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(The FASEB Journal. 1999;13:1315-1324.)
© 1999 FASEB

Transforming growth factor-ß1 as a regulator of the serpins/t-PA axis in cerebral ischemia

FABIAN DOCAGNE, OLIVIER NICOLE, HUGO H. MARTI*, ERIC T. MacKENZIE, ALAIN BUISSON and DENIS VIVIEN1

Université de Caen, CNRS UMR 6551, IFR 47, bd H. Becquerel, BP 5229, 14074 Caen Cedex, France; and
* Max-Planck-Institut, für physiologische und klinische Forschung, D-61231 Bad-Nauheim, Germany

1Correspondence: Université de CAEN-CNRS UMR 6551, Laboratoire de Neurosciences, BP 5229, 14074 Caen Cedex, France. E-mail: d.vivien{at}neuro.unicaen.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-ß1 (TGF-ß1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT-PCR and Western blotting, we observed that only the expression of PAI-1 was modulated by TGF-ß1 treatment via a TGF-ß-inducible element contained in the PAI-1 promoter (CAGA box). Finally, we compared the specificity of TGF-ß1 action with other members of the TGF-ß family by using luciferase reporter genes. These data show that TGF-ß and activin were able to induce the overexpression of PAI-1 in astrocytes, but that bone morphogenetic proteins, glial cell line-derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t-PA axis and the mechanism by which TGF-ß may be neuroprotective.—Docagne, F., Nicole, O., Marti, H. H., MacKenzie, E. T., Buisson, A., Vivien, D. Transforming growth factor-ß1 as a regulator of the serpins/t-PA axis in cerebral ischemia.


Key Words: TGF-ß • plasminogen activator • neuroserpin • NTN


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE BALANCE BETWEEN the accumulation and degradation of the extracellular matrix is essential to an array of physiological and pathological processes. Tissue type plasminogen activator (t-PA)2 is a serine protease that catalyzes the activation of the plasminogen into an active protease plasmin (1) , which plays a critical role in the metabolism of the extracellular matrix. t-PA expression has been correlated with hippocampal function, and its mRNA level is rapidly increased in relation with neuronal activity (2) . Furthermore, t-PA-deficient mice are resistant to neuronal cell death induced either by excitotoxins (3) or ischemic injury (4) . These data suggest that excessive neuronal activity can lead to neuronal death through the t-PA proteolytic loop.

Transforming growth factor-ß1 (TGF-ß1) is the prototype of a large family of peptides, including BMPs (bone morphogenetic proteins) and activin (5) . TGF-ßs, BMPs, and activin elicit their effects by binding to their own set of cell surface serine/threonine kinase receptors (6 , 7) expressed in cortical neurons and astrocytes (8) . The activated TGF-ß receptors (TßR-I and TßR-II) induce the phosphorylation of two intracellular proteins, Smad2 and Smad3 (9 , 10) , which form hetero-oligomeric complexes with Smad4 (11 , 12) . These complexes translocate to the nucleus, where they regulate transcriptional responses (13) . More recently, two novel members of the TGF-ß family were identified in the CNS as GDNF (glial cell line-derived neurotrophic factor) and neurturin (NTN), which transduce their signal through a tyrosine kinase receptor, c-Ret (14 , 15) .

TGF-ßs have been found to be abundantly expressed in the central nervous system (CNS) and are characterized as injury-related peptide growth factors (16 17 18 19 20 , 8) . In addition to its potentially neuroprotective effects (21 , 22) , TGF-ß1 regulates biological activity in astrocytes and microglia (18) . TGF-ß1 is known to activate the transcription of mammalian genes important for cell cycle regulation, (23 24 25) as well as some involved in the extracellular matrix metabolism, such as the plasminogen activator inhibitor type I (PAI-1), which controls the activity of t-PA (26 27 28 29) . Recently, a DNA element (CAGA box) in the PAI-1 promoter was identified as that with which the transcriptional factors Smad3 and Smad4 directly interact after TGF-ß treatment (30) . We demonstrated in vitro that an up-regulation of PAI-1 in astrocytes mediates the neuroprotective activity of TGF-ß1 against NMDA-induced excitotoxicity (31) .

