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PubMedDate not supplied

Differential regulation of stromelysin-1 (matrix metalloproteinase-3) and matrilysin (matrix metalloproteinase-7) in baboon endometrium.

OBJECTIVE: To investigate in vivo expression of matrix metalloproteinase enzymes (MMP), MMP-3 and MMP-7, by the baboon endometrium in relation to the process of tissue remodeling that accompanies menstruation in the primate. METHODS: Endometrial tissues and uterine fluids obtained from reproductively intact cycling baboons, cycling baboons treated with an anti-progestin, and ovariectomized steroid-treated baboons were evaluated for MMP mRNA expression and protein production by using multiple analytic techniques. RESULTS: The pattern of MMP protein production matched the pattern of MMP mRNA expression. In intact cycling baboons, MMP mRNA expression and protein production were specific to cell type and stage of the menstrual cycle. Endometrial stroma expressed minimal amounts of MMP-3 during the proliferative and secretory stages, whereas endometrial epithelia expressed MMP-7 during the proliferative stage. Expression of stromal MMP-3 and epithelial MMP-7 increased during early menses. Administration of anti-progestin beginning on the day of the LH surge increased MMP-7 but not MMP-3 expression at the mid secretory phase. Expression of MMP-3 and MMP-7 in the ovariectomized steroid-treated animals paralleled that of the intact cycling animals. CONCLUSIONS: These results highlight the similarity of human and baboon endometrial MMP-3 and MMP-7 production, providing further evidence for use of the baboon as a model for studying events associated with hormonally regulated changes during the menstrual cycle. These results also demonstrate that endometrial epithelial MMP-7 is suppressed by progesterone in the baboon endometrium, but endometrial stromal MMP-3 is regulated by a different mechanism.

Amino Acid Sequence↗
PubMed2000

Tumor cell-matrix interaction: pericellular matrix degradation and metastasis.

In order to determine key MMPs for invasion and metastasis in various human cancers, we examined the expression of ten MMPs (MMP-1, 2, 3, 7, 8, 9, 13 and MT1, 2, 3-MMPs) and tissue inhibitors of metalloproteinases (TIMP-1 and 2) in breast carcinomas, thyroid papillary carcinomas, endometrial carcinomas, ovarian carcinomas, gastric adenocarcinomas, oral squamous cell carcinomas and gliomas. Of the MMPs examined, the activation of proMMP-2 by MT1-MMP (membrane type 1-MMP) was commonly important for the invasion and metastasis of these cancers except for endometrial carcinomas. The MMP-2 and MT1-MMP were localized to the carcinoma cells and gelatinolytic activity was demonstrated within the carcinoma cell nests by in situ zymography. In endometrial carcinomas, production and activation of proMMP-7 were a key determinant of the lymph node metastasis. The activation of proMMP-2 in gliomas involved MT2-MMP as well as MT1-MMP, and a combination of decreased TIMP-2 production and enhanced MT1-MMP expression was important in the subarachnoidal dissemination of glioblastoma cells. Brevican, a major adult brain proteoglycan, was degraded with MMP-1, 2, 3, 7, 10 and ADAMTS4 (aggrecanase-1) by being cleaved at the MMP site (the Ala360-Phe361 bond) with the MMPs and ADAM site (the Glu395-Ser396 bond) with ADAMTS4. Since activated MMP-2 and ADAMTS4 are present in human glioma tissues, they may play a key role in the invasion of glioma cells through the brevican degradation. The data in the present study suggest that the extracellular matrix-degrading metalloproteinases acting probably on the cell membranes of cancer cells are essential to the invasion and metastasis of human cancers.

Extracellular Matrix↗
PubMedDate not supplied

Relationship between expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 and invasion ability of cervical cancer cells.

