The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cellsurface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

IntroductionA remarkable feature of the CNS is its ability to undergo rapid activity-dependent remodeling of neuronal connections in a process that is referred to as synaptic plasticity. This process appears to depend, at least in part, on the activity of specific proteinases, and on the balance between proteinase activity and inhibition. It is thought that controlled local proteolytic activity mediates the assembly and disassembly of different components needed for axonal path-finding, and for the establishment of new neuronal connections.Tissue-type plasminogen activator (tPA) is expressed at low levels within the CNS, but its synthesis is increased by events that require synaptic plasticity, such as long-term potentiation, kindling, seizures, and motor learning (1-3). The primary substrate for tPA in vivo is the zymogen plasminogen, which tPA activates to the broad-specificity proteinase plasmin. Outside the CNS, tPA is primarily a thrombolytic enzyme, since plasmin's principal substrate is fibrin (4). However, within the CNS the roles of tPA and plasmin are not well characterized, and their primary substrates are not known. For example, both tPA-deficient (tPA -/-) and plasminogen-deficient (Plg -/-) mice have been shown to be resistant to direct kainic acid-induced (KA-induced) neuronal death (5, 6), suggesting that tPA acts through the activation of plasmin. However, while tPA -/-mice show reduced seizure-dependent mossy fiber outgrowth, Plg -/-mice do not (7), suggesting that plasmin may not be the primary substrate for tPA within the CNS. Likewise, tPA -/-mice show a decrease in infarct volume and an increase in neuronal survival compared with wild-type animals following middle cerebral artery occlusion (8), while animals deficient in plasminogen show an increase in infarct volume, suggesting a plasminogen-independent function for tPA in cerebral ischemia (9). However, the precise roles of tPA and plasminogen in these events are unclear. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase expressed in the CNS during events that require neuronal plasticity. In this study we demonstrate that endogenous tPA mediates the progression of kainic acid-induced (KA-induced) seizures by promoting the synchronization of neuronal activity required for seizure spreading, and that, unlike KA-induced cell death, this activity is plasminogen-independent. Specifically, seizure induction by KA injection into the amygdala induces tPA activity and cell death in both hippocampi, and unilateral treatment of rats with neuroserpin, a natural inhibitor of tPA in the brain, enhances neuronal survival in both hippocampi. Inhibition of tPA within the hippocampus by neuroserpin treatment does not prevent seizure onset but instead markedly delays the progression of seizure activity in both rats and wild-type mice. In tPA-deficient mice, seizure progression is significantly delayed, and neuroserpin treatment does not further delay seizure spreading. In contrast, plasminogen-deficient mice show a pattern of se...

No abstract

Matrix metalloproteinase-14 is required for degradation of fibrillar collagen by mesenchymal cells. Here we show that keratinocytes use an alternative plasminogen and matrix metalloproteinase-13-dependent pathway for dissolution of collagen fibrils. Primary keratinocytes displayed an absolute requirement for serum to dissolve collagen. Dissolution of collagen was abolished in plasminogen-depleted serum and could be restored by the exogenous addition of plasminogen. Both plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase blocked collagen dissolution, demonstrating the requirement of both plasminogen activation and matrix metalloproteinase activity for degradation. Cell surface plasmin activity was critical for the degradation process as aprotinin, but not ␣ 2 -antiplasmin, prevented collagen dissolution. Keratinocytes with single deficiencies in either urokinase or tissue plasminogen activator retained the ability to dissolve collagen. However, collagen fibril dissolution was abolished in keratinocytes with a combined deficiency in both urokinase and tissue plasminogen activator. Combined, but not single, urokinase and tissue plasminogen activator deficiency also completely blocked the activation of the fibrillar collagenase, matrix metalloproteinase-13, by keratinocytes. The activation of matrix metalloproteinase-13 in normal keratinocytes was prevented by plasminogen activator inhibitor-1 and aprotinin but not by tissue inhibitor of metalloproteinase-1 and -2, suggesting that plasmin activates matrix metalloproteinase-13 directly. We propose that plasminogen activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases.

