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Endocrinology Vol. 143, No. 2 636-646
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Estradiol (E2) Enhances Neurite Outgrowth by Repressing Glial Fibrillary Acidic Protein Expression and Reorganizing Laminin

Irina Rozovsky, Min Wei, David J. Stone, Hadi Zanjani, Christopher P. Anderson, Todd E. Morgan and Caleb E. Finch

Neurogerontology Division (I.R., M.W., C.P.A., T.E.M., C.E.F.), Andrus Gerontology Center and Department of Biological Sciences (C.E.F.), University of Southern California, Los Angeles, California 90089-0191; Curagen Corporation (D.J.S.), Branford, Connecticut 06405; and Laboratoire de Developpement et Vieillissement du Systeme Nerveux (H.Z.), 75005 Paris, France

Address all correspondence and requests for reprints to: Irina Rozovsky, Andrus Gerontology Center, University of Southern California, 3715 McClintock Avenue, Los Angeles, California 90089-0191. E-mail: rozovsky{at}molbio.usc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuronal remodeling in response to deafferenting lesions in the brain can be enhanced by estradiol (E2). Astrocytes are among the targets of E2 in complex interactions with neurons and may support or inhibit neuronal remodeling. In ovariectomized female rats given entorhinal cortex lesions, E2 replacement inhibited the increase of glial fibrillary acidic protein (GFAP) protein. To model the role of E2 in these complex processes, we used the "wounding-in-a-dish" of astrocyte-neuron cocultures. Low physiological E2 (1 pM) blocks the wound-induced increase of GFAP expression (transcription and protein) and enhances neurite outgrowth. The transcriptional responses to E2 during wounding are mediated by sequences in the 5'-upstream region of the rat GFAP promoter. Concurrently, E2 reorganized astrocytic laminin into extracellular fibrillar arrays, which others have shown support neurite outgrowth. The inhibition of GFAP expression by E2 in this model is consistent with in vivo findings that E2 enhanced recovery from deafferenting cortical lesions by increased neurite outgrowth in association with decreased GFAP expression. More generally, we hypothesize that physiological variations in E2 levels modulate neuronal plasticity through direct effects on GFAP transcription that, in turn, modify GFAP-containing intermediate filaments and reorganize astrocytic laminin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTRADIOL (E2) ENHANCES neuronal responses to lesions. As shown in rodent models for the hippocampal deafferentation of Alzheimer’s disease, neuronal outgrowth is enhanced by E2 in ovariectomized (OVX) female rodents (1, 2, 3). Astrocytes are among the targets of E2 in complex interactions with neurons, which may support or inhibit neuronal remodeling (e.g. neurite outgrowth is stimulated by the astrocytic secretions) of laminin and other adhesion substrates (4–7). In contrast, reactive astrocytes can inhibit neurite outgrowth through the formation of glial scars, which contain fibrous astrocytes with high expression of glial fibrillary acidic protein (GFAP), a component of intermediate filaments in the cytoskeleton (8, 9, 10, 11).

Sex steroids may be mediators of neuronal sprouting through their effects on astrocyte activities. For example, in OVX female rats, E2 replacement suppressed the focal astrogliosis (GFAP immunoreactivity) induced in cerebral cortex by stab wounding (12). Similarly, castration of male rats increased GFAP expression in hippocampal astrocytes (13). The effect of E2 on astrocyte reactivity in vivo (12) is consistent with our findings that in confluent primary cultures of astrocytes, E2 regulates GFAP transcription through a functional estrogen response element in the rat upstream GFAP promoter (14).

