Regulation of LRP-1 expression: make the point.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a membrane receptor displaying both scavenging and signaling functions. The wide variety of extracellular ligands and of cytoplasmic scaffolding and signaling proteins interacting with LRP-1 gives it a major role not only in physiological processes, such as embryogenesis and development, but also in critical pathological situations, including cancer and neurological disorders. In this review, we describe the molecular mechanisms involved at distinct levels in the regulation of LRP-1, from its expression to the proper location and stability at the cell surface.

[Involvement of matrix metalloproteinases in obstetrics: from implantation to delivery]. / Implication des métalloprotéases matricielles en obstétrique: de la nidation à l'accouchement.

Matrix metalloproteinases, which remodel the extracellular matrix, are involved in all physiological and pathophysiological processes. In particular, they contribute to the success of a pregnancy: from embryo implantation in the endometrium to uterine cervical ripening and uterine involution. A misregulation of their expression and/or of their activity is observed in two major diseases in pregnancy such as spontaneous abortion and preeclampsia.

[Multiple involvements of LRP-1 receptor in tumor progression]. / Implications multiples du récepteur LRP-1 dans la progression tumorale.

Extensive proteolytic remodeling processes constitute a critical step during tumor progression. The endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1), by its function in the clearance of multiple extracellular proteases involved in metastatic spreading, has long been considered as a putative tumor suppressor. Moreover, the receptor is likely to control the peritumoral microenvironment by internalization of growth factors and matricial proteins and could therefore participate to the control of signaling events involved in survival and proliferation of cancer cells. Nevertheless, recent data lead to reconsider the initially attributed antitumor properties of LRP-1. A more complex model seems to emerge in which LRP-1 could constitute a sensor of pericellular environment and regulate the membrane proteome dynamics. By its control of focal adhesions composition and turn-over, regulation of the cytoskeleton organization and integrin endocytic recycling, LRP-1 appears as a crucial actor of the epithelial-mesenchymal transition, thereby reinforcing the aggressive phenotype of malignant cells. LRP-1 partitioning into rafts and association with tissue-type and tumor grade specific intracellular scaffold proteins appear crucial to determine its function in tumor progression. Those emerging aspects present numerous promising perspectives in oncology and allow envisaging the development of innovative strategies of control of tumor progression through the targeting of LRP-1.

Binding of matrilysin-1 to human epithelial cells promotes its activity.

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K(D) of approximately 2.5 nM) and capacity (approximately 260,000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2.

Fibronectin type II (FnII)-like modules regulate gelatinase A activity.

Gelatinase A, a member of the matrix metalloproteinase family, contains three fibronectin type II (FnII)-like modules that are inserted within its catalytic domain. These FnII modules, defined as exosites, play an essential role in targeting the enzyme to matrix macromolecules, a process which can down-regulate membrane-type metalloproteinase-driven progelatinase A activation. The exosite/substrate-directed gelatinase inhibitors has been proposed as an alternative approach to disappointing active site-directed inhibitors, to control gelatinase A activity. In preliminary experiments, we evidenced that long-chain unsaturated fatty acids could bind preferentially to the first FnII module of gelatinase A. This interaction inhibits the activity of this enzyme towards proteins (type I gelatin and collagen) and an octapeptide substrate, with K(i) in the micromolar range. Since gelatinase A-catalyzed matrix proteolysis might display a positive or negative influence (depending on the substrate cleaved), the design of exosite-specific compounds for noncatalytic targeting of gelatinase A would necessitate an extensive degradomic analysis.

[Effect of elastin peptides on the production of matrix metalloproteinase 2 by human skin fibroblasts in culture]. / Influence des peptides d'élastine sur la production de la métalloprotéinase matricielle-2 par les fibroblastes du derme humain en culture.

