 |
 Previous Article | Next Article 
The Journal of Neuroscience, November 15, 1999, 19(22):9913-9927
Neuregulins Signaling via a Glial erbB-2-erbB-4 Receptor Complex
Contribute to the Neuroendocrine Control of Mammalian Sexual
Development
Ying J.
Ma1,
Diane F.
Hill1,
Kimberly E.
Creswick1,
Maria E.
Costa1,
Anda
Cornea1,
Mario N.
Lioubin2,
Gregory D.
Plowman2, and
Sergio R.
Ojeda1
1 Division of Neuroscience, Oregon Regional Primate
Research Center, Beaverton, Oregon 97006, and 2 Sugen,
Inc., South San Francisco, California 94080
 |
ABSTRACT |
Activation of erbB-1 receptors by glial TGF has been shown to be
a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other
members of the erbB family may be required, however, for full signaling
capacity. Here, we show that activation of astrocytic erbB-2/erbB-4
receptors plays a significant role in the process by which the
hypothalamus controls the advent of mammalian sexual maturation.
Hypothalamic astrocytes express both the erbB-2 and erbB-4
genes, but no erbB-3, and respond to neuregulins (NRGs) by
releasing prostaglandin E2 (PGE2), which acts on
neurosecretory neurons to stimulate secretion of luteinizing
hormone-releasing hormone (LHRH), the neuropeptide controlling sexual
development. The actions of TGF and NRGs in glia are synergistic and
involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of
puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by
treatment with an antisense oligodeoxynucleotide inhibited the
astrocytic response to NRGs and, to a lesser extent, that to TGF and
blocked the erbB-dependent, glia-mediated, stimulation of LHRH release.
Intracerebral administration of the oligodeoxynucleotide to developing
animals delayed the initiation of puberty. Thus, activation of the
erbB-2-erbB-4 receptor complex appears to be a critical component of
the signaling process by which astrocytes facilitate the acquisition of
female reproductive capacity in mammals.
Key words:
astroglial cells; tyrosine kinase receptors; glial growth
factors; female sexual development; hypothalamus; puberty
| |
INTRODUCTION |
Mammalian sexual development and
adult reproductive function depend on the functional integrity of a
group of specialized neurosecretory neurons that produce the
neuropeptide luteinizing hormone-releasing hormone (LHRH).
LHRH-secreting neurons are located in the basal forebrain and send
their axons to the median eminence (ME) of the hypothalamus
(Silverman et al., 1994 ) in which they release their secretory products
into the portal vasculature for delivery to the anterior pituitary gland.
The regulation of LHRH secretion is exceedingly complex, because it
involves multiple transsynaptic inputs, the modulatory influence of
gonadal steroids, and the regulatory participation of astroglial cells
(Brann and Mahesh, 1994; Ojeda, 1994; Crowley et al., 1995; Ojeda and
Ma, 1995; Terasawa, 1995). Hypothalamic astrocytes are in intimate
contact with LHRH neurons, because astrocytic processes appose most of
the LHRH cell membrane, particularly along the secretory axons (Witkin
et al., 1991; King and Letourneau, 1994; Silverman et al., 1994).
Astroglial cells affect LHRH neuronal function by both remodeling the
delivery sites of LHRH neurosecretory axons in the median eminence
(Witkin et al., 1991; King and Letourneau, 1994) and the production of
trophic (Melcangi et al., 1995; Tsai et al., 1995; Voigt et al., 1996)
and neuroactive substances (Gallo et al., 1995; Ma et al., 1997a) able
to affect the release of LHRH.
Evidence now exists that trophic factors signaling through receptor
tyrosine kinases play a pivotal role in the cell-cell interactive
mechanism by which astrocytes regulate LHRH neuronal function (Olson et
al., 1995; Tsai et al., 1995; Voigt et al., 1996). For instance,
transforming growth factor (TGF), a member of the epidermal
growth factor (EGF) family (Massague, 1990), is synthesized in
hypothalamic astrocytes and specialized glioependymal cells lining the
third ventricle of the brain (Ma et al., 1992, 1994a), and promotes
LHRH release indirectly, via juxtacrine-paracrine stimulation of glial
cells containing EGF (erbB-1) receptors (Ma et al., 1994a, 1997a; Voigt
et al., 1996). The ME appears to be an important site at which TGF
exerts its neuroendocrine actions. When cells genetically modified to
secrete the growth factor are grafted into this region, puberty is
advanced (Rage et al., 1997). Conversely, puberty is delayed by
inhibition of erbB-1 tyrosine kinase activity targeted to the median
eminence (Ma et al., 1992).
Signaling through erbB receptors is not, however, an isolated process
involving the activation of a single receptor by a single ligand.
