Deficient release of plasminogen activator inhibitor-1 from astrocytes triggers apoptosis in neuronal cells

doi:10.1016/S0169-328X(01)00133-4

Molecular Brain Research

Volume 91, Issues 1–2, 13 July 2001, Pages 96-103

https://doi.org/10.1016/S0169-328X(01)00133-4 Get rights and content

Abstract

Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the processes of peripheral tissue remodeling and fibrinolysis through the regulation of plasminogen activation. We found that cultured human astrocytes efficiently released PAI-1, and that both mRNA expression and protein release of PAI-1 were suppressed by pretreatment of the cells with daunorubicin. To examine the role of PAI-1 in the nervous system, neuronally differentiated PC-12 cells (PC-12 neurons) were maintained in a PAI-1-deficient culture medium derived from daunorubicin-pretreated astrocytes. The deficiency of PAI-1 in the medium caused a significant reduction in Bcl-2 and Bcl-X_L mRNAs and an increase in Bcl-X_S and Bax mRNAs in PC-12 neurons at 3 h. The changes in balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins caused caspase-3 activation following the release of cytochrome c from mitochondria. Apoptotic morphological change and DNA fragmentation were also observed in the neuronal cells at 24 h. Addition of exogenous PAI-1 protein to the inhibitor-deficient medium blocked the apoptotic changes in PC-12 neurons. However, addition of PAI-1 antibodies to control medium caused similar apoptotic changes in PC-12 neurons. During the apoptotic processes, plasminogen activator (PA) activity in the PAI-1-deficient medium was as low as the control level. The present data suggest that PAI-1 has physiological functions other than its role as PA inhibitor for the survival of neurons.

Introduction

Plasminogen activators (PAs) are serine proteases that generate plasmin from the inactive zymogen, plasminogen. Two types of PA have been identified in neuronal and non-neuronal systems: tissue-type PA (t-PA) and urinary PA (u-PA). t-PA is the primary PA in the brain [10], [15]. During embryonic and neonatal development, t-PA may play important roles in cell migration and tissue remodeling. In the adult nervous system, t-PA is induced in the hippocampus after seizures, kindling and long-term potentiation [16]. Moreover, t-PA and plasminogen have been demonstrated to be involved in the regulation of neuronal survival in response to excitotoxin [22], [23], [24]. Thus, like the peripheral PA-plasmin system, the specific inhibitor(s) may exist to regulate this neural protease cascade.

Recently, neuroserpin has been identified in the brain as a novel serine protease inhibitor (serpin) family [9], [14], [20]. It is secreted axonally during embryogenesis and in adult nervous systems and inhibits PAs and plasmin, but not thrombin [13]. This serpin may play a regulatory role in motor learning and neuronal survival [4]. PAI-1 was also found in the nervous system [17], [18]. However, the neural PAI-1 is not co-expressed with PAs in a pattern suggestive of the role of physiological regulator.

During our studies on the active roles of astrocytes in the survival and regeneration of neurons, we found that normal human astrocytes efficiently released PAI-1 in culture media and that the PAI-1 release was decreased by pretreatment of the cells with daunorubicin [8]. Daunorubicin, an anthracycline used in the treatment of leukemia, causes an increase in ceramide levels in cells via activation of ceramide synthase [2] or of sphingomyelinase [7]. Therefore, the generated ceramide may play a regulatory role in the PAI-1 release from human astrocytes [8] and vascular endothelial cells [21]. Here, we examine how the insufficient release of PAI-1 from astrocytes affects the survival of neuronally differentiated PC-12 cells (PC-12 neurons) by maintaining the neuronal cells in the culture media derived from daunorubicin-treated and -untreated astrocytes. We report that sufficient PAI-1 in the extracellular environments of neurons prevents apoptotic changes and that the anti-apoptotic effect of PAI-1 is due to a function other than that of PA inhibitor.

Section snippets

Materials

The following materials were obtained commercially: daunorubicin from Sigma; nerve growth factor (NGF) from CHEMICON International, Temecula, CA, USA; rat PAI-1 and goat anti-human PAI-1 IgG from American Diagnostica, Greenwich, CT, USA; goat anti-human whole serum IgG from ICN Pharmaceuticals, Aurora, OH, USA; Ac-Asp-Glu-Val-Asp 4-methylcoumaryl-7-amide (Ac-Asp-Glu-Val-MCA), pyroGlu-Gly-Arg 4-methylcoumaryl-7-amide (pyroGlu-Gly-Arg-MCA) and 7-amino-4-methylcoumarine (AMC) from Peptide

Effect of daunorubicin on the release of PAI-1 from cultured human normal astrocytes

Astrocytes, maintained in a serum-free GIT medium, released PAI-1 in a time-dependent manner, and the PAI-1 antigen levels in the media at 24 h reached 68.1±3.54 ng/ml (Fig. 1A). On the other hand, t-PA antigen levels determined at 24 h were very low (4.43±0.15 ng/ml, data not shown), suggesting that the balance between t-PA (∼70 kDa) and PAI-1 (∼50 kDa) in the culture medium largely shifts to PA inhibition. Next, to examine the regulatory role of intracellular ceramide in astrocytes for the

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan and by funds from the Central Research Institute of Fukuoka University.