Constitutive overexpression of matrix metalloproteinases (MMPs) is frequently observed in malignant tumors. MMPs are a family of zinc endopeptidases consisting of at least 20 different members. In particular, MMP-2 and MMP-9 are reported to be closely associated with invasion and metastasis in several cancers. We investigated whether expression of MMP-2 and MMP-9 is associated with invasion ability of seven cervical cancer cells by administration of o-phenanthroline as MMP inhibitor. In two cell lines, Siha and Caski, MMP-2 mRNA and protein were expressed at high levels. After treatment with o-phenanthroline, the rate of invasion in these two cell lines was significantly decreased. In contrast, in the other two cell lines, HT-3 and Caski, high levels of MMP-9 mRNA and protein were expressed but there was no decrease in the rate of invasion in these cells after treatment with o-phenanthroline. The data suggest that expression level of MMP-2 mRNA may regulate with invasion ability of cervical cancer.

Female↗
PubMed1989

Structure of the gene for cartilage matrix protein, a modular protein of the extracellular matrix. Exon/intron organization, unusual splice sites, and relation to alpha chains of beta 2 integrins, von Willebrand factor, complement factors B and C2, and epidermal growth factor.

The entire gene for chicken cartilage matrix protein (CMP) has been isolated and characterized by restriction mapping, electron microscopy, nuclease S1 mapping, and sequence analysis. The gene, which is present in a single copy in the chicken genome, is 18 kilobase pairs long and comprises eight exons and seven introns. It has two transcription initiation sites, 8 base pairs from each other. A sequence very homologous to the consensus nuclear factor III binding-site sequence, a CAT- and a TATA-like sequence are found in the promoter region and ATTAAA is used as a polyadenylation signal. The nucleotide sequence defines a primary translation product of 493 amino acids which consists of a 23-amino acid signal peptide and two large repeated domains connected by an epidermal growth factor module. Amino acid sequences homologous to those of the repeated domains are present in the type A repeats of von Willebrand factor, complement factors B and C2, and in the alpha chains of the integrins Mac-1, p150,95, and LFA-1. The exon-intron structure indicates that the CMP gene may have arisen by exon duplication and exon shuffling during evolution. The GT-AG splice rule cannot be applied for the excision of the last intron of the CMP pre-mRNA. The donor splice site of intron G is basically different from the consensus sequence indicating that a novel type of splicing mechanism might exist in cartilage.

Amino Acid Sequence↗
PubMed1996

Membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation in primary human ovarian epithelial carcinoma cells.

Metastatic dissemination of epithelial ovarian carcinoma occurs primarily through exfoliation of cells from the primary tumor, with subsequent implantation, invasion, and growth throughout the organs within the peritoneal cavity. Previous studies have suggested a role for matrix metalloproteinases (MMPs), particularly MMP-2, in ovarian cancer invasion and metastasis. To characterize further the role of MMPs and their inhibitors in ovarian carcinoma, in this study the production and activation of MMPs by short-term primary cultures of human ovarian epithelial carcinoma cells were analyzed. We report that MMP-2 is the predominant gelatinolytic MMP secreted by primary ovarian cancer cells derived from both ovarian tumors and ascites fluid. Furthermore, zymographic analysis demonstrated that MMP-2 is present in conditioned media in both the latent and activated forms, indicating that primary ovarian cancer cells catalyze proMMP-2 activation. Presence of a proMMP-2 activator was confirmed by immunohistochemistry and immunoprecipitation studies which found membrane-type 1 MMP (MT1-MMP) in the membranes of unstimulated cells and levels of both MT1-MMP and tissue inhibitor of metalloproteinases-2 (TIMP-2) were enhanced by culturing cells in the presence of concanavalin A. In addition, interaction of MMP-2 with the ovarian carcinoma cell surface resulted in a 2.5- to 5-fold increase in invasiveness. These data suggest that MT1-MMP-catalyzed activation of proMMP-2 may play a physiologic role in intraperitoneal invasion of ovarian carcinoma cells.

Carcinoma↗
PubMed1997

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells.

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA-induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 microg/ml), with optimal effects seen at 25 microg/ml orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1-MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.

Concanavalin A↗
PubMed1996

Enhanced production of matrix metalloproteinases and activation of matrix metalloproteinase 2 (gelatinase A) in human gastric carcinomas.