Recent studies have shown that ligand binding to a particular erbB
receptor involves the recruitment of related erbB receptors (Carraway
and Cantley, 1994; Burden and Yarden, 1997). In addition to erbB-1 that
binds EGF, TGF, and four other EGF-related ligands (Carpenter and
Cohen, 1990), the family of erbB tyrosine kinase receptors includes
erbB-2 (Bargmann et al., 1986b), erbB-3 (Kraus et al., 1989; Plowman et
al., 1990), and erbB-4 (Plowman et al., 1993a). Whereas erbB-3 and
erbB-4 bind a large group of structurally related, EGF-like peptides
collectively known as neuregulins (NRGs) (Wen et al., 1992; Marchionni
et al., 1993; Burden and Yarden, 1997; Carraway et al., 1997; Chang et
al., 1997), no ligand for erbB-2 has yet been identified (Carraway and
Cantley, 1994). Rather than acting as a typical receptor, erbB-2
appears to function as an auxiliary molecule (Karunagaran et al., 1996)
recruited by ligand-induced activation of both erbB-1 and NRG (erbB-3
and erbB-4) receptors (Akiyama et al., 1988; Beerli et al., 1995; Karunagaran et al., 1996; Riese et al., 1996; Zhang et al., 1996). We
show here that expression of the genes encoding erbB-2 and erbB-4
within the hypothalamus is mostly astroglial and that it selectively
increases in this brain region during the phases of development that
precede and accompany the advent of female sexual maturation. This
increase in gene expression is sequentially determined by both
gonad-independent and sex steroid-regulated mechanisms. Both NRGs and
TGF are equally effective in stimulating astrocytic release of
PGE2, an eicosanoid involved in mediating
neurotransmitter-induced LHRH release. Disruption of erbB-2 synthesis
by an antisense oligodeoxynucleotide not only prevents
PGE2 release in response to NRG stimulation and
the ability of astrocytes to stimulate LHRH release via diffusible factors, but significantly, it also delays the onset of puberty.
Parts of this work have been published previously (Ma et al.,
1997b).
| |
MATERIALS AND METHODS |
Animals
Immature and pregnant female rats of the Sprague Dawley strain
were purchased from B & K Universal, Inc. (Fremont, CA). They were
housed in a room with controlled photoperiod (14/10 hr light/dark cycle; lights on from 5:00 A.M. to 7:00 P.M.) and temperature (23 25°C). Animals were allowed access to tap water and pelleted rat
chow ad libitum.
Cell culture
Hypothalamic and cerebrocortical astrocytes were purified from
1- to 2-d-old rats, as described previously (Ma et al., 1994a). In
brief, brain tissues were mechanically dissociated using a Stomacher 80 blender (Tekmar, Cincinnati, OH) at 80% power for 2-3 min. The cell
suspension was filtered first through a 230 µm metal sieve (Bellco,
Vineland, NJ) and then through a 130 µm nylon mesh filter (Nitex,
Elmsford, NJ). The cells were plated in 75 cm culture flasks (Corning
Costar Co., Acton, MA) and cultured in DMEM-F-12 medium (1:1,
vol/vol) supplemented with 10% calf serum under an atmosphere of 5%
CO2-95% air at 37°C. Upon reaching confluency
(8-10 d), the astrocytes were isolated from other contaminating cells
by first shaking the flasks at 250 rpm for 6 hr, replacing the medium,
and then shaking again for another 18 hr. Thereafter, the astrocytes
were replated in six-well plates at 800,000 cells per well. Upon
reaching 80-90% confluency, the medium was replaced with a
serum-free, astrocyte defined medium (ADM), and the astrocytes were
used 1-2 d later for the experiments. ADM consisted of DMEM (lacking
glutamate and phenol red) supplemented with L-glutamine (2 mM), HEPES (15 mM), insulin (5 µg/ml), and
putrescine (100 µM). The cultures were more than 95%
pure, as assessed by the number of cells immunopositive for the
astrocytic marker, glial fibrillary acidic protein (Ma et al.,
1994a).
The immortalized LHRH-producing cell line GT1-1 (kindly provided by
Dr. Richard Weiner, University of California at San Francisco, San
Francisco, CA) was used to assess the effect of astrocyte-derived substances on LHRH release. The cells were seeded in 24-well plates (100,000 cells per well) and cultured in DMEM containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). Upon
reaching 50-60% confluency, they were transferred to a serum-free, neuronal defined medium (NDM) for 24 hr, before being used for the
experiments. NDM consisted of glutamate-free DMEM supplemented with
transferrin (100 µg/ml), putrescine (100 µM),
L-glutamine (2 mM), sodium selenite (30 nM), and insulin (5 µg/ml) (Berg-von der Emde et al.,
1995).
MCF-7 cells (American Type Culture Collection, Manassas, VA; HTB-22,
human breast adenocarcinoma), used as a positive control to verify the
ability of NRG1 to induce erbB-2 tyrosine phosphorylation, were
cultured in DMEM supplemented with 10% fetal calf serum and 2 mM L-glutamine.
Cell treatments
Astrocytes and MCF-7 cells. (1) To document the
ability of TGF and NRG1 to induce erbB-1, erbB-2, or erbB-4 tyrosine
phosphorylation, hypothalamic and cortical astrocytes and MCF-7 cells
were seeded into 100 mm tissue culture dishes and grown to ~80%
confluency. The cells were then transferred to serum-free medium and
cultured for 24 hr before treating them with TGF or the NRG1, neu
differentiation factor- 2 (NDF-2; 100 ng/ml, 5 min). Thereafter,
the cells were washed with PBS and solubilized in
radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 0.5%
sodium deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 1 µM PMSF, 20 µg/ml aprotinin (0.09 IU/ml), and
1 mM sodium orthovanadate. The solubilized
proteins were stored at 85°C until immunoprecipitation and
electrophoretic separation of the tyrosine phosphorylated species (see
below). (2) The ability of NRG1 to stimulate the release of
PGE2 from hypothalamic astrocytes was examined by
treating the cells with the NDF isoforms 1, 2, and 2 and
comparing the response with that to TGF administered at a similar
dose. The possibility that NRG1 may facilitate the effect of TGF on
PGE2 release was also examined by treating
astrocytes with an ineffective concentration of NDF-2 (10 ng/ml) in
combination with increasing doses of TGF (5, 10, and, 20 ng/ml). (3)
To determine the importance of erbB-2 in erbB-4- mediated NRG signaling
in hypothalamic astrocytes, the astrocytes were treated with NDF-2
(50 ng/ml) for 16 hr in the presence of a 21-mer antisense
oligodeoxynucleotide [erbB-2 oligodeoxynucleotide (ODN);
5'-CATGATGATCATTGCGGCTCC-3'; 1 µM] encompassing the
translation initiation codon [nucleotides (nt) 9 to +12] of rat
erbB-2 mRNA (Suen and Hung, 1990). At the end of the treatment, the
medium was collected for PGE2 measurement (Campbell and Ojeda, 1987). An oligodeoxynucleotide containing the same
nucleotides but in a random order was used as a control. The sequence
of this oligodeoxynucleotide has no similarity to any other mammalian
sequence thus far deposited in GenBank. The effectiveness of the erbB-2
ODN to selectively disrupt erbB-2 synthesis was assessed by treating
hypothalamic astrocytes for 16 hr with either the ODN or the scrambled
sequence and then subjecting the cells to a 5 min exposure to NDF-2.
Thereafter, the cells were processed as outlined above for subsequent
immunoprecipitation of erbB-2 and erbB-4 and determination of
phosphorylated receptor content.
GT1-1 cells. These cells were used to determine whether NRG
is able to act directly on LHRH-secreting neurons to stimulate LHRH
release or whether it does so via a glial intermediacy. The cells were
treated with either NDF-2 (50 ng/ml) or a culture medium conditioned
by hypothalamic astrocytes treated for 16 hr with NDF-2 alone, or
NDF-2 in the presence of the above described erbB-2 antisense
oligodeoxynucleotide. After treating the GT1-1 cells for 30 min, the
medium was collected for LHRH measurement (Ojeda et al., 1986a). A
conditioned medium derived from hypothalamic astrocytes treated with
TGF, known to induce LHRH release via its content of
PGE2 (Ma et al., 1997a) was also applied to
GT1-1 cells as a positive control for LHRH release.
Cell transfection
Because NRG1 induces erbB-2 tyrosine phosphorylation in
hypothalamic astrocytes, which contain erbB-4 receptors, but not in cerebrocortical astrocytes, which lack these receptors, cortical astrocytes were transfected with cNHER4, a plasmid that encodes the
human erbB-4 receptor, to document the requirement of erbB-4 for NRG1
signaling in astrocytes. The cells were seeded into 60 mm plates, grown
to 80% confluency, and transfected for 5 hr with Lipofectamine (Life
Technologies, Grand Island, NY), as described previously (Mayerhofer et
al., 1996). Forty-eight hours after transfection, the astrocytes were
treated with NDF-2 (100 ng/ml, 5 min) and analyzed for erbB-2
tyrosine phosphorylation.
Reverse transcription-PCR of NRGs and erbB receptors
NRGs. A 251 bp NRG1 cDNA fragment was amplified from
total RNA from either hypothalamic tissue or hypothalamic astrocytes. The primers used were 20-mer oligodeoxynucleotides corresponding to nt
532-551 (5' primer) and 764-783 (3' primer) in the
extracellular-encoding region of the NRG1 gene (Wen et al., 1992) that
is common to all described NRG mRNA isoforms (Wen et al., 1994). To
obtain cDNAs complementary to the and forms of NRG2 mRNA (314 and 270 bp, respectively), we used 5' primers, specific for each form:
5'-AAACGGATTCTTC- GGACAGA-3', corresponding to nt 247-266 in the form; 5'-CGAAGGCATCAACCAACTCT-3', corresponding to nt 989-1008 in the
form; and a common 3' primer, 5'-TGGTGGGCCGGACA- CATGTT-3',
complementary to nt 541-560 in the form (Chang et al., 1997).
Total RNA isolated from the hypothalamus or hippocampus was used as the
template. To isolate an NRG3 cDNA, we used RNA from the hypothalamus,
hypothalamic astrocytes, human keratinocytes, and rat liver, and 21-mer
primers that amplify a region (nt 1260-1627) including the entire
transmembrane-encoding portion of murine NRG3 mRNA (Zhang et al.,
1997). The 5' primer used was 5'-CTACCAAGGAGTCCGTTGTGA-3'; the 3'
primer was 5'-TTGACTCCATTATTTT- CTCCA-3'.