References (25)

R. Bose et al.
Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals
Cell
(1995)
G.A. Hastings et al.
Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons: implication for the regulation of motor learning and neuronal survival
J. Biol. Chem.
(1997)
S.A. Hill et al.
Differential mechanisms targeting type 1 plasminogen activator inhibitor and vitronectin into the storage granules of a human megakaryocytic cell line
Blood
(1996)
P. Li et al.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade
Cell
(1997)
X. Liu et al.
DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis
Cell
(1997)
T. Osterwalder et al.
The axonally secreted serine proteinase inhibitor, neuroserpin, inhibits plasminogen activators and plasmin but not thrombin
J. Biol. Chem.
(1998)
R.N. Pittman
Release of plasminogen activator and a calcium-dependent metallo-protease from cultured sympathetic and sensory neurons
Dev. Biol.
(1985)
S.P. Schrimpf et al.
Human neuroserpin (PI 12): cDNA cloning and chromosomal localization to 3q 26
Genomics
(1997)
S. Soeda et al.
Tumor necrosis factor-α-induced release of plasminogen activator inhibitor-1 from human umbilical vein endothelial cells: involvement of intracellular ceramide signaling event
Biochim. Biophys. Acta
(1998)
J.M. Adams et al.
The Bcl-2 protein family: arbiters of cell survival
Nature
(1998)

M. Enari et al.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Nature

(1998)

Y. Hu et al.

Bcl-X_L interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Proc. Natl. Acad. Sci. USA

(1998)

Cited by (40)