As a model for effects of E2 on astrocyte-neuron interactions during brain lesions, we used the "wounding-in-a-dish," in which astrocyte or astrocyte-neuron cocultures are given scratch wounds (15, 16, 17). In monotypic astrocyte cultures, lesion-induced gliosis was attenuated by antisense GFAP introduced by a retroviral vector (16). Moreover, in astrocyte-neuron cocultures, treatment with antisense GFAP, which decreased GFAP protein, also enhanced neurite outgrowth in association with the reorganization of astrocytic laminin (17). Laminins are secreted by astrocytes and, as constituents of the basement membrane that interact with the cytoskeleton, can guide neurite sprouting and other cell movements (4, 18). Because E2 inhibits GFAP expression in vivo and in vitro (13, 14), we hypothesized that E2 effects would be similar to antisense GFAP treatment, by suppressing GFAP responses to wounding and enhancing neurite outgrowth in association with the reorganization of astrocytic laminin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, OVX, entorhinal cortex lesions, and estrogen replacement
F344 female rats, aged 3 months, were OVX under ketamine:xylazine anesthesia: (46 mg:4.6 mg/kg) and 7 d later were given unilateral entorhinal cortex lesions (ECL) under this anesthesia using a Scouten retractable wire knife (Kopf Instruments, Tujunga, CA); stereotaxic coordinates, relative to lambda were: 2.2 mm anterior, 5 mm lateral, and 1 mm ventral from bregma. The extended knife was lowered 4 mm ventrally at +30 degrees and at -135 degrees relative to the lateral plane (19). At the time of ECL, pellets containing placebo or 17ß-E2 (0.72 mg/pellet, Innovative Research of America, Sarasota, FL) were inserted sc. Animals were killed 3 or 7 d post lesioning; brains were snap frozen for immunocytochemistry.

Serum for E2 measurements were sent out to Harbor UCLA (Los Angeles, CA). Serum E2 levels in OVX + E2 pellets were maintained for 7 d in a high physiological range (Table 1Go).


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  		Table 1. Serum E2 (pg/ml)

 
Cell cultures
Primary astrocytes were originated from the cerebral cortex of 1- to 3-d-old F344 rat pups of mixed genders by mechanical dissociation (20, 21). The gender from which astrocytes were derived did not alter GFAP responses to E2 (14). Cells were plated in tissue culture dishes at 2 x 105 cell/cm2 and maintained in DMEM/F12 culture medium (Cellgro, Herndon, VA) supplemented with 10% FBS (Life Technologies, Inc., Rockville, MD), 100 U/ml penicillin, and 50 U/ml streptomycin (Sigma, St. Louis, MO) at 37 C under 5% CO2. Media were refreshed every 2–3 d until confluence at 10–12 d. Confluent cultures were freed from contaminating microglia and oligodendrocytes by shaking (22). To establish astrocyte-neuron cocultures ( 21, 23), we seeded cortical neurons from fetal (embryonic d 18–19) brains onto confluent monolayers of astrocytes (1:3, neurons:astrocytes), which were maintained 5–7 d before experimentation.

Transfection
Astrocytes or astrocyte-neuron cocultures were transfected with rat GFAP promoter constructs with Luciferase reporter using DOTAP (Roche Molecular Biochemicals, Indianapolis, IN) (14, 21). Luciferase was assayed in cell lysates (Promega Corp., Madison, WI) and normalized to total protein (Coomassie blue assay). To standardize transfection efficiencies, we cotransfected with a pSV-ß-galactosidase vector (Promega Corp.). The GFAP promoter was not active when transfected into neurons (not shown).

Wounding and E2 treatment
In the wounding-in-a-dish model, monolayers of astrocytes or astrocyte-neuron cocultures were given scratch wounds with a plastic pipette tip (15, 16, 17). E2 (Sigma) was introduced the day after transfection at various times in relation to wounding; the ethanol vehicle was included in controls (0.08%, final concentration). Cells were harvested for luciferase assay 24 h later. Transfections were done in duplicate for each treatment; experiments were repeated three to four times.

Immunocytochemistry, GFAP measurements in vivo, neurite outgrowth
Cryostat-cut 18-µm brain sections were fixed (4% paraformaldehyde, 30 min), rinsed in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM sodium phosphate [dibasic], 1.4 mM potassium phosphate [monobasic]), treated with normal serum for 30 min, and incubated overnight with monoclonal antibodies to GFAP at 2.5 µg/ml (1:500 dilution; Roche Molecular Biochemicals). After incubation with secondary antibodies, reaction products were visualized with diaminobenzidine (Vectastain kit, Vector Laboratories, Inc., Burlingame, CA).

GFAP-immunoreactive signal was measured in the outer molecular layer (OML) of the dentate gyrus in both ipsilateral to the ECL and contralateral to the lesion hippocampus 3 and 7 d post ECL with or without E2 replacement of OVX female rats. Quantification was based on the regional analysis of OML covered by GFAP-stained processes (three zones within each OML) of three horizontal slices of each brain. Each area in the OML was manually drawn, and signal above background was quantified using IPLab Spectrum Image analysis software (Scanalytics, Inc., Fairfax, VA). The differences between slices of the same brain were less than 5%. Data are based on four to six animals per each experimental group.

In cultures, reaction products were detected by using immunofluorescent secondary antibodies (rhodamine conjugated for laminin or GFAP and fluorescein isothiocyanate conjugated for microtubule-associated protein-5 [MAP-5], Sigma). Fixed astrocyte-neuron cocultures were double immunolabeled with polyclonal rabbit antilaminin antibody, (Sigma; 0.5 mg/ml, 1:50) or polyclonal rabbit anti-GFAP (DAKO Corp., Carpinteria, CA; 1:500) and monoclonal anti-MAP-5 (2.6 mg/ml) (1:1000).

We measured both the number and the length of MAP-5-immunopositive neurites extended into the empty wound zone beyond astrocytic cell bodies (8 µm as cut-off) 48 h after wounding with or without E2 (1 pM or 100 nM) or TGF-ß (5 ng/ml). The length was measured using IPLab Spectrum image analysis software. Measurements were taken in seven to eight randomized areas of the wound zone (100–120 µm wide in diameter). Data are obtained from four independent experiments, each of which included all experimental conditions.

Western blotting
Cellular proteins were obtained by lysing cells in 10 mM Tris, 2% SDS, 10% ß-mercaptoethanol, and 0.5 mM EDTA, followed by boiling for 5 min. Equal amount of proteins were electrophoresed on 10% acrylamide gels. After transfer to membranes, immunoblotting was done with polyclonal rabbit anti-GFAP (1:1000) followed by peroxidase-conjugated secondary antibodies and immunoperoxidase diaminobenzidine visualization. OD was measured by computer videodensitometry (IPLab gel, Signal Analytics Corp., Vienna, VA). Experiments were repeated three times.

Astrocyte proliferation
Proliferation was evaluated in cocultures with or without E2 (1 pM or 100 nM), or TGF-ß1 (5 ng/ml), 48 h after scratch wound. Cocultures were labeled with 10µCi [3H]-thymidine for an additional 24 h, washed twice in PBS, fixed in 10% buffered formalin, coated with NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed 4 d, developed, and stained with cresyl violet. Labeled cells were counted in 10 random areas of the wound zone (100 µm2) in each experimental group. Experiments were repeated three times.

Statistical analysis
Two-way ANOVA was run on raw data. Graphed data (Figs. 1–4GoGoGoGo) are presented as a percentage change for clarity.



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  		Figure 1. E2 replacement of OVX female rats inhibits the increased GFAP immunoreactivity in ipsilateral OML of the dentate gyrus 7 d post ECL. A, Contralateral hippocampus of OVX, 7 d post ECL (x200). B, Ipsilateral hippocampus of OVX, 7 d post ECL (x200). C, Ipsilateral hippocampus of OVX + E2, 7 d post ECL (x200). D, High-power (x400) microphotograph of OML from panel B. E, High-power (x400) microphotograph of OML from panel C; scale bars, 10 µm; g, granule neurons of the dentate gyrus; f, hippocampal fissure. F, GFAP immunoreactivity in OML of the dentate gyrus in ipsilateral to the ECL 3 and 7 d post ECL with or without E2 replacement of OVX female rats. Quantification is based on the regional analysis of the defined area covered by GFAP-stained processes (three areas within each OML) of three horizontal sections for each brain, and four to six animals per each experimental group. Data (mean ± SEM) are expressed as a percentage of sham, contralateral 3 d post ECL; *, P < 0.05. The relative changes were the same when expressed as a percentage of sham, contralateral 7 d post ECL.