ErbBs. A 322 bp erbB-2 and a 168 bp erbB-4 cDNA
fragment were generated from total RNA from either hypothalamic tissue
or hypothalamic astrocytes. A 331 bp erbB-3 DNA was obtained from liver
RNA. The 5' primer (5'-CAGTGTGTCAACTGCAGTCA-3') used to amplify erbB-2
corresponds to nucleotides 1610-1629 in the rat erbB-2 mRNA sequence
(Bargmann et al., 1986b). The 3' primer
(5'-CAGGAG- TGGGTGCAGTTGAT-3') is complementary to nucleotides
1913-1932. The primers used to amplify erbB-3 and erbB-4 DNA fragments
were synthesized based on human erbB-3 and erbB-4 sequences. The 5' primer is a common 20-mer oligonucleotide
(5'-AACTGCACCC- AGGGGTGTAA-3') corresponding to a highly conserved
region (nt 1891-1910 in the human erbB-4 gene) in the extracellular
domain-encoding portion of both genes (Kraus et al., 1989; Plowman et
al., 1993a). In the rat sequence, nt 12 of this primer (G) is
substituted for an A. The 3' primers used are specific to each mRNA.
The erbB-3 3' primer (20-mer; 5-AAATCCCCTTGTGGACAGTT-3') is
complementary to nt 2361-2380 in the intracellular domain of the
erbB-3 mRNA sequence (Kraus et al., 1989). The 20-mer 3' primer of
erbB-4 (5'-AACATAAACAGCAAATGTCA-3') is complementary to nt 2039-2058 in the transmembrane domain of human erbB-4 (Plowman et al., 1993a). Nucleotides 1 and 10 in this primer differ from the recently published rat erbB-4 sequence at positions 2049 and 2058 (GenBank
accession number AF041838).
RNase protection assay-solution hybridization
Immediately after decapitation of the rats, brains were removed,
and the medial basal hypothalamic area including the ME, arcuate
nucleus (ARC), and the ventromedial nuclei of the hypothalamus (VMH)
(referred to as ME-ARC) was collected, as described previously (Ma et
al., 1992). Cerebral cortex was used as a control. All dissected
tissues were quickly frozen on dry ice and stored at 85°C until RNA isolation.
Total RNA from brain tissue and cultured cells was isolated as reported
previously (Lara et al., 1990; Ojeda et al., 1991; Ma et al., 1994a).
The RNase protection assay used has been described previously in detail
(Ma et al., 1996). In brief,
32P-UTP-labeled rat erbB-2 and erbB-4
antisense RNA probes were purified using a Fullengther apparatus
(Biokey Co., Portland, OR), as recommended (Ma et al., 1996). Each
probe (500,000 cpm) was hybridized to RNA samples (5 µg/tube) or to
different amounts of in vitro synthesized sense RNA for
18-20 hr at 45°C. The tissue RNA samples were simultaneously
hybridized to 5000 cpm of a cyclophilin antisense cRNA probe to correct
for procedural variability (Ma et al., 1996), because cyclophilin mRNA
is constitutively expressed in brain and other tissues (Danielson et
al., 1988). Upon completion of the hybridization, the samples were
treated with ribonucleases A and T1 to digest unhybridized RNA species.
The protected cRNA fragments were separated by polyacrylamide gel
electrophoresis (5 or 7% acrylamide, 7 M urea),
and the hybridization signals were visualized by exposing the dried
gels to Reflection x-ray film (NEN, Boston, MA). Quantitation of the
signals was performed as described previously (Ma et al., 1994a), using
an edited version of the NIH Image program (Correa-Rotter et
al., 1992).
Probes
To prepare erbB riboprobes labeled with
32P-UTP (for RNase protection assay) or
35S-UTP (for hybridization
histochemistry), we used DNA templates corresponding to sequences
contained in the coding region of each erbB mRNA. An erbB-2 DNA
template was prepared by cloning a BamHI 422 bp fragment of
a rat erbB-2 cDNA (Bargmann et al., 1986a) (pSV2NeuT; a generous gift
of Dr. R. A. Weinberg, Whitehead Institute for Biomedical
Research, Cambridge, MA) into the BamHI site of pGEM-3Z.
ErbB-3 and erbB-4 cDNA templates generated by Reverse transcription
(RT)-PCR (see above) were cloned into the riboprobe vector pGEM-T. The
cyclophilin cDNA template used for the transcription of cyclophilin
cRNA probes was a 132 bp NcoI DNA fragment excised from a
rat cyclophilin cDNA (Danielson et al., 1988) and cloned into the
riboprobe vector pGEM-5zf().
Hybridization histochemistry
The procedure used (Simmons et al., 1989) has been reported in
detail previously (Ma et al., 1992, 1994b). Briefly, the brains were
transcardially perfused with 4% paraformaldehyde in borate buffer, pH
9.5. After an overnight post-fixation in the same fixative containing
10% sucrose, the brains were blocked and stored at 85°C until
being coronally sectioned at 20-25 µm using a sliding microtome. The
sections were then mounted on Superfrost Plus slides (Fisher
Scientific, Kent, WA) and dried overnight under vacuum before
hybridization. After prehybridization (Simmons et al., 1989), each
slide was overlaid with 70 µl of hybridization solution containing
50% formamide, 0.25 M NaCl, 10 mM Tris, pH
8.0, 10 mM EDTA, 2× Denhardt's solution, and the
riboprobe of interest (1 × 107
cpm/ml). Thereafter, the slides were hybridized for 18-20 hr at
55°C. Posthybridization washes were performed as reported previously (Simmons et al., 1989; Ma et al., 1992, 1994b). After dehydration in
graded alcohols, the slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) and developed after 3 weeks of exposure. Controls sections were hybridized with erbB-2 or erbB-4 sense
RNA probes.
Immunohistochemistry-confocal microscopy
The brains of peripubertal female rats were fixed by
transcardiac perfusion with Zamboni's fixative (Ma et al., 1994b) and subjected to double immunohistofluorescence using 50 µm floating vibratome sections and a procedure described previously (Jung et al.,
1997). ErbB-2 was detected with a monoclonal antibody (Ab)
(c-neu, Ab-3; Oncogene Research Products, Cambridge, MA) diluted 1:100.
ErbB-4 was detected with monoclonal antibody c-erbB-4, Ab-1 (also at a
1:100 dilution; NeoMarkers, Union City, CA). Astrocytes were identified
with a monoclonal antibody to GFAP (1:1000; Sigma, St Louis, MO). After
an overnight incubation at 4°C with either erbB-2 or erbB-4
antibodies, the reactions were developed with a Texas Red-conjugated
goat anti-mouse gamma globulin (1:200, 1 hr at room temperature;
Jackson ImmunoResearch, West Grove, PA). After extensive washes,
the sections were incubated (overnight at 4°C) with the GFAP
antibody. Because this antibody was also monoclonal, we did not use a
secondary antibody to develop the reaction, but instead labeled the
GFAP antibody directly with Oregon Green 488 (Molecular Probes, Eugene,
OR), according to the manufacturer's instructions, to a specific
activity of 2.7 molecules of dye per molecule of antibody protein.
Immunofluorescence controls consisted of sections incubated in the
absence of the primary antibodies.
Confocal images were acquired using a Leica (Nussloch, Germany) TCS NT
confocal system, with a 25× NA 0.75 PL FLUOTAR objective. FITC and
Texas Red were imaged simultaneously in most cases, using the 488 and
568 nm lines of an argon and krypton gas lasers, respectively, for excitation, a double dichroic at 488 and 568 nm, and a reflective mirror for wavelengths <580 nm in front of the first detection channel. A bandpass emission filter of 530 ± 30 nm was used for Oregon Green, and a long-pass filter at 590 nm was used for Texas Red.
The intensity of the excitation light in each channel was adjusted so
that the contribution of fluorescein to light detected in the Texas Red
channel was negligible. Typically, 16 optical sections 1-3 µm apart
were acquired for each image. Colors were merged and sections were
projected into a single plane using MetaMorph (Universal Imaging, West
Chester, PA). Images were further processed using Photoshop 5.0 (Adobe
Systems, San Jose, CA).
ErbB-1, erbB-2, and erbB-4 tyrosine phosphorylation
For erbB-1, erbB-2, and erbB-4 kinase assays, lysates from
treated cells were microfuged at 4°C for 5 min, and the supernatants were reacted for 3 hr at room temperature with a slurry of protein A-Sepharose that had been preabsorbed to antibodies against either erbB-1 (polyclonal Ab 1383; a gift from Shelton Earp, Department of
Pharmacology, University of North Carolina, Chapel Hill, NC), erbB-2
(polyclonal Ab 1275, provided by G. Clinton, Department of
Biochemistry, Oregon Health Sciences University, Portland, OR; or
SC-284, purchased from Santa Cruz Biotechnology, Santa Cruz, CA), or
erbB-4 (SC-283; Santa Cruz Biotechnology). Immunoprecipitated proteins
were electrophoresed on a 8% SDS-polyacrylamide minigel and then
transferred onto a nitrocellulose membrane. After blocking for 1 hr
with 2% BSA-0.2% Tween 20 in Tris-buffered saline (TBS), the
membranes were probed with a monoclonal phosphotyrosine antibody (4G10,
kindly provided by Dr. David Kaplan, Montreal Neurological Institute,
Montreal, Canada; or PY-20, purchased from Santa Cruz Biotechnology)
and then with an anti-mouse HRP-linked antibody (Boehringer Mannheim,
Indianapolis, IN), as described previously (Ma et al., 1994b). After
several extensive washes, tyrosine phosphorylated proteins were
detected using the Enhanced Chemiluminescence system from Amersham
Pharmacia Biotech (Arlington Heights, IL).
