Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells.
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The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels. (+info)
Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions.
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We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568-631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity. (+info)
Expression of macrophage MARCO receptor induces formation of dendritic plasma membrane processes.
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MARCO is a novel macrophage-specific receptor structurally related to macrophage class A scavenger receptors. It is constitutively expressed in macrophages of the marginal zone of the spleen and in lymph nodes and is up-regulated in other tissues during systemic bacterial infections. In this study, we show that ectopic expression of MARCO in cell lines such as Chinese hamster ovary, HeLa, NIH3T3, and 293 induces dramatic cell shape changes. Typically these changes include formation of large lamellipodia-like structures and of long dendritic processes. The morphological changes are accompanied by disassembly of actin stress fibers and often also by complete loss of focal adhesions. The MARCO-induced changes are dependent on cell adhesion and are inhibited, but not completely abolished, when the cells are plated on fibronectin-coated surfaces. Similarly, a dominant-negative mutant of the Rho family GTPase Rac1 partially inhibited the morphogenic effects of MARCO in Chinese hamster ovary cells, whereas a dominant-negative form of a related protein, Cdc42, did not. Expression studies with a variety of truncated MARCO forms indicated that the proximal segment of the cysteine-rich domain V is important for the morphoregulatory activity. The results indicate that expression of MARCO has a direct effect in generating the phenotype of activated macrophages necessary for the trapping and removal of pathogens and other foreign substances. (+info)
Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor.
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To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration. (+info)
Matrix degradation by chondrocytes cultured in alginate: IL-1 beta induces proteoglycan degradation and proMMP synthesis but does not result in collagen degradation.
(29/9343)
OBJECTIVE: To determine the role of interleukin-1 beta (IL-1 beta) in the degradation of proteoglycans and collagen by articular chondrocytes. DESIGN: Chondrocytes were cultured in alginate beads for 2 weeks to produce extracellular matrix, followed by the addition of IL-1 beta for 1 or 2 days. Breakdown of extracellular matrix (with and without activation of pro-matrix metalloproteinases (MMPs) by APMA) was monitored by release of glycosaminoglycans (GAG, proteoglycans) and hydroxyproline (collagen) from the beads into the medium, and by the amount of damaged collagen in the bead. Levels of (pro)MMPs in the beads were assayed by zymography and their activity was quantified fluorometrically. RESULTS: IL-1 beta induced a profound GAG release (approximately 80% after 2 days at 20 ng/ml IL-1 beta) that was both time and IL-1 beta concentration dependent. Under these conditions no increase in collagen release or damaged collagen in the bead was detected. Zymography demonstrated that the synthesis of a variety of proMMPs was induced by IL-1 beta, without a detectable increase of MMP-activity as measured in the activity assay. After activation of the proMMPs by APMA, a time and IL-1 beta concentration-dependent increase in MMP-activity was found, which resulted in almost complete deterioration of collagen already after 18 h of incubation. In the presence of APMA, GAG release from IL-1 beta treated beads was significantly increased from 24 to 31%. CONCLUSIONS: Our data suggest that proteoglycan and collagen degradation are regulated through different mechanisms: IL-1 beta induces the synthesis of active enzymes that degrade proteoglycans, such as 'aggrecanase', and inactive proMMPs. Thus, IL-1 beta alone is not sufficient to result in collagen-degrading MMPs. Once activated, MMPs may account for up to a quarter of the aggrecan degradation in this model. (+info)
Cell-extracellular matrix interactions and EGF are important regulators of the basal mammary epithelial cell phenotype.
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The mammary epithelium is composed of a luminal epithelium and a basal layer containing myoepithelial cells and undifferentiated precursors. Basal cells express specific protein markers, such as keratin 14 (K14) and P-cadherin. To study the factors that regulate the basal mammary epithelial cell phenotype, we have established two clonal derivatives of the mouse HC11 cell line, BC20 and BC44, expressing high levels of K14 and P-cadherin. Unlike the parental HC11 cells, these basal cells did not produce beta-casein in response to lactogenic hormone treatment; however their phenotype appeared to be plastic. Cultured in EGF-free medium, they exhibited enhanced cell-extracellular matrix adhesions and deficient cell-cell junctions, whereas long-term treatment with EGF induced a decrease of focal contact number and establishment of cell-cell junctions, resulting in downregulation of K14 and P-cadherin expression at the protein and mRNA levels. To determine whether cell-extracellular matrix interactions mediated by integrins have a role in the regulation of the expression of K14 and P-cadherin, the amounts of transcripts for the two proteins were analysed in the basal cells, which were plated on the function-blocking antibodies against beta1 and alpha6 integrin chains, on fibronectin and on laminin 5. The amount of P-cadherin transcript was 2- to 4-fold higher in cells plated on the function-blocking anti-integrin antibodies and on the extracellular matrix proteins, as compared to cells plated on poly-L-lysine, whereas the K14 transcript levels were not significantly modified in response to adhesion. The data demonstrate that integrin-mediated cell interaction with extracellular matrix is directly implicated in the control of P-cadherin expression, and that EGF and cell-extracellular matrix adhesion events are important regulators of the basal mammary epithelial cell phenotype. (+info)
Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix.
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Fibrillin-1, the main component of 10-12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin. (+info)
Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors.
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Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner. (+info)