Soluble elastin-derived peptides from alkaline or elastase hydrolysis of insoluble elastin, as well as tropoelastin, increase matrix metalloproteinase-2 (MMP-2) production by human skin fibroblasts in culture as determined by gelatin zymography and ELISA. Such an effect is time and concentration dependent; it can be reproduced by synthetic elastin: VGVAPG, PGAIPG, and laminin: LGTIPG, hexapeptides and inhibited by lactose and is therefore elastin receptor-mediated. The steady state levels of MMP-2 mRNAs are invariant following elastin-fibroblasts interaction. Inhibition of phospholipase C (D-609), ADP-ribosylation factor (brefeldin), protein kinase C (RO-318220) and phospholipase D (1-propanol) totally abolished the elastin-mediated increase of MMP-2 production. It suggested that the post-transcriptional mechanism controlling the elastin-mediated overproduction of MMP-2 involved a cascade leading to phospholipase D activation.

Involvement of fibronectin type II repeats in the efficient inhibition of gelatinases A and B by long-chain unsaturated fatty acids.

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

Involvement of the ceramide signaling pathway in modulating the differentiated state of porcine thyroid cells.

Neutral sphingomyelinase (Smase) is a cell membrane-associated phospholipase that hydrolyzes sphingomyelin to phosphocholine and ceramide, a lipid second messenger involved in cell differentiation and/or apoptosis. We first evidenced that porcine cultured thyroid cells could express neutral Smase activity even if thyrotropin (TStH), an essential hormone in thyroid cell differentiation, was found to induce a 1.7-fold decrease in Smase activity. Triggering the ceramide pathway by exogenous addition of neutral bacterial Smase (0.1 U/mL for 48 h), which transiently increased ceramide level by fourfold, drastically modified thyroid cell morphology. The follicle-like structures generated by TSH were disrupted, and the Smase-induced cell spreading was accompanied by a parallel loss of cell ability to iodinate proteins as well as a decrease of the adenylate cyclase system response. These inhibitory effects have been reproduced using short-chain exogenous ceramide analogs (C2-ceramides). Overall these data showed that ceramides emerged as potential mediators of dedifferentiation in thyroid cells.

Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

In the present study, we demonstrate that erythropoietin (Epo) induces the expression and the release of tissue inhibitors of metalloproteinase-1 (TIMP-1) in a time- and dose-dependent manner in Epo-dependent cell line UT-7 cells and in normal human erythroid progenitor cells from cord blood (CD36+) and required de novo protein synthesis. TIMP-1 was not expressed in the absence of Epo. Inhibition of the mitogen-activated protein kinase pathway by the specific inhibitors PD98059 and U0126 and of phosphatidylinositol 3-kinase by LY294002, strongly inhibited Epo-induced TIMP-1 expression and secretion. In the absence of Epo, both latent and active forms of matrix metalloproteinase-9 (MMP-9) were secreted into media. Upon Epo stimulation, MMP-9 and pro-MMP-9 secretion was inhibited in a dose-dependent manner parallel to TIMP-1 induction. The addition of PD98059, U0126, and LY294002 in the presence of Epo restored MMP-9 production in UT-7 and CD36+ cells. Our findings strongly suggest an inversely coordinated regulation of the TIMP-1 gene and MMP-9 production by Epo via mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ stimulates matrix metalloproteinase-2 expression by fibroblast cultures.

Glycyl-histidyl-lysine-Cu2+ (GHK-Cu) is a tripeptide-copper complex known to be a potent wound healing agent. We previously showed its ability to stimulate in vitro and in vivo the synthesis of extracellular matrix components. The aim of this study was to determine the effects of GHK-Cu on MMP-2 synthesis by dermal fibroblasts in culture. We showed that GHK-Cu increased MMP-2 levels in conditioned media of cultured fibroblasts. This effect was reproduced by copper ions but not by the tripeptide GHK alone. This stimulation was accompanied by an increase of MMP-2 mRNA level. We also showed that GHK-Cu increased the secretion of the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. Taken together, our results underline that GHK-Cu is not only an activator of connective tissue production but also of the remodeling of the extracellular matrix. It is able to modulate MMP expression by acting directly on wound fibroblasts.