We examined the production and tissue localization of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gastric carcinoma tissues. MMP-1 (tissue collagenase), MMP-9 (gelatinase B) and TIMP-2 were immunolocalized in carcinoma cells and MMP-2 (gelatinase A) on tumor cell membranes, whereas no or little immunostaining for MMP-3 (stromelysin-1) and TIMP-1 was seen in carcinoma cells. Stromal cells in carcinoma tissue were also positively stained for these MMPs and TIMPs. MMP-2 immunostaining was observed exclusively on advanced gastric carcinoma cells and correlated with vascular invasion by tumor cells. Sandwich enzyme immunoassays revealed enhanced production of MMP-1, MMP-2, MMP-3, MMP-9 and TIMP-1 by carcinoma tissues. Gelatinolytic activities were significantly higher in carcinoma samples than in normal controls. Using gelatin zymography, active forms of MMP-2 and MMP-9 were more frequently detected in carcinoma tissue, and the activation rate of the zymogen of MMP-2 (proMMP-2), but not that of proMMP-9, correlated well with degree of local invasion and lymphatic permeation. Our data indicate an enhanced production of 4 MMPs in gastric carcinoma tissue and suggest that activation of pro-MMP-2 may be a key step for spreading of gastric carcinoma cells.

Enzyme Activation↗
PubMed1997

Overexpression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 in liver from patients with gastrointestinal adenocarcinoma and no detectable metastasis.

Degradation of basement membranes is a key step in tumoral invasion, mainly mediated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Since the liver is a main target for metastases from gastrointestinal adenocarcinoma, we have investigated MMP2 and TIMP2 expression by RT-PCR, in situ hybridization and zymography in the liver of patients with gastrointestinal adenocarcinomas and no detectable hepatic metastasis (n = 12), in tumoral and nontumoral liver from patients with hepatic metastasis (n = 9) and in control liver (n = 4). MMP2 and TIMP2 mRNA levels were increased in liver from patients with gastrointestinal adenocarcinomas and no detectable metastasis, compared with those of either control liver (5-fold and 3.2-fold, respectively) or nontumoral areas of liver from patients with metastasis (7.8-fold and 3-fold, respectively). MMP2 and TIMP2 transcripts were located in mesenchymal cells of portal tracts and sinusoids. MMP2 was mainly in its latent form. In liver from patients with hepatic metastasis, the tumoral/nontumoral ratios for MMP2 and TIMP2 mRNA were 6.2 +/- 4 and 1.5 +/- 0.4, respectively. Both transcripts were localized in the stromal cells of liver metastases, and the active form of MMP2 was found only in the tumoral areas. In the matching nontumoral areas the signals for MMP2 and TIMP2 mRNA were restricted to mesenchymal cells in portal tracts and sinusoidal cells. Our data show that liver stromal cells express high levels of MMP2 and TIMP2 in patients with colonic carcinoma without liver metastasis, suggesting the distant induction of these transcripts by the primary tumor.

Adenocarcinoma↗
PubMed1997

Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7.

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.

Cell Adhesion↗
PubMed1996

Fibrolamellar carcinoma of the liver: composition of the extracellular matrix and expression of cell-matrix and cell-cell adhesion molecules.

We have analyzed the composition of the tumor stroma and the expression of cell-matrix and cell-cell adhesion molecules in 11 cases of fibrolamellar carcinoma of the liver (FLC), in comparison with 34 cases of hepatocellular carcinoma and 8 cases of focal nodular hyperplasia. Fibrolamellar carcinoma was characterized by the presence of large amounts of tenascin in tumor stroma and by the scarce expression of basement membrane components at the contact of neoplastic clusters. Like normal hepatocytes, neoplastic cells constantly expressed the alpha1 integrin chain, lacked the beta4 integrin chain, and coexpressed E-cadherin and the hepatocyte N-related cadherin. Abnormalities in the expression of cell adhesion molecules, including altered cadherin expression, alphaV integrin chain induction, and CD44 expression, were detected in the majority of cases. The composition of the tumor stroma and the pattern of expression of cell adhesion molecules in fibrolamellar carcinoma were reminiscent of those observed in grade III and grade IV hepatocellular carcinomas. Our results therefore show that, despite its slow local growth and good prognosis, fibrolamellar carcinoma expresses many characteristics usually associated with clinically aggressive malignancies. Further studies are needed to identify the factors responsible for the apparent dissociation between clinical behavior and biological characteristics in this tumor.

