Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.     

Glutathione restores collagen degradation in TGF-beta-treated fibroblasts by blocking plasminogen activator inhibitor-1 expression and activating plasminogen

Transforming growth factor (TGF)-beta plays an important role in tissue fibrogenesis. We previously demonstrated that reduced glutathione (GSH) supplementation blocked collagen accumulation induced by TGF-beta in NIH-3T3 cells. In the present study, we show that supplementation of GSH restores the collagen degradation rate in TGF-beta-treated NIH-3T3 cells. Restoration of collagen degradation by GSH is associated with a reduction of type I plasminogen activator inhibitor (PAI)-1 expression/activity as well as recovery of the activities of cell/extracellular matrix-associated tissue-type plasminogen activator and plasmin. Furthermore, we find that NIH-3T3 cells constitutively express plasminogen mRNA and possess plasmin activity. Blockade of cell surface binding of plasminogen/plasminogen activation with tranexamic acid (TXA) or inhibition of plasmin activity with aprotinin significantly reduces the basal level of collagen degradation both in the presence or absence of exogenous plasminogen. Most importantly, addition of TXA or active PAI-1 almost completely eliminates the restorative effects of GSH on collagen degradation in TGF-beta treated cells. Together, our results suggest that the major mechanism by which GSH restores collagen degradation in TGF-beta-treated cells is through blocking PAI-1 expression, leading to increased PA/plasmin activity and consequent proteolytic degradation of collagens. This study provides mechanistic evidence for GSH's putative therapeutic effect in the treatment of fibrotic disorders.     

PAI-1 in tissue fibrosis

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation-related disease. Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the ECM depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase/tissue type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities, and, thus, help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area, and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus, PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver, and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review, we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.     

A neuroprotective role for angiogenin in models of Parkinson's disease

We previously observed marked down-regulation of the mRNA for angiogenin, a potent inducer of neovascularization, in a mouse model of Parkinson's disease (PD) based on over-expression of alpha-synuclein. Angiogenin has also been recently implicated in the pathogenesis of amyotrophic lateral sclerosis. In this study, we confirmed that mouse angiogenin-1 protein is dramatically reduced in this transgenic alpha-synuclein mouse model of PD, and examined the effect of angiogenin in cellular models of PD. We found that endogenous angiogenin is present in two dopamine-producing neuroblastoma cell lines, SH-SY5Y and M17, and that exogenous angiogenin is taken up by these cells and leads to phosphorylation of Akt. Applied angiogenin protects against the cell death induced by the neurotoxins 1-methyl-4-phenylpyridinium and rotenone and reduces the activation of caspase 3. Together our data supports the importance of angiogenin in protecting against dopaminergic neuronal cell death and suggests its potential as a therapy for PD.     

Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.     

Proteolytic cleavage of extracellular α-synuclein by plasmin: implications for Parkinson disease

Parkinson disease (PD) is the second most common neurodegenerative disease characterized by a progressive dopaminergic neuronal loss in association with Lewy body inclusions. Gathering evidence indicates that α-synuclein (α-syn), a major component of the Lewy body, plays an important role in the pathogenesis of PD. Although α-syn is considered to be a cytoplasmic protein, it has been detected in extracellular biological fluids, including human cerebrospinal fluid and blood plasma of healthy and diseased individuals. In addition, a prion-like spread of α-syn aggregates has been recently proposed to contribute to the propagation of Lewy bodies throughout the nervous system during progression of PD, suggesting that the metabolism of extracellular α-syn might play a key role in the pathogenesis of PD. In the present study, we found that plasmin cleaved and degraded extracellular α-syn specifically in a dose- and time- dependent manner. Aggregated forms of α-syn as well as monomeric α-syn were also cleaved by plasmin. Plasmin cleaved mainly the N-terminal region of α-syn and also inhibited the translocation of extracellular α-syn into the neighboring cells in addition to the activation of microglia and astrocytes by extracellular α-syn. Further, extracellular α-syn regulated the plasmin system through up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression. These findings help to understand the molecular mechanism of PD and develop new therapeutic targets for PD.     