Relationship between cell-associated matrix metalloproteinase 9 and psoriatic keratinocyte growth.

Primary cultures of psoriatic keratinocytes proliferated at a higher rate and produced lower amounts of matrix metalloproteinase 9 than normal keratinocytes cultured under similar conditions. Sup- plementation of psoriatic keratinocyte cell culture medium with batimastat or the use of a matrix metalloproteinase 9 blocking antibody further stimulated psoriatic keratinocyte growth. An increase in intracellular ceramide level enhanced matrix metalloproteinase 9 production and inhibited cell proliferation in parallel. Whether cells were treated with sphingomyelinase or not, however, conditioned media from psoriatic keratinocytes contained higher levels of tissue inhibitor of metalloproteinase-1 compared with matrix metalloproteinase 9 and secreted only the proenzyme form. Pro-matrix metalloproteinase 9, as well as active matrix metalloproteinase 9, was identified in membrane preparations of psoriatic keratinocytes, and enzyme amounts were greatly elevated following sphingomyelinase action. As (i) tissue inhibitor of metalloproteinase-1 antibody nearly totally abrogated keratinocyte growth and (ii) complexes of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase 9 were recovered in membrane extracts of sphingomyelinase-treated psoriatic keratinocytes, we postulate that an increased level of cell-associated matrix metalloproteinase 9 might compete for tissue inhibitor of metalloproteinase-1 binding to its receptor. As a consequence, the increased levels of matrix metalloproteinase 9 will decrease keratinocyte growth.

Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry.

Cutaneous aging and chronic exposure to UV irradiation leads to alterations in the appearance and biochemical composition of the skin. Members of the MMP family have been involved in the destruction of the extracellular matrix. Among them, gelatinases A and B were found to display elastolytic activity, in vitro. In this study, we first determined the ex vivo elastolytic potential of both endopeptidases, using human skin tissue sections and computerized morphometric analyses, and compared it with those of neutrophil elastase. In such conditions, gelatinase B (50 nM) induced 50% elastolysis. The percentage of elastic fibers degraded by gelatinase A (10-100 nM) never exceeded 10%. Elastolysis by gelatinase B and leukocyte elastase was characterized by a decrease in fiber length and an increase in the average diameter of the fibers. In addition, gelatinase B exhibited fibrillin-degrading activities. On the contrary, gelatinase A (50 nM) elicited up to 50% hydrolysis of collagen fibers, preferentially degrading type III collagen fibers. Gelatinase B did not promote any collagen degrading activity. Our data suggested that in vivo gelatinases could disrupt most extracellular matrix structures of human skin. Gelatinase B and to a much lesser extent, gelatinase A would degrade components of the elastic fibers network while gelatinase A, but not gelatinase B, would alter mostly collagen fibers and also degrade constituents of the dermo-epidermal junction.

Contribution of the plasmin/matrix metalloproteinase cascade to the retraction of human fibroblast populated collagen lattices.

To assess the contribution of the plasmin/matrix metalloproteinase cascade in lattices retraction, human gingival fibroblast-populated collagen lattices were supplemented with plasminogen. The rate of lattice retraction was enhanced by addition of plasminogen. This effect was concomitant to plasmin generation, prostromelysin-1 and procollagenase activation. Plasminogen-mediated initiation of that proteolytic cascade was accompanied by conspicuous changes in cell morphology and collagen fibers organization. At day 1 of culture fibroblasts shifted from a rounded (control) to an elongated (in presence of plgn) shape. At the latest stage of retraction, intense vacuolization around fibroblasts was noticed in plgn-supplemented lattices which paralleled the increased collagen degradation. Plgn-enhancing influence on the initial phase of lattice retraction could be totally annihilated by either aprotinin or Batimastat. Those data emphasize the crucial importance of the plasmin-MMP proteolytic cascade in granulation tissue retraction in a healing wound.