Cross-linking of erbB-2 and erbB-4 receptors
Hypothalamic astrocytes were maintained in serum-containing
medium in 100 mm culture dishes until they reached ~ 90%
confluency. They were then cultured in ADM (see above) for 48 hr before
treatment with NDF2 (500 ng/ml) for 3 min at 37°C. At the end of
this treatment, the astrocytes were exposed to bis (sulfosuccinimidyl)
suberate (BS3) (2 mM; Pierce,
Rockford, IL), a noncleavable, amine-reactive cross-linker (Staros and
Kakkad, 1983), for 2 min at room temperature, followed by incubation on
ice for 30 min. Thereafter, the cells were washed with ice-cold PBS,
collected into PBS, pelleted by brief centrifugation, and lysed in RIPA
buffer. The lysates were immunoprecipitated overnight at 4°C using
rabbit polyclonal antibodies to either erbB-2 (C-Neu, Ab-1; Oncogene
Research Products) or erbB-4 (SC-283; Santa Cruz Biotechnology) at
1:200 dilution. For each sample tube, 80 µl of protein A-Sepharose
(1:1 slurry, in water) was added, and the tubes were tipped for an
additional 3 hr. Immunoprecipitates were pelleted by
microcentrifugation and washed once with ice-cold RIPA and once with
ice-cold PBS. Each immunoprecipitate was suspended in 30 µl of
H2O and 15 µl of a 3× concentrated sample
buffer (final concentration of 0.0625 M Tris, pH 6.8, 3%
SDS, 5% glycerol, and 5% -mercaptoethanol). The protein samples
were denatured at 100°C for 5 min, and 15 µl of each sample was
used for SDS-PAGE on a 6% polyacrylamide gel. Separated proteins were
electrophoretically transferred to a Hybond ECL nitrocellulose membrane
(Amersham Pharmacia Biotech, Piscataway, NJ), and blocked with 2% BSA
and 0.2% Tween 20, in TBS for 1 hr at room temperature. The membranes
were then incubated with antibodies, either erbB-2 (mouse monoclonal
C-Neu, Ab-3; Oncogene Research Products) or the same erbB-4 used
previously for immunoprecipitation (each at 1:200 dilution). After 4 hr
incubation at room temperature, the membranes were washed three times
(10 min each) with TBS-Tween 20 at room temperature and then incubated with a horseradish peroxidase-linked secondary antibody, diluted 1:5000
with TBS-Tween 20, for 1 hr at room temperature. After washing, the
reactions were developed using the SuperSignal Ultra Chemiluminescent
Substrate (Pierce).
Ovariectomy and steroid treatment
The ovaries from early juvenile (22-d-old) rats were removed via
a dorsal approach, as described previously (Andrews et al., 1981). Five
days after surgery, the rats received a subcutaneously SILASTIC capsule
containing corn oil or 17-E2 (Sigma) dissolved in corn oil at a concentration of 400 µg/ml (Andrews et al., 1981). These capsules have been shown (Andrews et al., 1981) to produce circulating levels of E2 similar to those that
precede the first preovulatory surge of gonadotropins at puberty in
female rats (Andrews et al., 1980). Some animals received a single
subcutaneous injection of progesterone (P) (1 mg/rat) 50 hr
(12:00 P.M.) after implantation of the
E2-containing capsules, to simulate the abrupt increase in plasma P that occurs in the afternoon of first proestrus, at the time of the first preovulatory surge of gonadotropins (Andrews et al., 1980). Brain tissues were collected 4 hr after the P injection, i.e., after 54 hr of estradiol exposure.
Intracerebroventricular infusion of an erbB-2
antisense oligodeoxynucleotide
To determine the importance of a functional hypothalamic erbB-2
for the timing of puberty, in vivo experiments were
performed. The same antisense oligodeoxynucleotide found to be
effective in inhibiting the astrocyte response to NRG1 in
vitro was chronically infused into the third ventricle of the
brain via a stereotaxically implanted infusion cannula (Plastic One,
Roanoke, VA) connected to a subcutaneously implanted Alzet mini-osmotic
pump (Alzet Corporation, Palo Alto, CA). The pumps (model 2002) have a
flow rate of 0.5 µl/hr and a capacity of 200 µl, resulting in a
delivery period of 14 d. Each pump was loaded with artificial
CSF (Dalva and Katz, 1994) containing either the erbB-2
antisense oligodeoxynucleotide or the scrambled sequence at 5 µg/ml.
Upon connection to the infusion devise and a 4 hr preincubation at
37°C, the assembly was implanted into 25-d-old juvenile intact
animals. At this time, the prepubertal increase in hypothalamic erbB-2
mRNA levels had not yet begun. Starting on day 30, the animals were
monitored daily for vaginal opening (see below). Once vaginal opening
occurred, vaginal lavages were obtained to estimate the time of first
ovulation (Ojeda and Urbanski, 1994). All animals were killed on
the day of first diestrus (defined by the presence of a predominance of
leukocytes in the vaginal lavage) after an estrous type of vaginal
cytology. This change in vaginal cytology has been shown to be an
accurate indication that the first ovulation has taken place (Rage et
al., 1997). In all cases, ovulation was confirmed by visual inspection
of the ovaries to verify the presence of corpora lutea.
Phases of puberty
The developmental changes in hypothalamic erbB-2 and erbB-4 gene
expression were examined at ages shown previously to correspond to key
stages of sexual maturation in the rat (for review, see Ojeda and
Urbanski, 1994). The different stages of puberty were defined according
to established criteria (Ojeda and Urbanski, 1994). According to these
criteria, the juvenile period in the female rat extends from postnatal
days 21-30. Thus, the juvenile animals used in this study can be
considered as mid-to-late juveniles. Their vaginae were closed, and
their uteri weighed 60 mg or less, with no accumulation of intrauterine
fluid. Animals with enlarged uteri and detectable intrauterine fluid
(an indication of E2 secretion) are considered to
be in the early phases of puberty and, thus, are classified as being in
an early proestrous (EP) stage, which precedes the day of the first
preovulatory surge of gonadotropins. Animals showing a uterus
"ballooned" with fluid and a uterine weight of at least 200 mg were
considered to be in late proestrus (LP), the phase of puberty during
which LHRH and gonadotropins are for the first time discharged as a
preovulatory surge. Ovulation occurs in the early morning of the next
day (first estrus; E). At this time, the vagina becomes patent and
exhibits a cytology of cornified cells. Formation of the first corpora
lutea leads to the first diestrus phase of puberty, characterized by a
vaginal cytology showing a predominance of leukocytes and the presence of fresh corpora lutea in the ovaries.
Statistics
Changes in erbB-2 or erbB-4 mRNA levels during different stages
of sexual development or in response to gonadal steroid treatments were
analyzed by a one-way ANOVA, followed by the Student-Neuman-Keuls multiple comparison test for unequal replications.
| |
RESULTS |
NRGs and erbB mRNAs are expressed in the hypothalamus of immature
female rats
To determine whether members of the NRG-erbB signaling complex
are expressed in the hypothalamus of prepubertal female rats, total RNA
from this region was subjected to RT-PCR using primers complementary to
sequences contained within the NRG (NRG1, NRG2, and NRG3), erbB-2,
erbB-3, and erbB-4 mRNA coding regions. With the exception of the NRG2
and erbB-3 genes, all other components of the signaling module were
found to be expressed in the hypothalamus, as evidenced by the
amplification of cDNA fragments of the expected size (Fig.
1) and their identification by sequencing
(data not shown). RT-PCR amplification of RNA from isolated
hypothalamic astrocytes yielded an identical expression profile (data
not shown). Although the primers used to detect NRG2 mRNA in
hypothalamic tissue yielded PCR products of a size similar to the
NRG2 and cDNAs amplified from hippocampus (Fig. 1), sequencing
of several of these hypothalamic DNA fragments revealed that they did
not contain the NRG2 sequence. On the other hand, failure to detect erbB-3 mRNA in the hypothalamus was not because of ineffective primers or inadequate PCR conditions, because an erbB-3 cDNA was readily amplified from tissues known to express the erbB-3 gene, such
as the liver and skin keratinocytes (Fig. 1). Thus, neither the
hypothalamus of immature rats nor isolated hypothalamic astrocytes contain the NRG2 and erbB-3 mRNAs, indicating that, in this region of
the brain, NRG-dependent signaling is effected exclusively via NRG1 and
NRG3 acting on erbB-2-erbB-4 receptors.
View larger version (87K):
[in this window]
[in a new window]
|
Figure 1.
Detection by RT-PCR of NRGs and erbB mRNAs in the
medial basal hypothalamus of late juvenile 28- to 30-d-old female rats.
M, DNA molecular size markers. NRG1, 268 bp; NRG2,
314 bp; NRG2, 270 bp; NRG3, 371 bp; erbB-2, 323 bp; erbB-3, 337 bp;
and erbB-4, 168 bp. H, Hypothalamus; Hc,
hippocampus; K, human keratinocytes; Lv,
rat liver; bp, base pairs.
|
|
Astroglial expression of erbB mRNAs is region-specific
It was shown previously that hypothalamic astroglial cells differ
both molecularly and functionally from astrocytes of regions not
involved in neuroendocrine regulation (Ma et al., 1992, 1994a). To
determine whether these differences are also manifested in the case of
NRG receptors, the relative abundance of the mRNAs encoding erbB-2,
erbB-3, and erbB-4 in hypothalamic and cerebrocortical astrocytes in
culture was assessed by RNase protection assay. Figure
2 shows that (1) erbB-2 mRNA is much more
abundant in hypothalamic than cortical astrocytes, (2) erbB-4 mRNA is
only detected in hypothalamic astrocytes, and (3) neither subpopulation
of astrocytes expresses the erbB-3 gene. Thus, as suggested by the PCR
data, NRG signaling in hypothalamic astrocytes appears to exclude an involvement of erbB-3 receptors.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 2.
Detection of erbB-2 and erbB-4 mRNAs and absence
of erbB-3 mRNA in isolated hypothalamic (Hypo) and
cerebrocortical (CTX) astrocytes (Astro.), as
assessed by RNase protection assay. M,
32P-UTP-labeled RNA molecular size marker;
UP, undigested cRNA probes; DP, digested
probes; cyclo, cyclophilin.
|
|
Localization of erbB-2 and erbB-4 mRNA in the peripubertal
female hypothalamus
Hybridization histochemistry of the brain from prepubertal (28-to
37-d-old) rats demonstrated that erbB-4 mRNA transcripts were more
clearly detected in the paraventricular (Fig.
3A, arrows), the arcuate nucleus (ARC), and the ventromedial (VMH) and dorsomedial (DMH) nuclei (B) of the hypothalamus. Although in all
of these regions some of the hybridization signal, analyzed under
bright-field illumination, was seen in neurons, a substantial fraction
of the signal was associated with small, dark nuclei, suggesting an
astroglial localization. Such a localization was clearly evident in the
subependymal region of the third ventricle (A, B,
small arrowheads) and the median eminence (B,
large arrowheads). This is a region devoid of neuronal cell
bodies that serve as a final common pathway for neurosecretory nerve
terminals converging to release their products into the portal
vasculature. ErbB-4 mRNA was detected in glial cells located in the
intermediate and external layer of the median eminence (B,
C, small and large arrowheads,
respectively). In addition to the hypothalamus, erbB-4 mRNA was
abundant in the piriform cortex and hippocampus (data not shown). In
both of these regions, the mRNA-containing cells appeared to be mostly
neurons.