Adult↗
PubMed1995

Extracellular matrix synthesized by clonal osteogenic cells is osteoinductive in vivo and in vitro: role of transforming growth factor-beta 1 in osteoblast cell-matrix interaction.

ROB-C26 (C26) is a multipotential, clonal cell line known to express several members of the TGF-beta superfamily and to become more osteoblastic (e.g., express higher levels of alkaline phosphatase) upon treatment with 10(-6)M retinoic acid (RA). We hypothesize that the expression of this more osteoblastic phenotype subsequent to RA exposure is the result of the treated cell's extracellular matrix (ECM) becoming a repository and active source of putative osteoinductive growth factors including, specifically, select members of the TGF-beta superfamily. To test this hypothesis, we isolated the ECM from RA-treated and untreated C26 cells and assessed them for their ability to promote osteogenic differentiation in vivo and in vitro. We then explored whether the latter activities could be attributed specifically to TGF-beta 1. We found that the ECM of treated cells isolated by cell lysis and extensive washing induced endochondral bone formation in vivo when implanted into the thigh muscles of athymic nude mice and stimulated alkaline phosphatase (ALP) activity in vitro in freshly plated C26 cells. This latter stimulation was comparable to levels observed with direct RA treatment. This latter in vitro activity was only very partially mimicked by the ECM prepared from untreated cells and not duplicated at all by RA-treated collagen or the ECM from another RA-treated multipotential cell line. Moreover, the in vivo osteoinductive effect of the treated C26 cell ECM was not duplicated by comparable ECM prepared from untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1996

Extracellular matrix components of the mouse thymus microenvironment. V. Interferon-gamma modulates thymic epithelial cell/thymocyte interactions via extracellular matrix ligands and receptors.

Extracellular matrix (ECM) proteins influence cell migration and differentiation in a variety of cell systems. Within the thymus, the ECM distribution pattern is conserved among various mammalian species, but its physiological role is not completely understood. Interferon-gamma (IFN-gamma), a cytokine produced by thymocytes, is able to in vitro modulate ECM production by thymic epithelial cells (TEC) in a dose-dependent biphasic pattern. In the same model, we determined herein that the expression of VLA-5 and VLA-6 (fibronectin and laminin receptors, respectively) were upregulated by low doses of IFN-gamma, whereas high doses of the cytokine induced an opposite effect. In a second in vitro system, we evidenced that thymocyte adhesion to a TEC line was also modulated by IFN-gamma. Importantly, such effects were due to the biphasic modulation of ECM ligands and receptors since they could be specifically prevented by preincubating the TEC cultures with anti-ECM or anti-ECM receptor antibodies. Additionally, spontaneous thymocyte release from thymic nurse cells was similarly biphasically modulated by IFN-gamma. Our data provide support for the notion that thymocyte-derived products can play a role in the dialogue that exists in the thymus, involving differentiating thymocytes and microenvironmental cells. Moreover, we bring arguments indicating that one of the implicated mechanisms involved is the modulation of ECM ligands and receptors by the thymic epithelium.

Animals↗
PubMed2000

Assembly and release of human immunodeficiency virus type 1 Gag proteins containing tandem repeats of the matrix protein coding sequences in the matrix domain.