IntroductionA remarkable feature of the CNS is its ability to undergo rapid activity-dependent remodeling of neuronal connections in a process that is referred to as synaptic plasticity. This process appears to depend, at least in part, on the activity of specific proteinases, and on the balance between proteinase activity and inhibition. It is thought that controlled local proteolytic activity mediates the assembly and disassembly of different components needed for axonal path-finding, and for the establishment of new neuronal connections.Tissue-type plasminogen activator (tPA) is expressed at low levels within the CNS, but its synthesis is increased by events that require synaptic plasticity, such as long-term potentiation, kindling, seizures, and motor learning (1-3). The primary substrate for tPA in vivo is the zymogen plasminogen, which tPA activates to the broad-specificity proteinase plasmin. Outside the CNS, tPA is primarily a thrombolytic enzyme, since plasmin's principal substrate is fibrin (4). However, within the CNS the roles of tPA and plasmin are not well characterized, and their primary substrates are not known. For example, both tPA-deficient (tPA -/-) and plasminogen-deficient (Plg -/-) mice have been shown to be resistant to direct kainic acid-induced (KA-induced) neuronal death (5, 6), suggesting that tPA acts through the activation of plasmin. However, while tPA -/-mice show reduced seizure-dependent mossy fiber outgrowth, Plg -/-mice do not (7), suggesting that plasmin may not be the primary substrate for tPA within the CNS. Likewise, tPA -/-mice show a decrease in infarct volume and an increase in neuronal survival compared with wild-type animals following middle cerebral artery occlusion (8), while animals deficient in plasminogen show an increase in infarct volume, suggesting a plasminogen-independent function for tPA in cerebral ischemia (9). However, the precise roles of tPA and plasminogen in these events are unclear. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase expressed in the CNS during events that require neuronal plasticity. In this study we demonstrate that endogenous tPA mediates the progression of kainic acid-induced (KA-induced) seizures by promoting the synchronization of neuronal activity required for seizure spreading, and that, unlike KA-induced cell death, this activity is plasminogen-independent. Specifically, seizure induction by KA injection into the amygdala induces tPA activity and cell death in both hippocampi, and unilateral treatment of rats with neuroserpin, a natural inhibitor of tPA in the brain, enhances neuronal survival in both hippocampi. Inhibition of tPA within the hippocampus by neuroserpin treatment does not prevent seizure onset but instead markedly delays the progression of seizure activity in both rats and wild-type mice. In tPA-deficient mice, seizure progression is significantly delayed, and neuroserpin treatment does not further delay seizure spreading. In contrast, plasminogen-deficient mice show a pattern of se...

The specific functions of plasminogen, stromal plasminogen activator, stromal plasminogen activator receptor, and stromal plasminogen activator inhibitor in the progression of the murine soft tissue sarcoma, T241 were investigated. Negation of plasminogen to the tumor blunted the orthotopic growth of the sarcoma in syngeneic mice. The reduced tumor growth was associated with a dramatic increase in tumor-infiltrating F4/80-positive macrophages and a diminution of vessel density, but not with obvious differences in fibrin and collagen deposition, or invasiveness of the tumor. Ablation of plasminogen activation by the tumor stroma only modestly impaired the prolonged growth of the sarcoma, suggesting that tumor cell-produced plasminogen activator is sufficient to mediate productive plasminogen activation. Plasminogen facilitated sarcoma progression, angiogenesis, and suppression of macrophage infiltration in the absence of either stromal urokinase plasminogen activator receptor or stromal plasminogen activator inhibitor. These data demonstrate that tumor cell-produced plasminogen activator and host plasminogen cooperate to facilitate soft tissue sarcoma growth and suppress the accumulation of tumor-infiltrating macrophages.

IntroductionA remarkable feature of the CNS is its ability to undergo rapid activity-dependent remodeling of neuronal connections in a process that is referred to as synaptic plasticity. This process appears to depend, at least in part, on the activity of specific proteinases, and on the balance between proteinase activity and inhibition. It is thought that controlled local proteolytic activity mediates the assembly and disassembly of different components needed for axonal path-finding, and for the establishment of new neuronal connections.Tissue-type plasminogen activator (tPA) is expressed at low levels within the CNS, but its synthesis is increased by events that require synaptic plasticity, such as long-term potentiation, kindling, seizures, and motor learning (1-3). The primary substrate for tPA in vivo is the zymogen plasminogen, which tPA activates to the broad-specificity proteinase plasmin. Outside the CNS, tPA is primarily a thrombolytic enzyme, since plasmin's principal substrate is fibrin (4). However, within the CNS the roles of tPA and plasmin are not well characterized, and their primary substrates are not known. For example, both tPA-deficient (tPA -/-) and plasminogen-deficient (Plg -/-) mice have been shown to be resistant to direct kainic acid-induced (KA-induced) neuronal death (5, 6), suggesting that tPA acts through the activation of plasmin. However, while tPA -/-mice show reduced seizure-dependent mossy fiber outgrowth, Plg -/-mice do not (7), suggesting that plasmin may not be the primary substrate for tPA within the CNS. Likewise, tPA -/-mice show a decrease in infarct volume and an increase in neuronal survival compared with wild-type animals following middle cerebral artery occlusion (8), while animals deficient in plasminogen show an increase in infarct volume, suggesting a plasminogen-independent function for tPA in cerebral ischemia (9). However, the precise roles of tPA and plasminogen in these events are unclear. Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase expressed in the CNS during events that require neuronal plasticity. In this study we demonstrate that endogenous tPA mediates the progression of kainic acid-induced (KA-induced) seizures by promoting the synchronization of neuronal activity required for seizure spreading, and that, unlike KA-induced cell death, this activity is plasminogen-independent. Specifically, seizure induction by KA injection into the amygdala induces tPA activity and cell death in both hippocampi, and unilateral treatment of rats with neuroserpin, a natural inhibitor of tPA in the brain, enhances neuronal survival in both hippocampi. Inhibition of tPA within the hippocampus by neuroserpin treatment does not prevent seizure onset but instead markedly delays the progression of seizure activity in both rats and wild-type mice. In tPA-deficient mice, seizure progression is significantly delayed, and neuroserpin treatment does not further delay seizure spreading. In contrast, plasminogen-deficient mice show a pattern of se...