1 and TIMP-1 in benign cells. The repressive action of TSH on MMP-1 mRNA was mimicked by forskolin and 8-bromo-cAMP and was abrogated by the PKA inhibitor, H-89, suggesting that the TSH inhibitory action is PKA-mediated. In conclusion, the present study provides novel data on MMP-1 and TIMP-1 gene expression and their modulation by the major signal transduction pathways operating in human thyroid cells. Similar and divergent patterns have emerged in the regulation of such gene expression in benign and malignant human thyrocytes, in many instances in accord with the concept of MMP playing the role of stimulating, and TIMP inhibiting, cell invasion. Although MMP-1 may be just one of the many factors responsible for tumor cell invasion, the present findings demonstrating the possibility, at least in vitro, of repressing MMP gene expression may have important clinical ramifications.
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PMID:Similar and divergent patterns in the regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 gene expression in benign and malignant human thyroid cells. 1048 6

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.
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PMID:Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat. 1091 13

Human breast cancer primary cultures are useful tools for the study of several aspects of cancer biology, including the effects of chemotherapy and acute gene expression in response to different hormonal/chemotherapy treatments. The present study reports the conditions for primary culture of breast cancer samples from untreated patients and the most effective collagenization method to dissociate human samples consisting in an overnight incubation with 1 mg/ml types II or IV collagenase and further incubation in DMEM:F12 (1:1) medium supplemented with glutamine, bovine insulin, penicillin-streptomycin, HEPES, estradiol, cortisol (F), tri-iodothyronine (T(3)), transferrine (TR), and 10% fetal calf serum (FCS). These conditions proved to be appropriate for both primary culture and the development of stable cell lines. Of the seven cell lines obtained, three fast growing and estrogen receptor (ER)+/progesterone receptor (PgR)+/EGF receptor (EGFR)+ have been characterized. The cells are able to grow both in soft agar and in nude mice, and express cytokeratins, all parameters characteristic of malignant epithelial cell lines. The cells also exhibit an increased proliferation rate in the presence of estradiol, progesterone, and EGF, suggesting the presence of the corresponding receptors. The mRNA expression of type alpha- and beta-ER as well as EGFR, was confirmed by RT-PCR. In conclusion, the novel cell lines described, arose from primary tumors and are sensitive to estradiol, progesterone, and EGF. This not only expands the repertoire of breast cancer cells available as potentially useful tools for examining most parameters in breast cancer "in vitro", but also provides unique new models to explore the complex regulation by steroids as well as growth factors in such cells.
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PMID:Three novel hormone-responsive cell lines derived from primary human breast carcinomas: functional characterization. 1509 93

EGF and type I collagen are known to play important roles in wound healing. In the present study, we demonstrated that EGF down-regulates the expression of type I procollagen protein as well as alpha2(I) collagen mRNA in cultured human dermal fibroblasts. EGF induced the degradation of type I procollagen protein in conditioned medium through the up-regulation of MMP-1 expression. EGF down-regulated alpha2(I) mRNA expression partially at the post-transcriptional level by reducing the mRNA stability. In contrast, EGF up-regulated MMP-1 mRNA expression mostly at the transcriptional level, in that it had a stimulatory effect on MMP-1 promoter activity, but no effect on MMP-1 mRNA stability. The MEK/ERK signaling pathway was shown to be involved in EGF-mediated type I collagen and MMP-1 expression.
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PMID:Epidermal growth factor affects the synthesis and degradation of type I collagen in cultured human dermal fibroblasts. 1641 67

Wound fluids, human serum from platelet-poor and platelet-rich plasma (SPPP and SPRP), contain various soluble factors involved in cell growth and proliferation. Levels of cytokines, chemokines, and matrix metalloproteinases (MMPs) in drainage fluids (DFs) harvested from subcutaneous wounds, punctured fluids (PF) from seroma, and SPPP were measured. SPPP and SPRP from four healthy volunteers were also subjected to the analysis. Biochemical profiles of DF reflected the sequential stages of wound healing. Early-phase DF contained high concentrations of basic fibroblast growth factor and platelet-derived growth factor and EGF. The levels of keratinocyte growth factor, interleukin-6, and MMP-8 in DF peaked on days 2-3, while vascular endothelial growth factor, hepatocyte growth factor, interleukin-8, and MMP-1 increased over time during days 0-6. Punctured fluids contained high levels of TGF-beta1, keratinocyte growth factor, vascular endothelial growth factor, hepatocyte growth factor, and MMP-1. Experiments using human adipose-derived stem cells and dermal fibroblasts cultured in media containing various concentrations of DF and fetal bovine serum suggested that for some cell types, DF-contained growth factors that are not obtained from SPRP could be used to supplement or substitute for serum in culture media. SPRP and DF are economical ready-made mixtures of serum and autologous soluble factors, and may be differentially useful for regenerative therapies.
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PMID:Characterization of wound drainage fluids as a source of soluble factors associated with wound healing: comparison with platelet-rich plasma and potential use in cell culture. 1765 95