These data indicate that 1) t-PA may play a critical role in neuronal death induced by excitotoxins; 2) TGF-ß1 regulates the neuronal cell death induced by excitotoxins; and 3) PAI-1 is a target for the action of TGF-ß. Based on this literature, as a first step we investigated the changes in serpins and t-PA expression induced by focal cerebral ischemia in mice. Second, we studied the effect of several members of the TGF-ß family such as TGF-ß, activins, BMPs, GDNF, and NTN on the regulation of these serpins in cultured cortical astrocytes and neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The polymerase chain reaction (PCR) kit was purchased from Life Technologies (Cergy Pontoise, France); the reverse transcriptase system kit was obtained from Promega (Paris, France). Protein-G Sepharose was from Pharmacia Biotech (Uppsala, Sweden). The luciferase assay kit was purchased from Promega; Lipofectamine was obtained from Life Technologies. [35S]Methionine and 35S-cysteine were obtained from NEN (Les Ulis, France). Modified Eagle's medium (MEM), cytosine arabinoside, horse serum, and fetal bovine serum were obtained from Sigma Chemical Co. (Isle D'Abeau, France), and poly-D-lysine was from Life Technologies. Anti-GFAP antibody was purchased from Sigma Chemical Co., and anti-PAI-1 antibody was from American Diagnostica (Greenwich, Conn.). All other chemicals used were obtained from Sigma Chemical Co.

Cell cultures
Cortical astrocyte cell cultures were prepared from 1- to 3-day postnatal mice (32) . Cerebral cortices were dissected and incubated for 20–30 min in 0.025% trypsin in stock media (MS, Eagle's MEM augmented with 2 mM glutamine and 25 mM glucose) and transferred to MS supplemented with 10% fetal bovine and 10% horse sera for trituration. Dissociated cells were plated at a density of ~3 x 105 cells per well. Cultures were kept at 37°C in a humidified 5% CO2-containing atmosphere. The medium was changed once weekly. Experiments were performed on cultured cortical astrocytes after 21 days in vitro.

Near-pure neuronal cell cultures, containing less than 5% astrocytes, were prepared as previously detailed (33) from 14- or 15-day-old embryonic mice. Dissociated cortical cells in MS supplemented with 5% fetal bovine and 5% horse sera were plated in multiwell vessels that had previously been coated with poly-D-lysine. After 3 to 5 days in vitro, cultures were treated with 3 µM cytosine arabinoside in order to inhibit the proliferation of astrocytes. There was no further exchange of the medium. Experiments were performed on neuronal cultures after 12–13 days in vitro.

Semiquantitative reverse transcriptase PCR
Total RNAs were prepared from cerebral cortex, cultured neurons, and astrocytes by a phenol/chloroform extraction method using the RNA Ble extraction kit (Eurobio, Paris, France). Samples (1 µg) of total mRNA were transcribed into cDNA using poly-dT oligonucleotides. An aliquot of the produced cDNAs was amplified using sense and antisense primers for PAI-1, t-PA, NSP, PN-1, and ß-actin, respectively (see Table 1 ). For the semiquantitative experiments, an aliquot of cDNA libraries (1 µl from 2O µl) was amplified by PCR with specific oligonucleotides for ß-actin (30 cycles of PCR were chosen corresponding to the 50% of the saturation curve of the PCR products). PCR products were cloned in pGEMT-4Z and their identity was confirmed by sequencing. Amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. PCR experiments for t-PA, PAI-1, NSP, and PN-1 were performed only if the expression pattern obtained for the housekeeping gene ß-actin showed no significant modification between our different samples.


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  		Table 1. Sense and antisense primers for PAI-1, t-PA, NSP, PN-1, and ß-actin

In situ hybridization
The technique used for in situ hybridization was previously described by Breier et al. (34) . Briefly, RNA probes were generated by in vitro transcription of the plasmid pGEMT4Z containing a partial mouse PAI-1 cDNA, spanning the base pairs from 466 to 772. This partial cDNA was derived from our own reverse transcriptase (RT) -PCR product, cloned in PGEMT4Z, and characterized by sequencing. Single-stranded antisense or sense RNA probes were generated using 100 µCi 35S-UTP and T7 or SP6 RNA polymerases, respectively, as described by the manufacturer (Stratagene, San Diego, Calif.). Frozen brains embedded in tissue tec were sectioned in cryostat. Sections (10 µM) were melted on silane-coated glass slides. Sections were incubated in 2x SSC at 70°C, digested with Pronase (40 µg/ml), fixed in 4% paraformaldehyde, and acetylated with acetic anhydride diluted 1:400 with 0.1 M triethylamine. Hybridization was performed in buffer containing 50% formamide, 10% dextran sulfate, 10 mM Tris-HCl (pH 7.5), 10 mM sodium phosphate (pH 6.8), 2x SSC, 5 mM EDTA, 150 µg/ml yeast tRNA, 0.1 mM UTP, 1 mM ADPßS, 1 mM ATP{gamma}S, 10 mM dithiothreitol, 10 mM 2-mercaptoethanol, and 2.5 x 104 cpm/ml 35S-labeled RNA probe overnight at 48°C. Sections were washed in 2x SSC/50% formamide at 37°C overnight and dehydrated in graded ethanol. Then slices were coated with Kodak NTB-2 emulsion (Eastman Kodak, Rochester, N.Y.) diluted 1:1 in water and exposed for 22 days. Slides were developed and counterstained with Toluidine blue, air dried, and mounted.

