Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Matrix”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,279 records · Page 71Linked to original sources
PubMed1994

Targeting of the immunoglobulin-binding domain of protein A to the extracellular matrix using a minifibronectin expression vector.

A truncated form of fibronectin consisting of the N-terminal 70-kDa and C-terminal 37-kDa regions, referred to as "minifibronectin," retains the ability to assemble into the extracellular matrix, even though it lacks the central approximately 120-kDa region containing most of the type III modules (Ichihara-Tanaka, K., Titani, K., and Sekiguchi, K., FEBS Lett. 299, 155-158, 1992). Taking advantage of the matrix assembly activity of minifibronectin, we developed a novel method to target non-matrix proteins to the extracellular matrix by inserting them between the N-terminal 70-kDa and the C-terminal 37-kDa regions of minifibronectin. Using the immunoglobulin-binding domain of Staphylococcal protein A as a model, we demonstrated that the bacterial protein expressed in mouse L cells as a chimeric protein with minifibronectin is secreted as disulfide-bonded dimers and successfully deposited onto the extracellular matrix of transfected cells. The chimeric protein retained the immunoglobulin-binding activity not only in solution but also after deposition at the matrix. This targeting strategy we developed will provide a means to manipulate the biological functions of the extracellular matrix through targeting of a wide variety of non-matrix proteins.

Animals↗
PubMed1998

Basal cell carcinoma cells resemble follicular matrix cells rather than follicular bulge cells: immunohistochemical and ultrastructural comparative studies.

To detail the histogenetic relationship between basal cell carcinoma (BCC) and hair follicles, we immunohistochemically compared BCC cells to follicular matrix cells and follicular bulge cells using a panel of monoclonal antibodies against melanocytes, cytokeratins, subepidermal extracellular matrix components, and bullous pemphigoid (BP) sera, as well as using electron microscopy. Cytokeratin expression patterns were not consistent with the variety in types of cytokeratins and in cases of BCC. The distribution of some extracellular matrix components was not only linear along the interfaces of BCC tumor nests and stroma, and follicular matrix and follicular papilla; granular deposits were also seen in the stroma and follicular papilla, whereas they were only linearly distributed along the follicular bulge. The BP antigens and integrin alpha 6, which were absent in BCC and follicular matrix, were expressed in the follicular bulge area. Electron microscopically, hemidesmosomes were poorly organized in these three tissues, but the lamina densa was incomplete in BCC and follicular matrix, whereas the lamina densa in the follicular bulge area was continuous. These morphologic similarities between BCC and follicular matrix cells, and coexistence of melanocytes in the BCC tumor nest strongly suggest the differentiation of BCC toward the follicular matrix cells.

Basal Cell Carcinoma↗
PubMed2002

Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1.

STUDY DESIGN: A study of the mechanisms involved in matrix repair by intervertebral disc cells cultured in alginate gel was performed. OBJECTIVES: To determine the effects of osteogenic protein-1 on the extracellular matrix of intervertebral disc cells previously exposed to interleukin-1, which is an in vitro model for degraded extracellular matrix. SUMMARY OF BACKGROUND DATA: Disc degeneration is accompanied by a decrease in the content of negatively charged proteoglycans in the matrix. No previous attempt has been made to repair the degraded matrix of the disc. METHODS: Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar discs of New Zealand white rabbits and were separately encapsulated in alginate beads. The alginate beads were cultured with or without osteogenic protein-1 after previous exposure to interleukin-1alpha in the presence of 10% fetal bovine serum. The total contents of proteoglycan, collagen, and DNA in the alginate beads were measured. The rate of proteoglycan synthesis by the encapsulated cells was also determined. RESULTS: Treatment with interleukin-1alpha resulted in a significant decrease in proteoglycan and collagen contents in the matrix formed by both the nucleus pulposus and anulus fibrosus. However, subsequent treatment with osteogenic protein-1 led in both cases to rapid recovery of proteoglycans and collagens, whose contents returned to the levels seen in cells not previously exposed to interleukin-1alpha. By the end of the culture period (day 21), those values reached levels higher than those found in beads containing cells never exposed to interleukin-1alpha. Further, the rate of proteoglycan synthesis by both cell types in beads treated with osteogenic protein-1 after previous exposure to interleukin-1alpha was significantly higher than in beads whose cells were not treated with osteogenic protein-1 after previous exposure to interleukin-1alpha. CONCLUSION: Disc cells that have been previously exposed to interleukin-1alpha have lost none of their potential to upregulate proteoglycan synthesis in response to stimulation with osteogenic protein-1. On stimulation with osteogenic protein-1, these disc cells not only replenished the matrix with proteoglycans that had been lost during interleukin-1alpha treatment but proceeded to reform a matrix that was richer in these resilient molecules than that formed by disc cells never exposed to interleukin-1alpha.