The acinar cell culture plays a very important role in research of pancreatic pathophysiology. The aim of this study was to establish a long-term culture of human (foetal) pancreatic acinar cells in standardized nutrient media with supplements. Acinar cells were prepared from pancreatic tissues obtained from aborted foetus (> or =35 weeks) with no prior pancreatic complications by collagenase digestion and cultured using different media and supplements. The purity and phenotype of acinar cells was confirmed by various staining techniques and FACS. The acinar cell proliferation was determined at different time intervals by Bromo-deoxyuridine (BrdU) incorporation, and metabolic enzyme activity was analysed. The acini could be cultured and maintained in Ham's F-12 K/M199 media in the presence of 5% BSA, 0.1 mg/ml STI, 10 ng/ml EGF, and 10% FCS with the same morphological appearance as that of freshly prepared for 12 days with maximum viability of 80-85% and formation of monolayer without extracellular matrix. A significant BrdU incorporation of acinar cells in primary culture was observed which was maximum (105%) at day four. Higher amylase and lipase activity was seen in freshly isolated acinar cells which decreased with time of the culture. The established human pancreatic acinar cell culture may act as an excellent model to study exocrine dysfunction or pancreatitis in response to acinar cell injury.
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PMID:Primary culture of pancreatic (human) acinar cells. 1824 27

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered--inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca2+ content formed monolayers, which after elevation of Ca2+ produced multilayers with cells containing keratohyalin-like granules. Ca2+ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca2+ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.
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PMID:Keratinization of outer root sheath cells is prevented by contact with inner root sheath of rat hair follicles. 1864 26

Rat small hepatocytes (SHs) are committed progenitor cells that can differentiate into mature hepatocytes and can selectively proliferate in serum-free medium when they are cultured on hyaluronic acid (HA)-coated dishes. In this study we examined the separation of human SHs from adult human livers. We obtained liver tissues from the resected liver of 16 patients who underwent hepatic resections. Extracted liver specimens were clearly separate from the tumor regions with sufficient margins. Hepatic cells were isolated using the modified method of two-step collagenase perfusion. A low-speed centrifugation was performed and cells in the supernatant were finally cultured on HA-coated dishes in serum-free DMEM/F12 medium including nicotinamide, EGF, and HGF. Small-sized hepatocytes selectively proliferated to form colonies and many colonies continued growing for more than 3 weeks. The average number of cells in a colony was 38.6 +/- 18.0, 79.0 +/- 54.0, and 101.5 +/- 115.7 at day 7, 14, and 21, respectively. About 0.04% of plated cells could form an SH colony. Immunocytochemistry showed that the cells forming a colony were positive for albumin, transferrin, keratin 8, and CD44. The results of RT-PCR showed that colony-forming cells expressed albumin, transferrin, alpha1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3alpha, HNF4alpha, C/EBPalpha, and C/EBPbeta). Furthermore, the cells expressed not only the genes of hepatic differentiated functions but also those of both hepatic stem cell marker (Thy1.1, EpCAM, AFP) and SH marker (CD44, D6.1A, BRI3). Albumin secretion into culture medium was also observed. Our results demonstrate the existence of hepatocyte progenitor cells in human adult livers, and the cells can grow in a serum-free medium on HA-coated dishes. Human SHs may be a useful source for cell transplantation as well as pharmaceutical and toxicological investigations.
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PMID:Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium. 1918 Dec 16

The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y(573)F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics.
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PMID:Epidermal growth factor receptor-mediated membrane type 1 matrix metalloproteinase endocytosis regulates the transition between invasive versus expansive growth of ovarian carcinoma cells in three-dimensional collagen. 1950 14

We have shown previously that the vasoactive peptide bradykinin (BK) stimulates proliferation of a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) via transactivation of epidermal growth factor receptor (EGFR) by a mechanism that involves matrix metalloproteinases (collagenase-2 and -3). Because collagenases lack an integral membrane domain, we hypothesized that receptors for extracellular matrix proteins, integrins, may play a role in BK-induced signaling by targeting collagenases to the membrane, thus forming a functional signaling complex. BK-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) in mIMCD-3 cells was reduced by approximately 65% by synthetic peptides containing an Arg-Gly-Asp sequence, supporting roles for integrins in BK-induced signaling. Neutralizing antibody against alpha5beta1 integrin partially (approximately 60%) blocked BK-induced ERK activation but did not affect EGF-induced ERK activation. Silencing of alpha5 and beta1 expression by transfecting cells with small interfering RNAs (siRNA) significantly decreased BK-induced ERK activation (approximately 80%) and EGFR phosphorylation (approximately 50%). This effect was even more pronounced in cells that were cotransfected with siRNAs directed against both collagenases and alpha5beta1 integrin. On the basis of our results, we suggested that integrin alpha5beta1 is involved in BK-induced signaling in mIMCD-3 cells. Using immunoprecipitation/Western blotting, we demonstrated association of BK B(2) receptor with alpha5beta1 integrin upon BK treatment. Furthermore, BK induced association of alpha5beta1 integrin with EGFR. These data provide the first evidence that specific integrins are involved in BK B(2) receptor-induced signaling in kidney cells, and ultimately might lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.
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PMID:Bradykinin B2 receptor interacts with integrin alpha5beta1 to transactivate epidermal growth factor receptor in kidney cells. 2038 9


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