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.     

ATP-sensitive potassium channel opener iptakalim protected against the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level

Mounting evidence reveals that ATP-sensitive potassium (K(ATP)) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinson's disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH-SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of K(ATP) channels. Moreover, our results showed that 1-methyl-4-phenyl-pyridinium ion (MPP+) induced up-regulation of mRNA for the Kir6.2 subunit and down-regulation of SUR1. It was further found that pretreatment with iptakalim, a novel K(ATP) channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH-SY5Y cell culture after exposure to MPP+. Trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+-induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-HD. These results suggest (i) K(ATP) channel dysfunction may be involved in the mechanisms of MPP+-induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoK(ATP) channels, thereby protecting SH-SY5Y cells against MPP+-induced cytotoxicity.     

Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells

Polyclonal antibodies against plasminogen activator inhibitor type-I (PAI-1) caused rapid retraction and rounding of substrate-attached HT- 1080 cells. The kinetics and extent of antibody-mediated cell rounding were not dependent on either urokinase or plasmin activity. Cells adherent to vitronectin-coated substrates detached within 2 h of antibody addition. Cells adherent to fibronectin were unaffected by the antibodies. Immunoblotting of substrate-attached material indicated that HT-1080 cells deposited PAI-1 into vitronectin, but not fibronectin, dependent contacts. These data suggest that the antibody- mediated cell rounding resulted from a steric disruption of vitronectin- dependent adhesions, indicating that the binding site on vitronectin for PAI-1 is near, but does not overlap, the binding site for vitronectin receptor. The accumulation of PAI-1 into vitronectin- dependent adhesion sites correlated temporally with the preferential degradation of fibronectin from the substrate. HT-1080 cells adherent to either fibronectin or vitronectin were able to activate exogenous plasminogen to plasmin. Plasmin levels were increased 200% on cells adherent to fibronectin and 100% on cells adherent to vitronectin. In the presence of a neutralizing antibody against PAI-1, vitronectin adherent cells activated plasminogen to the same extent as fibronectin adherent cells. Plasmin levels of 200% above baseline were associated with retraction of cells from the substrate. The ability of vitronectin adherent cells to activate exogenous plasmin was completely blocked in the presence of neutralizing antibodies against urokinase. These data represent the first demonstration that vitronectin-associated PAI-1 regulates urokinase in focal contact areas.     

TET2在帕金森病细胞模型中的表达及机制研究

目的:迄今为止,帕金森病(PD)发生的分子机制尚未完全阐明,本研究旨在体外细胞模型中寻找PD新型表观遗传标志物,探索其发病机制。方法:本次研究使用的细胞为神经母细胞瘤细胞系SH-SY5Y。首先,我们用CCK-8检测细胞活力,选取合适浓度的MPP+构建PD细胞损伤模型。再用PBS和MPP+分别处理SH-SY5Y细胞,用RT-qPCR检测了几个甲基化酶与去甲基化酶DNMT1,DNMT3A,DNMT3B及TET1, TET2, TET3的mRNA的表达水平,并用蛋白印迹检测TET2蛋白水平,免疫荧光检测了TET2蛋白定位。进一步用慢病毒转染SH-SY5Y细胞敲低TET2后,检测细胞增殖。结果:本研究发现,MPP+对SH-SY5Y细胞增殖的抑制具有时间与浓度依赖性,我们最终选择2.5 mM MPP+作为后续的细胞处理浓度。与对照组相比,MPP+处理细胞TET2的mRNA及蛋白水平表达均增加,且蛋白进入细胞核增加;同时发现,敲低TET2表达可以延缓MPP+对SH-SY5Y细胞增殖的抑制作用。结论:在当前的研究中,我们报道了TET2蛋白可能是PD新型的表观遗传学标志物,提示我们将来也许可以使用TET2抑制剂来治疗PD,因此本研究有可能为PD提供新的治疗方向和靶点。     