Animals↗
PubMed2004

Role of matrix metalloproteinases in hypertension-associated cardiac fibrosis.

PURPOSE OF REVIEW: The potential contribution of alterations in matrix metalloproteinase activity to the development of myocardial fibrosis in hypertensive heart disease is reviewed. RECENT FINDINGS: A number of experimental and clinical studies provide information on alterations in the balance between matrix metalloproteinase-1 or collagenase and tissue inhibitor of matrix metalloproteinases-1, which result in depressed proteolytic activity of the enzyme in animals and humans with hypertensive heart disease. While some recent data point to a genetic origin of such an imbalance, other findings suggest that depressed collagenase activity may contribute to disturbances of cardiac function via facilitation of myocardial fibrosis. On the other hand, emerging information is providing the basis for the notion that other matrix metalloproteinases, namely gelatinases, may participate in the process of myocardial fibrosis through stimulation of fibrillar collagen synthesis. Some fragmented matrix peptides or matrikines may be the mediators of the profibrotic action of these matrix metalloproteinases. SUMMARY: The matrix metalloproteinases represent an important biological system within the myocardium designed to maintain the complex and dynamic microenvironment of the extracellular matrix. Improved understanding of how this system is dysregulated in hypertensive heart disease will probably provide new insights into, and strategies for, heart failure.

Fibrosis↗
PubMed2004

Matrix metalloproteinases and their clinical applications in orthopaedics.

Imbalance in the expression of matrix metalloproteinases and their inhibitors contribute considerably to abnormal connective tissue degradation prevalent in various orthopaedic joint diseases such as rheumatoid arthritis and osteoarthritis. Matrix metalloproteinase expression has been detected in ligament, tendon, and cartilage tissues in the joint. They are known to contribute to the development, remodeling, and maintenance of healthy tissue through their ability to cleave a wide range of extracellular matrix substrates. Their role has been extended to cell growth, migration, differentiation, and apoptosis. In orthopaedics, their clinical applications constantly are being explored. The multiple steps in matrix metalloproteinase regulation offer potential targets for inhibition, useful in drug therapy. The correlation between matrix metalloproteinases and progression in joint erosion presents potential prognostic and diagnostic tools in rheumatoid arthritis. Matrix metalloproteinases also can be incorporated into scaffold design to control the degradation rate of engineered tissue constructs. This current review aims to summarize and emphasize the importance of matrix metalloproteinases and their natural inhibitors in the maturation of musculoskeletal tissue through matrix remodeling and, therefore, in the generation of a new clinical potential in orthopaedics.

Humans↗
PubMed2003

Urothlelium facilitates the recruitment and trans-differentiation of fibroblasts into smooth muscle in acellular matrix.