 


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  		Figure 2. GFAP expression increases after wounding in a dish. A, One hour after the focal injury of confluent astrocytes. B, Twenty-four hours after wounding; scale bar, 10 µm (x200). C, GFAP promoter activity: astrocytes transfected with a full length of the rat GFAP promoter with luciferase reporter were scratch wounded 24 h later. GFAP promoter activity was measured 1, 6, 24, and 48 h later. Data (mean ± SEM) are expressed as a percentage of uninjured controls in three independent experiments; *, P < 0.05. E, The rat GFAP promoter constructs and relative luciferase activity affected by scratch wounding in cultured astrocytes (24 h after wounding). The promoter sequences required to support transcriptional responses to wounding were identified by progressive deletions of the full-length construct A. Wounding responses were absent with proximal promoter constructs (A1–A4), which lacked sequences upstream of -641 bp. However, constructs with further upstream sequences from -1342 bp (A5–A7) gave as strong wounding responses as the full-length construct. Data (mean ± SEM) are expressed as a percentage of uninjured control in four independent experiments; *, P < 0.03. The diagram shows several response elements in the 5'-upstream region of the rat GFAP: NF-1, nuclear factor-1; NF-kB, nuclear factor-kB; GRE, glucocorticoid response element; TRE, (AP1 binding site)-tetra-phorbolacetate response element (24 ).

 


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  		Figure 3. E2 controls the wound-mediated increase of GFAP transcription in primary astrocyte cultures: role of ERE-1 and ERE-2 in the rat GFAP promoter. A, Astrocytes were transfected with wild-type full-length construct A (WT), mERE1, or construct A7, which does not contain ERE-2, and then were subjected to wounding with or without 1 pM E2. Asterisks signify responses to wounding without E2. Thus, transcriptional control of GFAP in response to wounding, without E2, does not depend on ERE-1 or ERE-2. On the other hand, construct with mERE1 was not effective during E2-wound interactions (P < 0.09), showing the role of ERE-1. Construct A7, which does not contain ERE-2 but contains wild-type ERE-1, also did not respond to E2 treatment during wounding (P < 0.1). Data (mean ± SEM) are expressed as a percentage of uninjured controls (four independent experiments); *, P < 0.05. B, Primary astrocytes were transfected with the full-length GFAP promoter, and E2 (1 pM) was added at 6 h before wounding, simultaneously, or 24 h after. Data (mean ± SEM) are expressed as a percentage of uninjured controls (four independent experiments); *, P < 0.05.

 


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  		Figure 4. E2 controls GFAP responses to wounding in astrocyte-neuron cocultures. A, Monotypic primary astrocyte cultures or astrocyte-neuron cocultures were transfected with the full-length GFAP promoter and were scratch wounded, with or without E2 (1 pM) for 24 h. Data (mean ± SEM) are expressed as a percentage of uninjured controls (three independent experiments); *, P < 0.05. B, Western blot of GFAP protein from astrocyte-neuron cocultures scratch wounded with or without E2 (1 pM). Data (mean ± SEM) are expressed as a percentage of unlesioned control; *, P < 0.01 (wound vs. unlesioned); **, P < 0.0008 (wound vs. wound+E2) (four independent experiments).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
First, we extended prior in vivo findings on GFAP mRNA during ECL-E2 interactions (3) to verify that GFAP protein is also repressed. This information was needed to validate our models because GFAP transcription and translation can be independently regulated (24, 25). We then examined the regions of the rat GFAP promoter that mediate transcription in the wounding-in-a-dish model to define effects of E2 on responses to lesions. Lastly, we examined effects of E2 on neurite outgrowth and laminin organization.

E2 attenuates GFAP induction by deafferenting lesions in vivo
ECL activates astrocytes distal to the lesion in the zone of degenerating neuron terminals. In prior studies, E2 attenuated the induction of GFAP mRNA in the zone of degenerating terminals in the outer molecular layer of the hippocampal dentate gyrus (3). We now show that E2 also attenuates the increase of GFAP protein after ECL. At 7 d after ECL, when GFAP induction and astrocytic fibrosis are maximum in the hippocampus, OVX rats showed a 3-fold increase of GFAP protein relative to the unlesioned side. E2 replacement during lesions reduced the increase of GFAP by 65% (Fig. 1Go).

GFAP induction by wounding in monotypic astrocyte cultures
We first examined the wounding response in confluent astrocytes alone (Fig. 2Go). After 24 h, the wound zone was partially filled with reactive astrocytes facing the scratch line (Fig. 2BGo). As shown in prior studies (15, 16), these cells extended their hypertrophic GFAP-immunoreactive processes into the lesion site.

We examined the role of transcription in these responses because, as noted above, GFAP transcription and translation can change independently. Confluent astrocyte cultures were transfected with 1876-bp construct of 5'-upstream rat GFAP promoter (full length, construct A) with a luciferase reporter (14, 21) and scratch wounded 24 h later. The wounding-induced GFAP transcription was significant by 12 h and maximal by 24 h (Fig. 2CGo). The transient dip of GFAP transcription at 2–4 h was repeatedly observed.

The promoter sequences required to support transcriptional responses to wounding were identified by progressive deletions of the full-length construct A (Fig. 2DGo). Wounding responses were absent with proximal promoter constructs (A1–A4), which lacked sequences upstream of -641 bp. However, constructs with further upstream sequences from -1342 bp (A5–A7) gave as strong wounding responses as the full-length construct. These data suggest that GFAP transcription in response to wounding in astrocyte cultures does not depend on estrogen response elements (EREs) within the promoter. We then defined the effects of E2 on GFAP expression during wounding in vitro.

Interactions of E2 and wounding on GFAP transcription in astrocyte cultures
E2 (1 pM) robustly inhibited the induction of the wild-type full-length GFAP promoter (Fig. 3AGo) in response to wounding. Effects of E2 on wounding operate within a broad window of time. Treatment with E2 for 6 h before, or simultaneously with wounding, blocked the increase of GFAP transcription as measured 24 h later. Even when introduced 24 h after wounding and measured at 48 h, E2 inhibited GFAP transcription to basal levels (Fig. 3BGo). Thus, E2 repressed the wounding-induced transcriptional induction of GFAP even after induction had persisted for at least 12 h.

The role of EREs in the E2-wounding interactions was evaluated. The GFAP upstream promoter includes two canonical EREs: ERE-1 (at -149 bp) and ERE-2 (-1817 bp) (Fig. 2DGo and Refs. 14 and24). The near upstream ERE-1 binds the ER-{alpha} and was shown by site-directed mutagenesis to be required for regulation of GFAP transcription by E2 (14). This mutated construct (mERE-1) was not effective in the inhibitory effect of E2 on wound-mediated increase of GFAP transcription (Fig. 3AGo). The deletion of ERE-2 at -1817 bp (construct A7, without ERE2 but with wild-type ERE1) was also not effective in the inhibitory effect of E2 on wound-mediated GFAP induction (Fig. 3AGo). Thus, E2 control of GFAP transcription induced by wounding requires sequences that contain EREs.

Interactions of E2 and wounding in astrocyte- neuron cocultures
We then examined whether GFAP was also induced by wounding in astrocyte-neuron cocultures because unlesioned cultures show "transcriptional inversion" (14, 21), in which the direction of transcriptional responses of GFAP to E2 can change from induction in monotypic astrocyte cultures to repression in astrocyte-neuron cocultures (Fig. 4AGo). In either pure astrocytes or astrocyte-neuron cocultures, GFAP transcription was similarly increased in the response to wounding (Fig. 4AGo). In both cell systems, 1 pM E2 completely blocked the injury-mediated increase of GFAP transcription (Fig. 4AGo). Moreover, the wound-mediated induction of GFAP protein was decreased by 50% by E2 treatment. (Fig. 4BGo). Note that the E2-induced repression of GFAP transcription in astrocyte-neuron cocultures was accompanied by decreased GFAP protein (Fig. 4Go, A and B).

E2, neurite outgrowth, and laminin organization
Lastly, we examined the effects of E2 on neurite outgrowth and laminin organization in the wounding-in-a-dish model. Because Lefrançois et al. (17) showed that antisense GFAP treatment of astrocyte-neuron cocultures enhanced neurite outgrowth, we predicted that E2, which decreases GFAP, would also enhance the outgrowth of neurites after scratch wounding. This experiment was not designed to evaluate whether E2 acted on neurons either directly, or independently of, its effects on astrocytes.