PEP-1-HO-1 prevents MPTP-induced degeneration of dopaminergic neurons in a Parkinson’s disease mouse model

Heme oxygenase-1 (HO-1) degrades heme to carbon dioxide, biliverdin, and Fe²⁺, which play important roles in various biochemical processes. In this study, we examined the protective function of HO-1 against oxidative stress in SH-SY5Y cells and in a Parkinson’s disease mouse model. Western blot and fluorescence microscopy analysis demonstrated that PEP-1-HO-1, fused with a PEP-1 peptide can cross the cellular membranes of human neuroblastoma SH-SY5Y cells. In addition, the transduced PEP-1-HO-1 inhibited generation of reactive oxygen species (ROS) and cell death caused by 1-methyl-4-phenylpyridinium ion (MPP⁺). In contrast, HO-1, which has no ability to transduce into SH-SY5Y cells, failed to reduce MPP⁺-induced cellular toxicity and ROS production. Furthermore, intraperitoneal injected PEP-1-HO-1 crossed the blood-brain barrier in mouse brains. In a PD mouse model, PEP-1-HO-1 significantly protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity and dopaminergic neuronal death. Therefore, PEP-1-HO-1 could be a useful agent in treating oxidative stress induced ailments including PD. [BMB Reports 2014; 47(10): 569-574]     

P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

Abnormalities of alpha-synuclein (ASN), the main component of protein deposits (Lewy bodies), were observed in Parkinson’s disease (PD), dementia with Lewy bodies, Alzheimer’s disease, and other neurodegenerative disorders. These alterations include increase in the levels of soluble ASN oligomers in the extracellular space. Numerous works have identified several mechanisms of their toxicity, including stimulation of the microglial P2X7 receptor leading to oxidative stress. While the significant role of purinergic signaling—particularly, P2 family receptors—in neurodegenerative disorders is well known, the interaction of extracellular soluble ASN with neuronal purinergic receptors is yet to be studied. Therefore, in this study, we have investigated the effect of ASN on P2 purinergic receptors and ATP-dependent signaling. We used neuroblastoma SH-SY5Y cell line and rat synaptoneurosomes treated with exogenous soluble ASN. The experiments were performed using spectrofluorometric, radiochemical, and immunochemical methods. We found the following: (i) ASN-induced intracellular free calcium mobilization in neuronal cells and nerve endings depends on the activation of purinergic P2X7 receptors; (ii) activation of P2X7 receptors leads to pannexin 1 recruitment to form an active complex responsible for ATP release; and (iii) ASN greatly decreases the activity of extracellular ecto-ATPase responsible for ATP degradation. Thus, it is concluded that purinergic receptors might be putative pharmacological targets in the molecular mechanism of extracellular ASN toxicity. Interference with P2X7 signaling seems to be a promising strategy for the prevention or therapy of PD and other neurodegenerative disorders.     

Biological control of tissue plasminogen activator-mediated fibrinolysis

Fibrinolysis, the body's ability to degrade fibrin, is an integrated part of hemostasis. Overactivity in the fibrinolytic system causes bleeding and underactivity causes thrombosis. Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), alpha 2-antiplasmin (alpha 2-AP) and plasminogen are definitely involved in fibrinolysis because: (1) these components can be assigned a fibrinolytic role in purified systems, i.e. in vitro, and (2) abnormal structural variants and abnormal levels of these components give rise to bleeding or to thrombosis. The biological control of tPA-mediated fibrinolysis is both cellular and humoral. The cellular regulation compasses synthesis of tPA and PAI-1 and release/uptake of these components. The humoral regulation involves: (1) the reaction between tPA and PAI-1; (2) the fibrin-stimulated plasminogen activation; (3) the reaction between plasmin and alpha 2-AP and (4) plasmin degradation of fibrin. The highly developed biological control of tPA-mediated fibrinolysis is indicative of its physiological importance.     