Western blot experiments
Cells were incubated for 6 h at 37°C in the presence of TGF-ß1 (1 ng/ml). Cells were removed by washing three times in a solution containing 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate with 1 mM phenylmethylsulfonyl fluoride. Cell matrix proteins were extracted and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of dithiothreitol (DTT) and ß-mercaptoethanol; thereafter, the proteins were transferred to a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Bedford, Mass.). Membranes were blocked and probed with an antibody raised against PAI-1 (sheep anti-mouse PAI-1; Orthoclinical, France) and revealed by a combination of both biotinylated rabbit anti-sheep antibody and a peroxidase-conjugated streptavidin reagent. Blots were finally developed with an enhanced chemiluminescence Western blotting detection system (NEN).

PAI-1 metabolic labeling
Cells were incubated in methionine/cysteine-free MEM containing 50 mCi/ml of [35S]methionine/35S-cysteine (Trans-label, NEN, 1100 Ci/mmol) for 6 h at 37°C in the presence of TGF-ß1 (1 ng/ml). Cells were removed by washing three times in a solution containing 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate with 1 mM phenylmethylsulfonyl fluoride. Cell matrix proteins were extracted and SDS-PAGE electrophoresis was performed in the presence of DTT and ß-mercaptoethanol.

Immunoprecipitation
Metabolically labeled cells were solubilized in 1 ml of lytic buffer in the presence of protease inhibitors at 4°C for 30 min. Through the use of specific antibodies, immunoprecipitations were performed from the supernatant of the lysis solution for 2 h at 4°C and followed by adsorption to protein-G-Sepharose (Pharmacia Biotech). Bound proteins were eluted in DTT and ß-mercaptoethanol containing a loading sample buffer; SDS-PAGE electrophoresis was then performed.

Transfection and luciferase assays
Cells were transiently transfected with the indicated constructs using the lipofection method (Lipofectamine, Life Technologies) and stimulated with 1 ng/ml of human recombinant TGF-ß1 (R&D, Abingdon, U.K.). Luciferase activities were quantified 24 h later with a commercial kit (Promega). Values were normalized with respect to the Renilla luciferase activity as expressed by the cotransfected control vector pRL-TK (Promega).

Immunocytochemistry
Cell cultures were fixed in the presence of 4% paraformaldehyde/phosphate-buffered saline (PBS). The preparations were then washed in PBS, followed by 20 min of treatment with PBS containing 1% of bovine serum albumin and 0.1% of Tween-20. Primary antibodies were incubated overnight at 4°C, and the appropriate biotinylated secondary antibodies were added for an additional 2 h. The cell culture was incubated in the VECTASTIN ABC reagent and developed in a substrate for peroxidase. Controls were performed by omitting primary antibodies.

Surgical procedure
Surgical procedures were conducted according to national legislation and as described previously by Gotti et al. (35) . Briefly, male wild-type mice (OF-1) weighing 20 to 25 g were anesthetized with an intraperitoneal chloral hydrate injection (500 mg/kg, i.p.); then a skin incision was made between the eye and the ear. The parotid gland and surrounding soft tissues were reflected downward and the underlying temporalis was incised. The tissue was retracted until the MCA (left middle cerebral artery) was visible through the surface of the skull. A craniectomy was performed and the MCA was coagulated. The temporalis and the parotid gland were replaced and the incision sutured. Mice were placed in a warm environment until they recovered from anesthesia. Extraction of total RNA was performed as described above in both ipsilateral and contralateral cerebral cortices and compared with sham-operated mice. The infarcted volume estimated 24 h after the beginning of the occlusion represents a reproducible lesion of 30 mm3 restricted to the cortex, with a standard deviation of less than 10% in a group of 10 animals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression pattern of serpins and t-PA mRNAs after cerebral ischemia in mice
Several serine protease inhibitors (serpins) have been characterized in the CNS such as NSP (36) , PN-1, (37) , and PAI-1, potent inhibitors for t-PA (38) . We studied the expression of these serpins and t-PA after cerebral ischemia in mice (Fig. 1 ). After middle cerebral artery occlusion, total RNAs from both ipsilateral and contralateral cortices were harvested. mRNA samples (1 µg) were reverse transcribed with poly-dT oligonucleotides. An aliquot of cDNA libraries (1 µl from 20 µl) was amplified by PCR with specific oligonucleotides for ß-actin. Then 1 µl of the same cDNA libraries was amplified with sets of specific oligonucleotides. As shown in Fig. 1 , all our PCR reactions showed the same level of expression of the chosen housekeeping gene (ß-actin). Each set of the oligonucleotides used gave us products of the expected size (252 bp for t-PA; 322 bp for PAI-1; 224 bp for PN-1; 400 bp for NSP; and 539 bp for ß-actin). To test the specificity of the PCR products, each was cloned and sequenced. Furthermore, a PCR control using ß-actin-specific primers was performed with a representative RNA sample harvested from the cerebral cortices of mice to rule out any genomic DNA contamination. The PAI-1 cDNA products obtained from both ipsilateral and contralateral cortices from the 12 h sham-operated mice failed to show any significant modification. In contrast, the expression of the PAI-1 mRNAs was dramatically enhanced between 24 h and 3 days, whereas no significant modification of the expression level of the mRNAs coding for PN-1, NSP, or t-PA was observed. By using our cloned PAI-1-PCR product, in situ hybridization using 35S-PAI-1 antisense cRNA probe was performed. Figure 2 B shows that the PAI-1 gene is weakly expressed in healthy cerebral cortex of adult mice, as previously observed in the semiquantitative RT-PCR experiments. Similarly, the expression pattern of PAI-1 was similar to that described in Fig. 1 , with an overexpression of the PAI-1 gene in the injured hemisphere (Fig. 2C, E ) compared to the contralateral hemisphere (Fig. 2D ). Based on our PCR experiments, this overexpression of PAI-1 occurred between 24 h and 3 days after cerebral ischemia (Fig. 2C, E ). Using an adjacent section immunostained with the anti-glial fibrillary-associated protein (GFAP) antibody, we established that the expression of PAI-1 is located in the region of GFAP positive astrocytes (Fig. 2A ).



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  		Figure 1. Modulation of the expression of serpins and t-PA mRNAs after permanent focal ischemia in mouse cortex. Total RNA from lesioned (I) and contralateral (C) brain cortices were isolated at the times indicated and analyzed by RT-PCR for PAI-1, t-PA, NSP, PN-1, and ß-actin as described in Materials and Methods. An individual animal was used for each time. The RT-PCR presented are representative of results obtained from 3 individual experiments. Sham 12 h: mRNA from control-operated mice without occlusion of the middle cerebral artery harvested 12 h after the surgery.



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  		Figure 2. In situ localization of PAI-1 mRNA expression in the ischemic cortex. A) Immunohistochemical staining of GFAP expression in ischemic cortex. Scale bars: 600 µm. B) Dark-field emulsion photomicrographs of coronal brain sections of a 24 h ischemic cortex show in situ hybridization to a sense 35S-labeled PAI-1 cRNA probe. C, D) Dark-field emulsion photomicrographs show in situ hybridization to an antisense 35S-labeled cRNA probe in the border of the ischemic core (C) and in the contralateral cortex (D). Scale bars: 150 µm. E) Bright-field emulsion photomicrograph show in situ hybridization to an antisense 35S-labeled PAI-1 cRNA probe in the border of the ischemic core. Scale bars 150 µM.

Serpins and t-PA expression pattern in cultured astrocytes and neurons: modulation by TGF-ß1
We further studied the cellular distribution of t-PA and serpins (NSP, PN-1, and PAI-1) in both glial cultures and pure cultures of cortical neurons. By using RT-PCR, we showed that t-PA mRNA was expressed in both neurons and astrocytes. Astrocytes and neurons both exhibited PN-1 and NSP, but the mRNA expression of PAI-1 was restricted to the astrocytes (Fig. 3 ). Although the expression of t-PA, PN-1, and NSP mRNAs was not influenced by TGF-ß1 treatment in astrocytic and neuronal cultures, an enhancement of the expression of PAI-1 mRNA after TGF-ß1 treatment was observed in the astrocytes but not in pure neuronal cultures. Immunocytochemistry shows an expression of PAI-1 in cultured astrocytes but not in pure neuronal cultures (Fig. 4 ).



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  		Figure 3. PAI-1 expression is restricted to astrocytes and enhanced after TGF-ß1 treatment. Total mRNA from TGF-ß1 (1 ng/ml) -treated astrocytes or near pure neurones cultured in serum-free medium were harvested after 24 h of treatment. Total RNA were isolated and analyzed by RT-PCR for t-PA, PAI-1, NSP, PN-1 and ß-actin as described in Materials and Methods. RT-PCR are representative of results obtained from 3 individual experiments. Control: C; TGF-ß1-treated cells: T.



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  		Figure 4. Immunocytochemistry for PAI-1 in cultured astrocytes and neurones. Bright-field photomicrographs of either pure cortical neurons (scale bars: 400 µM) and pure cortical astrocytes in culture show fields after fixation and peroxidase staining for antibodies raised against PAI-1. Scale bars: 100 µm; original magnification (x1000).

TGF-ß1-induced up-regulation of PAI-1 in astrocytes involves the activation of an Smad3/Smad4-inducible element
PAI-1 expression in astrocytes subsequent to TGF-ß1 treatment was studied at the transcriptional level. To quantify PAI-1 transcriptional activity that depends on TGF-ß1, we developed two luciferase reporter genes driven either by the full-length PAI-1 promoter (PAI-luciferase) or the TGF-ß-responsive element recently described (CAGA box) (30) . This latter sequence specifically mediates the TGF-ß signal through binding of the Smad3/Smad4 complex. The luciferase activity was normalized with respect to a control vector and directly reflects the level of the PAI-1 transcription. TGF-ß1 increased luciferase activity by around 75% for the full-length promoter of PAI-1 and to up to 3.6-fold for the TGF-ß-inducible element. Cotransfection of both reporter genes with the TßR-IIEI dominant negative receptor (39) totally prevented these responses (Fig. 5 ). These data were confirmed by RT-PCR and showed that the up-regulation of PAI-1 transcriptional activity in astrocytes occurred as early as 3 h after TGF-ß1 addition (Fig. 6 ) and was maintained as long as TGF-ß1 was present in the incubating bath. Moreover, overexpression of PAI-1 after 6 h of TGF-ß1 treatment was noted at the protein level by SDS-PAGE of extracellular matrix-associated proteins, followed by Western blot analysis through the use of an antibody raised against PAI-1 (Fig. 7 A). Similar experiments, performed after metabolic labeling using 35S methionine/cysteine, confirmed that TGF-ß1 treatment induced a neosynthesis of PAI-1 in astrocytes (Fig. 7B ).



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  		Figure 5. TGF-ß1-dependent transcriptional activity of PAI-1 occurs through an Smad3/Smad4-inducible element. Luciferase activity of astrocytes transiently cotransfected with either the PAI-luciferase (A) or CAGA-luciferase reporter (B) vectors in the presence of the empty pCMV5 (lightly shaded bar) or the pCMV5 containing the TßR-IIEI cDNA (darkly shaded bar) (mean, n=6). C) The CAGA box is a TGF-ß-inducible element contained in the PAI-1 promoter; the sequences of the three CAGA box found in this promoter are given. The results are representative of data obtained from 8 individual experiments.



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  		Figure 6. Time course of PAI-1 expression in astrocytes after TGF-ß1 treatment. Total mRNA from TGF-ß1 (1 ng/ml) -treated astrocytes were harvested at the times indicated and analyzed by RT-PCR for PAI-1 and ß-actin as described in Materials and Methods. RT-PCR represent results from 3 individual experiments. Control: C; TGF-ß1-treated cells: T.



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  		Figure 7. TGF-ß1 induces an overexpression of the protein PAI-1 in astrocytes. A) Control (C) and TGF-ß1-treated (T) astrocytes were harvested after 6 h of treatment and Western blot analysis with an antibody raised against PAI-1 performed after SDS-PAGE of the cell layer. B) Control (C) and 24 h-TGF-ß1-treated (T) astrocytes were metabolically labeled in serum-free medium containing [35S]methionine and 35S-cysteine. SDS-PAGE electrophoresis and autoradiography were performed without (total) or with (IP) a previous immunoprecipitation by an antibody raised against PAI-1.