Activation of fibroblasts in collagen lattices by mast cell extract: a model of fibrosis.

BACKGROUND: Mast cells are resident connective tissue cells able to secrete numerous inflammatory mediators in response to tissue aggression and might be implicated in the fibrotic processes. OBJECTIVES: To study the effects of mast cell products on fibroblast activity in connective tissues. METHODS: Mast cell extract was prepared by sonication of pure mast cell preparations obtained by peritoneal lavage of rats and added to the culture medium of fibroblast-populated collagen lattices. RESULTS: Mast cell extract was able to decrease the contraction of the collagen lattices, to stimulate total protein and collagen synthesis, and to increase the expression and activation of gelatinase A/MMP-2. CONCLUSION: These data are consistent with the hypothesis that mast cells in connective tissue may be responsible for fibroblast activation at the early phases of tissue repair and fibrosis.

Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro: a role for transforming growth factor beta (TGF-beta).

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([(3)H]thymidine) and collagen synthesis ([(3)H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56 degrees C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor beta (TGF-beta) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-beta neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-beta. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-beta. These data corroborate the importance of eosinophils in skin and lung fibrosis.

Involvement of the 92-kDa gelatinase (matrix metalloproteinase-9) in the ceramide-mediated inhibition of human keratinocyte growth.

Triggering the ceramide pathway by exogenous treatment with neutral sphingomyelinase (Smase) inhibited human keratinocyte growth rate, while having no influence on cell apoptosis. Increasing the ceramide content of keratinocytes with Smase (100 U/ml) or C6-ceramide (1 microM) enhanced matrix metalloproteinase (MMP)-9 production. On the contrary, levels of MMP-2 secretion were unchanged. The inhibition of keratinocyte growth rate induced by ceramide could be annihilated by a peptide hydroxamate MMP inhibitor or an MMP-9 blocking antibody. In addition, inhibiting MMP-9 activity in control keratinocyte culture was found to stimulate keratinocyte proliferation. These data suggest a pivotal function of MMP-9 in the control of keratinocyte growth.

Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.

We investigated the expression and activation of matrix metalloproteinases in a model of experimental wounds in rats, and their modulation by glycyl-L-histidyl-L-lysine-Cu(II), a potent activator of wound repair. Wound chambers were inserted under the skin of Sprague-Dawley rats and received serial injections of either 2 mg glycyl-L-histidyl-L-lysine-Cu(II) or the same volume of saline. The wound fluid and the neosynthetized connective tissue deposited in the chambers were collected and analyzed for matrix metalloproteinase expression and/or activity. Interstitial collagenase increased progressively in the wound fluid throughout the experiment. Glycyl-L-histidyl-L-lysine-Cu(II) treatment did not alter its activity. Matrix metalloproteinase-9 (gelatinase B) and matrix metalloproteinase-2 (gelatinase A) were the two main gelatinolytic activities expressed during the healing process. Pro-matrix metalloproteinase (pro-form of matrix metalloproteinase)-9 was strongly expressed during the early stages of wound healing (day 3). In the wound fluid, it decreased rapidly and disappeared after day 18, whereas in the wound tissue, matrix metalloproteinase-9 expression persisted in the glycyl-L-histidyl-L-lysine-Cu(II) injected chamber until day 22. Pro-matrix metalloproteinase-2 was expressed at low levels at the beginning of the healing process, increased progressively until day 7, then decreased until day 18. Activated matrix metalloproteinase-2 was present in wound fluid and wound tissue. It increased until day 12, then decreased progressively. Glycyl-L-histidyl-L-lysine-Cu(II) injections increased pro-matrix metalloproteinase-2 and activated matrix metalloproteinase-2 during the later stages of healing (days 18 and/or 22). These results demonstrate that various types of matrix metalloproteinases are selectively expressed or activated at the various periods of wound healing. Glycyl-L-histidyl-L-lysine-Cu(II) is able to modulate their expression and might significantly alter wound remodeling.