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma
2020, Pathology Research and Practice
The urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.
UPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.
UPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ² = 8.372, P =  0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P =  0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025).
Our data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.
uPA affects the CRSsNP nasal mucosa epithelium apoptosis by regulating WIF1
2019, Experimental Cell Research
Citation Excerpt :
Finally, uPA–uPAR system could enhance T-cell effector function such as latent proinflammatory cytokines (pro-TGF-b, pro-IL-1 and pro-IL-6) [14]. In addition, several studies have demonstrated that the uPA–uPAR system participates in tumorigenesis and inhibition of apoptosis [15–18]. Recently, researchers have shown that there is a clear increase in the uPA expression level in CRS [19].
Chronic rhinosinusitis without nasal polyps (CRSsNP) is the main type of Chronic rhinosinusitis (CRS) and is a common otorhinolaryngologic disease worldwide. However, the mechanisms of CRSsNP remain poorly understood. In this study, C57BL/6J wild-type and urokinase-type plasminogen activator (uPA) gene knockout (uPA^-/-) mice were used to construct the CRSsNP model. Primary human nasal epithelial cells (HNEC) were isolated from CRSsNP patient and treated with uPA knockdown/overexpression lentivirus. CCK-8 and Annexin-V/PI staining were used to detected cell proliferation and apoptosis. In vivo, we found that uPA depletion alleviated mucosal inflammation in the CRSsNP mice model. Wnt inhibitory factor 1 (WIF1) was upregulated in the uPA^-/- CRSsNP mice model. In vitro, inhibition of uPA increased cell proliferation and decreased cell apoptosis. Mechanistically, uPA depletion upregulated WIF1 and BCL2 expression, and reduced the expression level of BAX in CRSsNP HNEC. In contrast, decreased cell proliferation and increased cell apoptosis were observed after uPA overexpression. Consistently, a reduction in WIF1 and BCL2 expression levels and an increase in the BAX expression level were observed upon uPA ectopic expression. Furthermore, WIF1 overexpression rescued the effects caused by uPA overexpression in vitro. In conclusion, uPA affects the CRSsNP nasal mucosal epithelium cell apoptosis by upregulating WIF1. To our knowledge, this is the first study to explore the role of uPA in CRSsNP to date.
Novel Cell Surface Targets for the Plasminogen Activating System in the Brain: Implications for Human Disease
2018, Cell Surface GRP78, a New Paradigm in Signal Transduction Biology
The plasminogen (Pg) activator system plays an important role in the central nervous system (CNS), including processes of neuronal migration, neurite outgrowth, and neuronal plasticity. Pg and tissue-type plasminogen activator (t-PA) are primarily involved in fibrinolysis, however, they also play critical nonfibrinolytic functions in the CNS. Activation of Pg in the CNS is stimulated by unique cell surface protein complexes. One of such complexes involves the voltage-dependent anion channel 1 (VDAC1) in association with the Glucose Regulated Protein 78,000 (GRP78), t-PA, and Pg. VDAC1 is localized both at the mitochondrial and cell plasma membranes. In the mitochondria, VDAC1 transports ions, cholesterol, and metabolites across the outer mitochondrial and participates in the release of mitochondrial proapoptotic proteins to the cytosol. In cell plasma membranes, VDAC1 participates in the maintenance of redox homeostasis in normal cells and the promotion of anion efflux in apoptotic cells. VDAC and GRP78 are major players in the physical association between the endoplasmic reticulum (ER) and mitochondria which enables highly efficient transmission of Ca²⁺ from the ER to mitochondria under both physiological and pathological conditions. GRP78 associates with VDAC1 both at the outer mitochondrial and plasma membranes. The brain is an organ devoid of fibrin, a protein essential for efficient Pg activation, however, GRP78 and VDAC mimic fibrin to enhance the activation of locally synthesized Pg and t-PA. These functions make VDAC1 and GRP78 a point of convergence for a large variety of cell survival and death signals.
Obesity and dementia: Adipokines interact with the brain
2014, European Neuropsychopharmacology
Citation Excerpt :
In the periphery, PAI-1 inhibits fibrinolysis and regulates cell migration and angiogenesis (see Figure 1/Table 1). Even today, PAI-1 transport mechanisms across the BBB are unknown, so only PAI-1 produced by microglia has been shown to regulate apoptosis, energy intake, survival of neurites, microglia migration, neuroinflammation and the brain׳s immune system (Ahn et al., 1999; Jeon et al., 2012; Masos and Miskin, 1997; Miskin and Masos, 1997; Soeda et al., 2008, 2001). Although, it should be mentioned that these results are primarily obtained from in vitro experiments, thus it is still questionable if these processes are affected in human obesity.
Obesity is a pandemic and a serious global health concern. Obesity is a risk factor for multiple conditions and contributes to multi-morbidities, resulting in increased health costs and millions of deaths each year. Obesity has been associated with changes in brain structure, cognitive deficits, dementia and Alzheimer׳s disease. Adipokines, defined as hormones, cytokines and peptides secreted by adipose tissue, may have more widespread influence and functionality in the brain than previously thought. In this review, six adipokines, and their actions in the obese and non-obese conditions will be discussed. Included are: plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), tumor necrosis factors alpha (TNF-α), angiotensinogen (AGT), adiponectin and leptin. Their functionality in the periphery, their ability to cross the blood brain barrier (BBB) and their influence on dementia processes within the brain will be discussed.
Adipokines: A link between obesity and dementia?
2014, The Lancet Neurology
Being overweight or obese, as measured with body-mass index or central adiposity (waist circumference), and the trajectory of body-mass index over the life course have been associated with brain atrophy, white matter changes, disturbances of blood–brain barrier integrity, and risk of all-cause late-onset dementia and Alzheimer's disease. This observation leads us to question what it is about body-mass index that is associated with health of the brain and dementia risk. If high body-mass index and central adiposity represent an increase in adipose tissue, then the endocrine function of adipose tissue, mediated by adipose tissue hormones and adipokines, could be a clue to mechanisms that underlie the association with dementia and Alzheimer's disease. Hundreds of adipokines have been identified, creating a complexity that is a challenge to simplify. Nonetheless, adipokines are being investigated in association with clinical dementia outcomes, and with imaging-based measures of brain volume, structure, and function in human beings and in preclinical models of clinical dementia.
uPA and PAI-1-related signaling pathways differ between primary breast cancers and lymph node metastases
2012, Translational Oncology
The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell- related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β).Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node- negative breast cancer tissues.

View all citing articles on Scopus

View full text

Research reportDeficient release of plasminogen activator inhibitor-1 from astrocytes triggers apoptosis in neuronal cells

Abstract

Introduction

Section snippets

Materials

Effect of daunorubicin on the release of PAI-1 from cultured human normal astrocytes

Acknowledgements

Cell

J. Biol. Chem.

Blood

Cell

Cell

J. Biol. Chem.

Dev. Biol.

Genomics

Biochim. Biophys. Acta

The Bcl-2 protein family: arbiters of cell survival

Nature

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Nature

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Proc. Natl. Acad. Sci. USA

Research report
Deficient release of plasminogen activator inhibitor-1 from astrocytes triggers apoptosis in neuronal cells

Bcl-X_L interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation