HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/2/277 02-0403fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GABRIEL, C. Articles by VIVIEN, D. Search for Related Content PubMed PubMed Citation Articles by GABRIEL, C. Articles by VIVIEN, D.

Transforming growth factor -induced expression of type 1 plasminogen activator inhibitor in astrocytes rescues neurons from excitotoxicity¹

Université de CAEN, UMR CNRS 6551, IFR 47, Centre CYCERON, Bd H. Becquerel, BP 5229, 14074 CAEN Cedex, France

⁵Correspondence: Université de CAEN, UMR CNRS 6551, Centre CYCERON, 14074 CAEN Cedex, France. E-mail: d.vivien{at}neuro.unicaen.fr; a.buisson{at}neuro.unicaen.fr

SPECIFIC AIMS

Excitotoxicity is a hallmark of neuronal cell death in acute brain pathologies, including stroke. Transforming growth factor (TGF-), a member of the epidermal growth factor (EGF) family, has been suggested to protect neurons against excitotoxic and ischemic brain injuries. In the present study, we have investigated the molecular mechanism by which TGF- could protect neurons against excitotoxicity-induced neuronal death in vitro.

PRINCIPAL FINDINGS

1. Neuroprotective activity of TGF- against NMDA-induced neuronal death is mediated by astrocytes
We tested whether TGF- modulated neuronal death in primary cortical cell cultures subjected to apoptotic or necrotic paradigms. We observed that TGF- (1–50 ng/mL) failed to protect neurons against apoptosis induced by either serum deprivation in cortical neuronal cultures (7 days in vitro, [DIV 7]) or exposure of mixed cortical cultures (DIV14) to nifedipine (50 µM). We studied the influence of TGF- on necrotic neuronal death induced by exposure to NMDA (12.5 µM for 24 h). Although the presence of TGF- (from 1 to 50 ng/mL) reduced NMDA-induced neuronal death in mixed cultures of neurons and astrocytes (Fig. 1 A), it failed in pure cultures of neurons (Fig. 1B ). Overall, these data show that TGF- exerts its neuroprotective activity against NMDA-mediated excitotoxicity in the obligatory presence of astrocytes.

View larger version (40K): [in this window] [in a new window]   Figure 1. TGF- protects neurons against NMDA-induced excitotoxicity in cortical cultures containing neurons; astrocytes are associated with a decrease on NMDA-induced t-PA activity. A, B) Neuronal death (%) was estimated by LDH release in the bathing medium of mixed cortical cultures (A) (mean±SE, n=16) or pure cultures of neurons (B) (mean±SE, n=12) exposed to NMDA in the presence or absence of TGF-. *Significantly different from NMDA by one-way ANOVA, followed by Bonferroni Dunn’s test (P<0.0001). C, D) Bathing medium harvested from mixed cortical cultures (C) or pure cultures of neurons (D) exposed to NMDA in the presence or absence of TGF- was subjected to a PAGE-plasminogen zymography in order to measure the activity of t-PA.

2. The neuroprotective activity of TGF- is associated with a decrease in t-PA activity
Previous data have reported that endogenous tissue-type plasminogen activator (t-PA) released by neurons could potentiate the NMDA-induced excitotoxicity by enhancing NMDA receptor-mediated calcium influx. To investigate whether the effect of TGF- could be mediated by an inhibition of t-PA activity, we performed a plasminogen zymography assay on bathing medium of mixed cultures or neuronal cultures exposed to NMDA in the presence of TGF-. Although TGF- pretreatment (50 ng/mL for 6 h) in mixed cultures blocked NMDA-induced (100 µM for 1 h) t-PA activity in the bathing media (Fig. 1C ), it failed in pure neuronal cultures (Fig. 1D ). Thus, TGF--mediated neuroprotection against excitotoxicity is associated with a decrease of t-PA activity, a mechanism that involves astrocytes.

3. TGF- induces ERKs phosphorylation in astrocytes
We have attempted to identify the signal transduction pathway by which TGF- could mediate its neuroprotective activity. Although in astrocytes TGF- (10 ng/mL) induced a rapid increase in the phosphorylation of p44 (ERK1) and p42 (ERK2) MAPKs, it was unable to induce modulation of ERKs activity in neuronal cultures. Moreover, coincubation of astrocytes with a MAPK kinase (MEK) inhibitor, U0126 (1 µM), prevented TGF--induced phosphorylation of ERKs.

4. PAI-1 mediates the neuroprotective activity of TGF- against NMDA-induced neuronal death
Based on the above data, we have hypothesized that the type 1 inhibitor of the tissue type plasminogen activator (PAI-1) could mediate the neuroprotective activity of TGF-. We observed an enhanced expression of PAI-1 mRNAs (Fig. 2 A) and protein (Fig. 2B ) in astrocytes after TGF- (10 ng/mL) treatment for 6 h. We showed that inhibition of ERKs activation by using U0126 prevented TGF--induced PAI-1 expression.