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP⁺) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP⁺-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]     

Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP⁺) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP⁺ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.     

Detoxified Extract of <Emphasis Type="Italic">Rhus verniciflua</Emphasis> Stokes Inhibits Rotenone-Induced Apoptosis in Human Dopaminergic Cells,SH-SY5Y

Rhus verniciflua Stokes (RVS), traditionally used as a food supplement and in traditional herbal medicine for centuries in Korea, is known to possess various pharmacological properties. Environmental neurotoxins such as rotenone, a specific inhibitor of complex I provide models of Parkinson’s disease (PD) both in vivo and in vitro. In this study, we investigated the neuroprotective effect of RVS against rotenone-induced toxicity in human dopaminergic cells, SH-SY5Y. Cells exposed to rotenone for 24 h-induced cellular injury and apoptotic cell death. Pretreatment of cells with RVS provided significant protection to SH-SY5Y cells. Further, RVS offered remarkable protection against rotenone-induced oxidative stress and markedly inhibited mitochondrial membrane potential (MMP) disruption. RVS also attenuated the up-regulation of Bax, Caspase-9 and Caspase-3 and down-regulation of Bcl-2. Moreover, pretreatment with RVS prevented the decrease in tyrosine hydroxylase (TH) levels in SH-SY5Y cells. Interestingly, RVS conferred profound protection to human dopaminergic cells by preventing the downregulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). These results suggest that RVS may protect dopaminergic neurons against rotenone-induced apoptosis by multiple functions and contribute to neuroprotection in neurodegenerative diseases, such as PD.     

Extracellular matrix degradation by cultured mesangial cells: mediators and modulators

Decreased degradation of the glomerular extracellular matrix (ECM) is thought to contribute to the accumulation of glomerular ECM that occurs in diabetic nephropathy and other chronic renal diseases. Several lines of evidence indicate a key role for the plasminogen activator/plasminogen/plasmin system in glomerular ECM degradation. However, which of the two plasminogen activators (PAs) present in renal tissue, tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is responsible for plasmin generation and those factors that modulate the activity of this system remain unclear. This study utilized mesangial cells isolated from mice with gene deletions for tPA, uPA, and plasminogen activator inhibitor 1 (PAI-1) to further delineate the role of the PA/plasminogen/plasmin system in ECM accumulation. ECM degradation by uPA-null mesangial cells was not significantly different from controls (92% +/- 1%, n = 12). In contrast, ECM degradation by tPA-null mesangial cells was markedly reduced (-78 +/- 1%, n = 12, P < 0.05) compared with controls, whereas tPA/uPA double-null mesangial cells degraded virtually no ECM. Previous studies from this laboratory have established that transforming growth factor-beta1 (TGFbeta1) inhibits ECM degradation by cultured mesangial cells by increasing the production of PAI-1, the major physiological PA inhibitor. In keeping with this observation, TGFbeta1 (1 ng/ml) had no effect on ECM degradation by PAI-1-null MC. High glucose levels (30 mM) in the presence or absence of insulin (0.1 mM) caused a moderate increase in ECM degradation by normal human mesangial cells. In contrast, glycated albumin, whose concentration is known to increase in diabetes, produced a dose-dependent (0.2-0.5 mg/ml) inhibition of ECM degradation by normal human mesangial cells. Taken together, these results document the importance of tPA versus uPA in renal plasmin production and indicate that in contrast to elevated glucose, glycated albumin may contribute to ECM accumulation in diabetic nephropathy.     

The AAA-ATPase VPS4 regulates extracellular secretion and lysosomal targeting of α-synuclein

Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that α-synuclein (αSYN), a principal culprit for Lewy pathology in Parkinson's disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular αSYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of αSYN but facilitated αSYN secretion. The hypersecretion of αSYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of αSYN and thereby contribute to the intercellular propagation of Lewy pathology in PD.