PURPOSE: Tissue engineering has shown promise for patients requiring bladder reconstruction secondary to diverse diseases such as spina bifida and tuberculosis. In this study we addressed whether urothelium in conjunction with an acellular bladder matrix is necessary for the recruitment of host cells to repopulate the graft and whether the urothelium facilitates trans-differentiation of host fibroblastic cells into bladder smooth muscle type cells. MATERIALS AND METHODS: Urothelium from embryonic mice and rats (E18) was microdissected from bladder stroma after enzymatic digestion with 10 mM ethylenediaminetetraacetic acid. Acellular bladder matrix was prepared from postnatal day 1 rat and mouse pups using a combination of membrane disruption and DNA digestion. Urothelium was placed inside the bladder matrix and then grown either underneath the renal capsule or subcutaneously in athymic mice for 2 to 24 weeks. Grafts were sectioned and immunocytochemistry was performed with antibodies for alpha-actin smooth muscle, uroplakin, cytokeratins and vimentin to assess for the development of detrusor muscle. RESULTS: Embryonic bladder urothelium resulted in ingrowth of fibroblasts into acellular matrix in greater than 85% of 60 grafts consisting of acellular matrix and bladder urothelium. Moreover, there was apparent trans-differentiation of these fibroblasts into a smooth muscle phenotype. This smooth muscle development occurred in a graded, incremental fashion starting as early as 3 weeks and resulting in almost complete repopulation of the matrix at 6 months. CONCLUSIONS: These data support the hypothesis that epithelial mesenchymal signaling is important for the differentiation of bladder smooth muscle. Smooth muscle development during augmentation with acellular matrix is facilitated by the placement of epithelia onto the surface of the matrix.

Animals↗
PubMed2003

Cross-talk between RhoGTPases and stress activated kinases for matrix metalloproteinase-9 induction in response to keratinocytes injury.

Cell migration and extracellular matrix remodeling are two essential processes of wound healing, regulated by extracellular metalloproteinases such as matrix metalloproteinase-2 (Gelatinase A) and matrix metalloproteinase-9 (Gelatinase B). Expression of matrix metalloproteinase-9 is deregulated in numerous wound healing pathologies. To date the mechanisms regulating matrix metalloproteinase-9 during normal wound healing are poorly documented. Using both primary cultures of normal human keratinocytes and a wounding device especially designed to dissect the molecular events during the healing process in vitro, we show that matrix metalloproteinase-9 is stimulated by injury in normal human keratinocytes. This upregulation results from the mechanical stress created by injury and not from a soluble factor, secreted by wounded normal human keratinocytes. We also demonstrate that the Rho family of small GTPases, p38[MAPK] and JNK together play a key part in the signaling pathways controlling the stimulation of matrix metalloproteinase-9 in wounded cells. We provide lines of evidence indicating that in wounded keratinocytes, upregulation of matrix metalloproteinase-9 depends on two distinct pathways. The first involves Rac1 and/or Cdc42 that control the activation of p38[MAPK]. The second depends on RhoA activation that is required for stimulation of JNK.

Gene Expression Regulation↗
PubMed1998

The capacity of polyomavirus enhancer binding protein 2alphaB (AML1/Cbfa2) to stimulate polyomavirus DNA replication is related to its affinity for the nuclear matrix.

The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking. A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2alphaB1 (PEBP2alphaB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site. The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD). In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication. A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2alphaB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2alphaB1 and GAL4-RAD. Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2alphaB1 and GAL4-RAD. The inhibition was specific for replication mediated by PEBP2alphaB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD. These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment.

Animals↗
PubMed1999

Altered telomere nuclear matrix interactions and nucleosomal periodicity in ataxia telangiectasia cells before and after ionizing radiation treatment.

Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.

Adolescent↗
PubMed1988

Extracellular matrix assembly.

Fibronectin remains unique among matrix components because it exists in both soluble and matrix forms. It seems likely that the necessity to prevent inappropriate matrix assembly from soluble fibronectin results in a strict requirement for catalysis by specific cells to deposit fibronectin fibrils. Moreover, many cell types possess adhesive receptors for fibronectin (Buck & Horwitz 1987), but only certain mesenchymal cells appear capable of depositing organized matrices. These professional matrix-organizing cells somehow interact with the aminoterminus of fibronectin and construct fibrils, preventing diffusion of fibronectin away from sites of synthesis and forming an extracellular matrix with a very high concentration of fibronectin. The resulting matrix should facilitate the attachment and migration of macrophages and neural crest cells that possess adhesive receptors but lack matrix-forming ability. Thus, it is possible that this system of dual cell interactive cells evolved in order to allow careful modulation of cell interactions with fibronectin leading primarily to synthesis and deposition (fibroblasts), or to cell recognition (neural crest, macrophages). Elucidation of the cell surface molecules interacting with fibronectin's aminoterminus is a critical first step in further understanding this adhesive and matrix assembly system.