Astrocyte-neuron cocultures were wounded and double immunostained for GFAP and MAP-5, a marker for neurites (Fig. 5Go). Unlesioned cocultures (Fig. 5AGo) display a prominent network of MAP-5-positive neurites on the astrocyte monolayer. One hour after wounding, there is a clearly defined open area without cellular GFAP or MAP-2 (Fig. 5BGo); 48 h after wounding, reactive astrocytes facing the wound are immunopositive for GFAP, and MAP-5-immunopositive neurites are not seen in the wound area beyond the astrocytic cell bodies (Fig. 5CGo). The addition of E2 (1 pM) at the time of wounding allowed neurites to outgrow beyond the astrocytic cell bodies as early as 24 h (not shown). At 48 h, robust neurite outgrowth in the wound zone was seen (Fig. 5DGo).



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  		Figure 5. E2 enhances neurite outgrowth. Astrocyte-neuron cocultures were double immunolabeled with anti-GFAP and anti-MAP-5 after scratch wounding with or without E2. A, Unlesioned astrocyte-neuron coculture. MAP-5-positive neuronal processes (white pseudocolor) are prominent on the monolayer of GFAP-positive (green pseudocolor) astrocytes. B, One hour after wounding. C, Forty-eight hours after wounding. Reactive astrocytes facing the wound are immunopositive for GFAP (green pseudocolor); MAP-5-immunopositive neurites (white pseudocolor) are not seen in the wound area beyond the astrocytic cell bodies. D, The addition of E2 (1 pM) at the time of wounding allowed robust neurite outgrowth into the wound zone beyond the astrocytic cell bodies (white pseudocolor). Initial wound position is marked with asterisk; scale bar,10 µm (x200). E, Number of neurites and neurite length beyond the astrocytic cell bodies (longer than 8 µm) 48 h after wounding with E2 (1 pM and 100 nM) or TGF-ß1 (5 ng/ml). Neurite lengths are shown for more than 8 µm because shorter segments were highly variable. Lefrançois et al. (17 ) used a 5-µm cut-off. No neurites in the control (without E2) wounded cultures extended beyond 13 µm, whereas the hormone-treated cultures showed strong increases in number of neurites in the 13- to 18-µm range. We do not show longer neurites, which were sporadic, up to 40 µm, in hormone-treated cultures.

 
The number and length of neurites that extended into the wound zone 48 h later were increased by E2 (Fig. 5EGo). Effects of E2 on neurite outgrowth were greater at 100 nM E2 than 1 pM; this much higher dose was chosen for comparison with other studies of E2 effects on neurite outgrowth (26). TGF-ß1 treatment, which is known to promote neurite outgrowth (27), induced even greater neurite outgrowth than E2 (Fig. 5EGo). Neurite lengths (Fig. 5EGo) are shown for more than 8 µm because shorter segments were highly variable. Lefrançois et al. (17) used a 5-µm cut-off. No neurites in the control (non-E2) wounded cultures extended beyond 13 µm, whereas the hormone-treated cultures showed strong increases in number of neurites in the 13- to 18-µm range. We do not show longer neurites, which were sporadic, up to 40 µm, in hormone-treated cultures.

Concurrently with enhanced neurite outgrowth, E2 also induced a reorganization of astrocytic laminin (Fig. 6Go). Astrocyte-neuron cocultures were wounded with or without 1 pM E2 and 48 h later double immunostained for laminin and MAP-5. At 48 h after wounding, cultures without E2 lacked MAP-5immunopositive neurites in the wound zone (Fig. 6AGo). Moreover, the astrocytes contained abundant intracellular puncta of laminin in the perinuclear cytoplasm, whereas there was little extracellular laminin (Fig. 6Go, A and B). The overlying neurons had few if any MAP-5-positive neurites growing beyond the astrocytic cell bodies. The addition of E2 before wounding caused a profound reorganization of laminin, with a sharp decrease of intracellular laminin in astrocyte cell bodies and a marked increase of extracellular laminin that appeared fibrillar and was associated with robust extension of MAP-5-positive neurites (Fig. 6Go, C and D).