Overexpression of PAI-1 in astrocytes is induced by TGF-ß and activin but not by BMP, GDNF, and NTN
Specific serine/threonine kinase type I receptors transduce the intracellular signaling of various members of the TGF-ß family such as BMPs, TGF-ßs, and activins (40) . GDNF and NTN are novel members of the TGF-ß family that transduce their signal through activation of a common tyrosine kinase receptor, c-Ret. To test the specificity of TGF-ß on the expression of PAI-1, we cotransfected astrocytes with expression vectors encoding for constitutively activated versions of the type I receptors (Fig. 8 C) (41) in the presence of either the full-length PAI-1 promoter or the TGF-ß-responsive element (CAGA box) contained in the full-length PAI-1 promoter driving the luciferase reporter gene. All of these experiments were normalized according to the activity of a cotransfected control vector as described in Materials and Methods. As shown in Fig. 8 , expression of ALK-4/T206D and ALK-5/T204D led to transcriptional activation of the PAI-1 full-length promoter and of the TGF-ß-responsive element (CAGA box). In contrast, expression of ALK-2/Q207D, ALK3/Q233D ,and ALK-6/Q204D did not show any significant effect on the activation of the full-length promoter, demonstrating that the PAI-1 promotor is activated by TGF-ß and activin but not by BMP. We also tested the effect of both GDNF and NTN on the transcriptional activity of the PAI-1 full-length promoter and the CAGA box, with no significant activation (Fig. 9 ).



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  		Figure 8. TGF-ß1 and activin are able to induce PAI-1 overexpression in astrocytes, but not BMPs. Luciferase activity of astrocytes transiently cotransfected with either the PAI-luciferase (A) or CAGA-luciferase reporter vectors in the presence of the empty pCMV5 (lightly shaded bar) or the pCMV5 containing either autoactivated BMPs, activin, or TGF-ß type I receptors (darkly shaded bars) (mean, n=6). C) This table depicts the correspondence between each type I receptor used and its respective ligand (Yamashita et al., 1995; ref 41 ). The results are representative of data obtained from 5 individual experiments.



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  		Figure 9. GDNF and NTN did not influence PAI-1 expression in astrocytes. Luciferase activity of astrocytes transiently cotransfected with either the PAI-luciferase (A) or CAGA-luciferase (B) reporter vectors and pRL-TK control vector incubated 24 h with either TGF-ß1 (1 ng/ml), GDNF (10 ng/ml), or NTN (10 ng/ml) (mean, n=6). This figure represents the data obtained from a representative experiment. Results are representative of data obtained from 3 individual experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several studies have shown that TGF-ß1 mRNA is induced after hypoxic/ischemic brain injury (19 , 22 , 42) . Similarly, in a model of focal permanent ischemia in mice, we confirmed an overexpression of TGF-ß1 mRNAs after 24 h and up to 3 days (8) . Overall, these findings suggest that TGF-ß could play a critical role in the neuropathological consequences of ischemia.

TGF-ß1 has been shown to protect central neurons against diverse metabolic and excitotoxic challenges (17 , 21) . However, others have reported that under certain conditions TGF-ß1 was able to potentiate glutamate-induced neuronal injury (43 44 45) . We previously investigated the influence of TGF-ß1 in apoptosis and necrosis, two mechanisms involved in ischemic neuronal death. We showed that TGF-ß1 exerts a beneficial effect against NMDA- but not against AMPA- or kainate-induced necrosis. We extended this observation by demonstrating that PAI-1, a serine protease inhibitor, expressed in astrocytes and overexpressed after TGF-ß1 treatment, mediated the neuroprotective effect of TGF-ß1 (31) . These results raised the question of the implication of serine proteases and their inhibitors in the pathophysiology of ischemia.