Animals↗
PubMed1998

TNF-alpha-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis.

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-alpha causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-alpha have not been defined. Because TNF-alpha can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-alpha (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface alpha5beta1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-alpha treatment of endothelial monolayers with matrices prelabeled with 125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-alpha exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-alpha increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-alpha-induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-alpha appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased alpha5beta1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-alpha exposure.

Animals↗
PubMed2000

Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.

Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.

Animals↗
PubMed1984

Alveolar macrophage modulation of proteolysis by neutrophil elastase in extracellular matrix.

An extracellular connective tissue matrix, made up of components found in the pulmonary alveolar interstitium, was generated in vitro and used as a culture surface and substrate for proteolysis by human alveolar macrophages (AM) and neutrophil elastase (NE). The ability of human AM to modulate NE-mediated degradation of elastin and collagen in the surrounding matrix was studied to gain insights into the inflammatory process that accompanies the pathogenesis of emphysema in humans. Neutrophil elastase that had been internalized by AM showed a diminished but more prolonged time course of matrix proteolysis than did a similar amount of NE added to the matrix in the absence of AM. Collagen and elastin degradation were quantitated by release of hydroxylysine and desmosine, respectively, into the culture medium. Significantly more hydroxylysine and desmosine were released by AM that had internalized NE than by AM or by culture medium alone. When 14 X 10(6) AM were added to the extracellular matrix, followed 2 h later by addition of 2 micrograms of NE, collagen and elastin degradation measured at 24 h were not significantly different from that which occurred when matrix was incubated with NE in the absence of AM. Collagen degradation, determined in the same cultures during the period from 24 to 96 h, was significantly greater when matrix was incubated with both AM and NE. These findings suggest that AM can release previously internalized NE in an enzymatically active form and that AM may enhance collagen degradation in matrix that was also exposed to NE.

Animals↗
PubMed1985

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.

Animals↗
PubMed1996

Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils.

We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.

Carrier Proteins↗
PubMed2006

Insulin-like growth factor binding protein-3 in extracellular matrix stimulates adhesion of breast epithelial cells and activation of p44/42 mitogen-activated protein kinase.

IGF-binding protein-3 (IGFBP-3) is a multifunctional protein that regulates the potent mitogenic and antiapoptotic effects of IGF-I and IGF-II and exerts bioactivity independent of modulating IGF receptor activation. Previous studies have shown that in solution, IGFBP-3 binds constituent proteins of the extracellular matrix (ECM) such as fibronectin and collagen and is present in ECM deposited by fibroblasts in vitro; however, binding of IGFBP-3 to matrix has not been characterized, nor has its function in this environment been investigated. In this study, we show that IGFBP-3 binds to ECM deposited by human breast epithelial and cancer cells and neonatal human fibroblasts. IGF-I and heparin blocked binding of IGFBP-3 to matrix when added with the binding protein but were unable to displace IGFBP-3 already bound to the matrix. IGF-I bound to matrix-immobilized IGFBP-3 with approximately 25-fold reduced affinity compared with IGFBP-3 in solution. Mutation of the C-terminal basic domain of IGFBP-3 (228KGRKR-->MDGEA) resulted in markedly reduced binding to matrix compared with wild-type IGFBP-3, whereas mutation of the adjacent consensus heparin-binding domain (220KKK-->HSR) had relatively little effect. In the presence of matrix-bound IGFBP-3, adhesion of breast epithelial cells was increased by approximately 25%, and activation of the signaling pathway intermediate p44/42 MAPK was enhanced greater than 3-fold. These results indicate a previously unrecognized and potentially important role for IGFBP-3 in the extracellular matrix.

Breast↗
PubMed2001

Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation.

During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.

Animals↗
PubMed1993

ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner.

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

Animals↗