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  		Figure 6. E2 reorganizes astrocytic laminin into linear extracellular arrays permissive for neurite outgrowth. Astrocyte-neuron cocultures were double immunolabeled for laminin (red fluorescence) and MAP-5 (green fluorescence) after wounding with or without E2 (1 pM). A (x200) and B (x400; squared area in A), 48 h after wounding, no E2. Reactive astrocytes facing the wound zone contained abundant intracellular puncta of laminin immunodeposits in perinuclear cytoplasm (marked with asterisks in B). Overlying neurons had few or any MAP-5-positive neurites extended beyond the astrocytic cell bodies. C (x200) and D (x400; squared area in C), Addition of E2 at the time of the wounding caused profound reorganization of astrocytic laminin, with sharply decreased intracellular laminin and marked increase of extracellular pattern, which appeared as fibrillar arrays. Extension of MAP-5-immunopositive neurites is seen in association with laminin reorganization. Initial wound position is marked with asterisk; scale bar, 10 µm.

 
We examined the effects of E2 treatment on astrocyte proliferation because E2 can accelerate skin wound healing in association with increased TGF-ß1 production (28), which is known to be antiproliferative for astrocytes (27, 29). The mitotic index (ratio of 3[H]-thymidine-labeled cells to total number of cells, expressed as a percentage) was not altered by 48 h of E2 exposure of wounded astrocyte-neuron cocultures. TGF-ß1 treatment, however, inhibited astrocyte proliferation in the wound zone. Mitotic index: 89 ± 2, in wound; 90 ± 1, in wound with1 pM E2; 88 ± 3, in wound with 100 nM E2; and 63 ± 4 (P < 0.001), in wound with TGF-ß1, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Laminin organization after wounding was profoundly altered in association with decreased GFAP expression, whether caused by physiological levels of E2 in this study or by treatment with antisense GFAP (17). In both studies that used the wounding-in-a-dish model, the diminution of GFAP expression was associated with similar effects on laminin reorganization into extracellular fibrillar arrays supportive of neurite outgrowth. These results support the hypothesis that E2 enhances neurite outgrowth through a cascade of events within astrocytes, in which an early event is the repression of GFAP expression. The ensuing decreases of GFAP protein, through yet unknown interactions with GFAP-containing intermediate filaments, then modify laminin organization, with increased extracellular fibrillar forms that in turn support enhanced axonal outgrowth. These data identify estrogens as a new potential hormonal regulator of astrocytic laminin organization in vivo. Thyroid hormones also enhance the formation of linear arrays of extracellular laminin as well as modulating the organization of microfilaments (30).

The inverse associations between GFAP and laminin expression implicate synergistic interactions between the cytoskeleton and cell surface laminin, which could be mediated by integrins. In myotube cultures, disruption of intracellular actin filaments by cytochalasin or genestein also disrupted the cell surface laminin network (31). The latter study used 25 µM genestein as a general inhibitor of protein tyrosine kinases; however, an additional mechanism could involve transcriptional effects because genestein interacts with ERß complexes (32, 33).

Astrocytes most strongly express ERß, localized to cell nuclei, perikarya, and glial processes (34); however, ER{alpha} was recently localized to cell membranes of a subpopulation of hippocampal astrocytes, some of which contained glial filaments (35). ER{alpha} was also found in lesioned primate brain with marked induction in reactive astrocytes in the vicinity of the lesion (36) (ERß was not determined because of the lack of specific antisera). Thus, the present actions of E2 could be mediated by either or both ER{alpha} or ERß.

In view of the inverse association of GFAP expression and neurite-promoting organizations of laminin, the expression of GFAP may have a broader role than usually considered in the context of glial scarring. Particular subsets of integrin and laminin genes in astrocytes may be of significance to hormonal regulation of neurite outgrowth and, more generally, to the hormonal modulation of synaptic plasticity. It is unclear which laminins are produced by astrocytes. Kedar et al. (37) detected only the laminin B2 chain, which is sufficient to stimulate neurite outgrowth. In addition, Wagner and Gardner (38) detected laminin-5, a heterotrimer consisting of the {alpha}3ß3{gamma}2-chains (formerly known as kalinin [39 ]).