View larger version (61K): [in this window] [in a new window]   Figure 2. ERKs activation mediates TGF--induced up-regulation of PAI-1 in astrocytes and subsequent neuroprotective activity. A, B) Cortical astrocytes were treated with TGF- (10 ng/mL) for 6 h in the presence of the MAPK inhibitor U0126 (1 µM). A) Expression of PAI-1 mRNAs was evaluated by semiquantitative RT-PCR. B) Extracellular concentrated medium was subjected to SDS-PAGE, transferred to membranes, and revealed with an antibody raised against PAI-1. Control: C; TGF--treated astrocytes: T; (TGF-+U0126)-treated astrocytes: T+U. C) Neuronal death (%) was estimated by LDH release (mean±SE, n=16) in the bathing medium of mixed cortical cultures after exposure to NMDA in the presence or absence of either TGF- or an antibody raised against PAI-1. *Significantly different from NMDA alone; #significantly different from NMDA + TGF- by one-way ANOVA, followed by Bonferroni Dunn’s test (P<0.0001). D) Immunocytochemistry performed with an antibody raised against MAP-2 in mixed cortical cultures after 24 h of exposure to NMDA 12.5 µM: Scale bars: 100 µm. E) Mixed cultures containing either wild-type (WT) or PAI-1-deficient (PAI-/-) astrocytes were exposed to NMDA in the presence of TGF- (50 ng/mL). Neuronal death (%) was estimated by measurement of LDH release in the bathing medium (mean±SE, n=16). *Significantly different from NMDA alone by multiparametric ANOVA, followed by Bonferroni Dunn’s test (P<0.0001). F) Bathing medium, harvested from mixed cortical cultures, exposed to (6 h of pretreatment) TGF- alone or with the MAPK inhibitor U0126 in the presence or in the absence of NMDA for 1 h, was subjected to a PAGE plasminogen zymography in order to measure the activity of t-PA. Data presented are representative of 3 independent experiments.

To clarify the involvement of PAI-1 in the neuroprotective activity of TGF-, NMDA-induced toxicity in mixed cultures was performed in the presence of an antibody raised against PAI-1. This antibody blocked the neuroprotective effect of TGF- as estimated by measurement of LDH release (Fig. 2C ) and by immunocytochemistry performed with an antibody raised against MAP-2 (Fig. 2D ). Although TGF- (50 ng/mL) significantly rescued neurons from NMDA-induced death in wild-type mixed cultures, it failed to protect neurons in mixed cultures containing PAI-1-deficient astrocytes (Fig. 2E ). To validate that the neuroprotective activity of TGF- requires the activity of PAI-1 and ERKs, additional zymogram assays from mixed cultures treated with U0126 were performed. Addition of U0126 (1 µM) prevented the effect of TGF- on t-PA activity after NMDA exposure in mixed cultures (Fig. 2F ).

Taken together, these data demonstrate that the neuroprotective effect of TGF- against NMDA-induced excitotoxicity requires overexpression of PAI-1 in astrocytes by an ERK signaling pathway-dependent mechanism and is associated with a decrease of t-PA activity.

CONCLUSIONS

In the present study, we noted that TGF- mediates a protective effect against NMDA-induced excitotoxic neuronal death through a mechanism requiring the obligatory presence of astrocytes. In vivo, TGF- is neuroprotective after permanent or transient focal ischemia and intraparenchymal injection of quinolinic acid in rats. Previous studies have shown that TGF- was able to promote neuronal survival for dopaminergic neurons in vitro. Until now, the effects of TGF- observed in vitro required the participation of astrocytes, and only few studies reported a direct biological effect of TGF- in neurons.

In this work, we demonstrate a neuroprotective role of TGF- against NMDA-induced excitotoxicity in mixed cortical cultures. We observed that TGF- activates the ERKs pathway only in cultured astrocytes. This lack of neuronal response explains the selective neuroprotective activity of TGF- against NMDA-mediated death. Finally, we saw that this neuroprotective effect was due to an ERK-dependent up-regulation of PAI-1.

PAI-1 is characterized as a potent t-PA inhibitor in many systems. The serine protease t-PA plays a key role in the metabolism of the extracellular matrix by converting plasminogen into plasmin. In the brain, use of recombinant t-PA has been approved by the Food and Drug Administration as treatment for stroke. Although t-PA is produced mainly by endothelial cells, it has also been detected in neurons. t-PA-deficient mice are resistant to glutamate-induced excitotoxicity, which occurs after ischemia. t-PA potentiates NMDA-induced calcium influx in neurons.

In conclusion, our results suggest that the neuroprotective activity of TGF- against NMDA-induced neuronal death could be a consequence of the inhibition of the effect of t-PA on the glutamatergic neurotransmission. Altogether, these data underscore the role of astrocytes as key elements in the control of neuronal outcome after acute neurodegenerative disorders such as cerebral ischemia. The role of serine protease inhibitors and their respective serine proteases, such as the PAI-1/t-PA axis, in the control of excitotoxic neuronal death is clearly highlighted by the present study.

View larger version (61K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0403fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0403fje

² Equal contribution to this work.

³ Present address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

⁴ Present address: School of Biological Sciences, Molecular Neuropatholology Department, University of Southampton, Southampton S0167PX, UK.

This article has been cited by other articles:

				K. M. Dhandapani, F. M. Wade, V. B. Mahesh, and D. W. Brann Astrocyte-Derived Transforming Growth Factor-{beta} Mediates the Neuroprotective Effects of 17{beta}-Estradiol: Involvement of Nonclassical Genomic Signaling Pathways Endocrinology, June 1, 2005; 146(6): 2749 - 2759. [Abstract] [Full Text] [PDF]

				E. O. Lee, J. L. Kang, and Y. H. Chong The Amyloid-{beta} Peptide Suppresses Transforming Growth Factor-{beta}1-induced Matrix Metalloproteinase-2 Production via Smad7 Expression in Human Monocytic THP-1 Cells J. Biol. Chem., March 4, 2005; 280(9): 7845 - 7853. [Abstract] [Full Text] [PDF]

				J. Liu, S. Yin, N. Reddy, C. Spencer, and S. Sheng Bax Mediates the Apoptosis-Sensitizing Effect of Maspin Cancer Res., March 1, 2004; 64(5): 1703 - 1711. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/2/277 02-0403fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GABRIEL, C. Articles by VIVIEN, D. Search for Related Content PubMed PubMed Citation Articles by GABRIEL, C. Articles by VIVIEN, D.