We have constructed human immunodeficiency virus (HIV) gag mutants by increasing the matrix protein (MA) sequences via tandemly repeated duplication of the central 107-MA codons. Instead of a total of 132 amino acid residues for the wild-type MA, the resultant mutants designated as MA2, MA3, and MA4 contained a total of 242, 352, and 462 codons in the MA domains, respectively. Analysis indicated that the addition of 110 or 220 amino acid residues to the MA did not significantly affect the assembly, release, and processing of particles; however, particle production was markedly reduced when another copy of 110 residues was added to the MA. Subcellular fractionation analysis suggested that the MA tandem repeat mutations enhanced the Gag membrane affinity, in a manner which correlated with the copy number of MA sequences. The effects of enhanced membrane affinity were substantially reduced when sequences downstream of the capsid (CA) domain were deleted. Sucrose density gradient fractionation analysis showed that particles produced by the large insertion mutants possessed wild-type (wt) HIV particle density. Truncation of sequences downstream of the nucleocapsid (NC) domains of the mutants did not influence the budding of particles. In contrast, particle budding was severely impaired when sequences downstream of the CA domain were truncated. Particle densities for the large Gag proteins, which were truncated at the C-terminus of CA, were about 1.12-1.14 g/ml lower than that for wt. Our results suggest that the HIV MA domain could adopt insertions of large protein sequences, and strongly support the proposal that the NC and p2 domains play a crucial role in the process of correct Gag protein packing.

Cell Line↗
PubMed1993

Renin-angiotensin system and myocardial collagen matrix remodeling in hypertensive heart disease: in vivo and in vitro studies on collagen matrix regulation.

The interstitial space of the myocardium is composed of nonmyocyte cells and a highly organized collagen network which serves to maintain the architecture and mechanical behavior of the myocardial walls. It is the myocardial collagen matrix that determines myocardial stiffness in the normal and structurally remodeled myocardium. In hypertensive heart disease, the heterogeneity in myocardial structure, created by the altered behavior of nonmyocyte cells, particularly cardiac fibroblasts which are responsible for collagen synthesis and degradation, explains the appearance of diastolic and/or systolic dysfunction of the left ventricle that leads to symptomatic heart failure. Several lines of evidence suggest that circulating and myocardial renin-angiotensin systems (RAS) are involved in the regulation of the structural remodeling of the nonmyocyte compartment, including the cardioprotective effects of angiotensin converting enzyme (ACE) inhibition that was found to prevent myocardial fibrosis in the rat with renovascular hypertension. In cultured adult rat cardiac fibroblasts angiotensin II was shown to directly stimulate collagen synthesis and to inhibit collagenase activity, which is the key enzyme for collagen degradation, that would lead to collagen accumulation. In the spontaneously hypertensive rat, an appropriate experimental model for primary hypertension in man, left ventricular hypertrophy could be regressed and abnormal myocardial diastolic stiffness due to interstitial fibrosis could be restored to normal by inhibition of the myocardial RAS. These antifibrotic or cardioreparative effects of ACE inhibition that occurred irrespective of blood pressure normalization may be valuable in reversing left ventricular diastolic dysfunction in hypertensive heart disease.

Collagen↗
PubMed1985

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules.

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.

Animals↗
PubMed1989

Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 ('gelatinase') by human neutrophil elastase and cathepsin G.

The ability of human neutrophil elastase and cathepsin G to activate matrix metalloproteinase 3 (MMP-3 = stromelysin) and MMP-2 ('gelatinase') purified from human rheumatoid synovial fibroblasts in culture was examined. The zymogen of MMP-3 (proMMP-3) was activated to full activity with elastase and cathepsin G by limited proteolysis of the molecule into two active forms of Mr approximately 45,000 and Mr approximately 25,000. In contrast, proMMP-2 was not activated at all by these neutrophil serine proteinases, although it was degraded into small fragments. These data suggest that neutrophil elastase and cathepsin G may play an important role in the activation of proMMP-3 in vivo in various inflammatory conditions, but proMMP-2 may be activated in different ways.

Cathepsin G↗
PubMed1993

In vitro Ca deposition by rat matrix vesicles: is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media. 2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment. 3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment. 4. The specific activity of the released ALP was at least 5-fold higher than the residual activity. 5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or beta GP-dependent calcium deposition. 6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

Alkaline Phosphatase↗
PubMed1999

Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.

Adenosine Triphosphate↗