The complexity of GFAP control by sex steroids is well documented by us and others. In particular, the direction of GFAP responses to sex steroids changes from induction to repression (transcriptional inversion) depending on brain region in vivo (13, 14, 40), and in vitro, in glial cultures with neurons derived from different brain regions (14, 41) (Table 2Go). The ability of neurons in coculture with astrocytes to induce transcriptional inversion in GFAP responses to E2 (14) suggest that local activities of neurons and/or astrocyte-neuron interactions in vivo might mediate the brain region differences in GFAP control by sex steroids. The developmental age may also be pertinent to regulation of GFAP by E2. Transcriptional inversion of GFAP in response to glucocorticoids has opposite directions in astrocytes cultured from neonatal brains (as in this study) vs. long-term cultures of neonatal astrocytes or of astrocytes from adult brains (42). We do not know whether these different aspects of age influence responses of GFAP to E2.


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  		Table 2. Bidirectional control of GFAP expression by sex steroids in vivo and in vitro

 
About 2 kb of the 5'-upstream GFAP promoter appears to suffice for transcriptional responses to wounding in the present studies of rat GFAP and in prior studies of the human and mouse promoters in transgenic mice (43, 44). The transcriptional controls that modulate wound interactions with E2, as shown here in vitro, are also likely to mediate the attenuation of GFAP expression by E2 after wounding in vivo, shown in Fig. 1Go and by Garcia-Estrada et al. (12). Mechanisms include the near 5'-upstream ERE-1. We showed that mutations of ERE-1 that blocked regulation of GFAP by E2 alone also blocked the effects of E2 on GFAP induction by wounding. This mutation in ERE-1 was also shown to block binding of ER{alpha} (14). Because ER{alpha} is required for neuroprotection by E2 in a rodent stroke model (45), it is possible that its role in GFAP regulation by E2 has broader significance to neuroprotection and neuronal sprouting. The far-upstream ERE-2 in GFAP promoter is also required for blockade of GFAP induction by E2 during wounding. These data suggest direct interactions between both EREs and other transcription factor complexes that mediate wounding responses, possibly through the formation of DNA loops. As a precedent, the activation of the rat PRL promoter by E2 requires loop formation between a distal ERE and the proximal promoter (46).

Although downstream GFAP gene elements are important for development (47, 48), no role is indicated in the regulation by wounding or steroids observed so far in differentiated astrocytes. The upstream region necessary for wounding responses is highly conserved, with 84–95% similarity between mouse and rat (-642 to 1867 bp) and more than 70% similarity of rat, mouse, and human promoter for -1000 to -1800 bp (24). This extensive similarity is unusual for far-upstream sequences without coding functions, which implies a conserved set of regulatory responses to integrate steroidal and inflammatory mechanisms (24).

In summary, the wounding-in-a-dish model shows that down-regulation of GFAP protein is sufficient to promote laminin reorganization, whether mediated by E2 in the present studies or by antisense GFAP (17) (antisense GFAP is unlikely to have interacted directly with neurite outgrowth). We hypothesize that the effects of E2 in promoting neurite outgrowth are driven by the transcriptional repression of GFAP. The ensuing decrease of GFAP protein modifies GFAP-containing intermediate filaments that, in turn, modify laminin organization to increase extracellular fibrillar arrays that support axonal outgrowth. According to this model, the effects of E2 on increasing apolipoprotein E (49), synaptophysin, and other presynaptic markers (50) is downstream of or secondary to the reorganization of astrocytic laminin that is required for neurite outgrowth.

This schema has several major targets for further study. The duration of sensitivity to E2 extends from at least 6 h before wounding to at least 24 h after wounding, when GFAP induction is already strong, which implies complex signaling events that may be investigated by experimental modulation of various kinases. The role of GFAP in the modulation of laminin expression by intermediate filaments may be investigated with mice deficient in GFAP and/or vimentin.


   Footnotes
 
This work was supported by NIA Grant AG-14751 (to C. E. F.).

Abbreviations: ECL, Entorhinal cortex lesion; ERE, estrogen response element; GFAP, glial fibrillary acidic protein; MAP-5, microtubule- associated protein-5; OML, outer molecular layer; OVX; ovariectomized.

Received July 25, 2001.

Accepted for publication October 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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