Serine proteases have been involved in a variety of processes during the development of the nervous system as well as in the physiology of adult brain. For example, thrombin and plasminogen activators (PAs) are known to modulate the neurite outgrowth in cell culture (46) . More recently a function for PAs in synaptic plasticity and memory consolidation has been suggested, as plasminogen activator (t-PA) mRNA is increased in the hippocampus during the induction of long-term potentiation (LTP) (2) and in the cerebellum after the learning of a complex motor task (47) . Furthermore, membrane depolarization in PC12 cells induces the release of t-PA (48) , and mice deficient for t-PA are resistant to neuronal loss after the intrahippocampal injection of excitotoxins (3) or ischemic insult (4) . These data suggest that serine proteases such as t-PA are involved in the physiological processes and pathological mechanisms that occur during neuronal degeneration. Serine proteases with a role in the nervous system are thought to be regulated by serine protease inhibitors, termed serpins. The expression pattern of serpins (PAI-1, NSP, and PN-1) seems to demonstrate a restricted overexpression of PAI-1 after permanent focal cerebral ischemia. An equilibrium between serine proteases and their specific inhibitors (serpins) exists in many tissues where these agents are involved in physiological processes associated with inflammation or connective tissue turnover; in contrast, no clear involvement of serpins has been proposed for either physiological or pathological processes within the central nervous system.

We have shown that both NSP and PN-1 are expressed by cortical neurons and astrocytes in culture, as has been demonstrated for t-PA. In contrast, the expression of PAI-1 mRNA and protein is clearly restricted to astrocytes. Moreover, TGF-ß1 is able to induce an up-regulation of PAI-1 in astrocytes without any effect on the expression of the others serpins expressed in the CNS. The time course of the expression of PAI-1 after middle cerebral artery occlusion in mice matched the expression pattern of TGF-ß1 under the same conditions (8) , which might suggest that the overexpression of PAI-1 after TGF-ß1 treatment, observed in vitro, could reflect the in vivo process occurring during cerebral ischemia.

The bone morphogenetic proteins BMP-2, BMP-4, BMP-6, and BMP-7 (49) and their receptors, BMPR-IA, BMPR-IB, and BMPR-II mRNAs, are constitutively expressed in the developing mouse CNS (50) and the adult rat brain (51) . Moreover, BMPs are involved in both morphogenesis and neurogenesis in the mouse (52) . BMPs are also able to inhibit the proliferation of astrocytes and to induce their differentiation (53 , 54) . Messenger RNAs for activin and the activin receptors ActR-II, ActR-I, and ActR-IB are found in the developing rat spinal cord and cortex (51) and the adult rat brain (55) . Activin is known to exert a neurotrophic effect on cultured hippocampal neurons (56) . In addition to these TGF-ß related cytokines, two other members of the same family—the glial-derived neurotrophic factor (14) and neurturin (15) —which are associated with tyrosine kinase receptors, have recently been identified. These two members have been characterized as neurotrophic cytokines able to prevent neurodegeneration of dopaminergic neurons. In the present study, we found that the expression of PAI-1 in astrocytes is enhanced by TGF-ß and potentially by activin, but not by other members of the family such as BMPs, GDNF, or NTN. Moreover, our results obtained with the TGF-ß-responsive element (CAGA box) suggest that the TGF-ß signaling pathway in astrocytes involves Smad3 and Smad4 complexes, as previously characterized in peripheric tissues.

PAI-1 is a serpin that regulates the proteinase cascade initiated by t-PA. In a previous study, an increased expression of PAI-1 has been observed in the cerebrospinal fluid from patients with various neurological diseases such as Alzheimer's disease, cerebral infarction, CNS infection, and CNS neoplasia (57) . More recently, the serpins/t-PA axis has been shown to be involved in the excitotoxic cascade induced after cerebral ischemia (3 , 4) . Our present data suggest that the overexpression of TGF-ß1 that occurs after cerebral ischemia may specifically promote an increased expression of PAI-1.


   ACKNOWLEDGMENTS
 
This work was supported in part (O.N.) by a fellowship from the Commissariat à l'Energie Atomique (CEA, France).


   FOOTNOTES
 
2 Abbreviations: CNS, central nervous system; DTT, dithiothreitol; GFAP, glial fibrillary-associated protein; TGF-ß1, transforming growth factor-ß1; MEM, Eagle's minimal essential medium; PAI-1, plasminogen activator inhibitor; PAs, plasminogen activators; t-PA, tissue type plasminogen activator; PN-1, protease nexin-1; NSP, neuroserpin; BMPs, bone morphogenetic proteins; GDNF, glial cell line-derived neutrophic factor; MS, stock media; NTN, neurturin; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Received for publication January 11, 1999. Revision received March 6, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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