View larger version (145K):
[in this window]
[in a new window]
|
Figure 3.
Cellular localization of erbB-4 and erbB-2 mRNAs
in the brain of peripubertal female rats, as detected by in
situ hybridization using 35S-UTP-labeled cRNA
probes. Within hypothalamic nuclei, the mRNA was found to be more
abundant in cells of the paraventricular nuclei (A,
arrows) and the ARC, VMH, and DMH nuclei
(B). ErbB-4 mRNA is also diffusely
detected throughout the medial basal hypothalamus (B,
C) and is more abundantly present in cells of the
subependymal region (A, B, small
arrowheads) and glial cells in the intermediate and external
layers of the median eminence (B and C,
small and large arrowheads,
respectively). ErbB-2 transcripts were more abundantly expressed in
ependymal cells lining the third ventricle (E,
F, arrowheads) and glial cells of the
median eminence (F, arrows).
D and G depict sections adjacent to
C and F hybridized with sense erbB-4
(D) and erbB-2 (G) RNA
probes. Scale bars: B, 200 µm; A,
C-G, 100 µm.
|
|
In contrast to erbB-4 mRNA, the hypothalamic distribution of erbB-2
mRNA transcripts was more circumscribed. ErbB-2 mRNA was predominantly
detected in ependymal cells lining the third ventricle (E,
F, arrowheads) and glia of the median eminence
itself (F, arrows). Adjacent tissue sections
incubated with the respective sense RNA probes showed no hybridization
signals (D, G).
Localization of erbB-2 and erbB-4 proteins in the peripubertal
female hypothalamus
Because the medial basal hypothalamic-median eminence region
showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined
this region for the presence of erbB-2- and erbB-4-immunoreactive cells
using immunohistofluorescence followed by confocal microscopy. Both
receptor proteins were found in glial cells, in addition to some
neurons. ErbB-2 immunoreactivity was abundant in tanycytes lining the
wall of the third ventricle (Fig.
4A,B,F),
with immunoreactive material distributed throughout the length of the
processes that these cells send to the base of the brain (B,
arrows). Astrocytes of the ventral aspect of the median
eminence, identified by their content of GFAP
(green), were also rich in erbB-2 immunoreactivity (C-E), as were astrocytes located along the walls of
the third ventricle adjacent to tanycytes, which were negative for GFAP (F-I). In addition to this location, erbB-2
immunoreactivity was also observed in astrocytes of the medial basal
hypothalamus away from the median eminence, especially those
surrounding blood vessels (J-L). Scattered neurons
containing erbB-2 were also observed (A, B,
short arrows).
View larger version (103K):
[in this window]
[in a new window]
|
Figure 4.
Immunofluorescent confocal microcopy localization
of erbB-2 in the medial basal hypothalamus-median eminence region of
peripubertal female rats. ErbB-2 was visualized with a monoclonal
antibody to an amino acid sequence contained in the human protein, and
the reaction was developed with a second antibody conjugated to
the fluorochrome Texas Red (red). Astrocytes were
identified with a monoclonal antibody to GFAP directly labeled with the
fluorochrome Oregon Green 488 (green). A,
B, Low- and high-magnification views of the medial basal
hypothalamus-median eminence region showing abundant erbB-2
immunoreactivity in tanycytes lining the wall of the third ventricle
(A, arrowheads) and the processes that
these cells send to the median eminence (B,
arrows). Some neurons are also labeled
(A, B, short arrows).
C-E, Presence of erbB-2-immunoreactive material
(D, red) in astrocytes (C,
green) of the ventrolateral aspect of the median
eminence (images merged in E). F,
Presence of erbB-2 in astrocytes adjacent to ependymal cells of the
third ventricle. G-I, Higher magnification view of
erbB-2-positive (H) astrocytes
(G) adjacent to the third ventricle (images
merged in I). Notice the abundant erbB-2
immunoreactivity in GFAP-negative tanycytes. J-L,
Detection of erbB-2 in astrocytes of the medial basal hypothalamus
associated with blood vessels. Scale bars: A, 100 µm;
B, 25 µm; C-E, G-L, 10 µm; F, 40 µm.
|
|
In agreement with the mRNA localization, erbB-4 immunoreactivity was
observed in glial cells of the median eminence (Fig. 5A,C,
arrowheads) and neurons of the arcuate nucleus
(A, B, arrows). As in the case of
erbB-2, astrocytes of the ventral aspect of the median eminence were
found to contain erbB-4 (D-F), as were astrocytes
adjacent to the wall of the third ventricle (G-I), and astrocytes of the medial basal hypothalamus
(J-L), including those associated with blood vessels
(M). No erbB-4 immunoreactivity was detected in
tanycytes (G-I), a predominant site of erbB-2 expression.
View larger version (100K):
[in this window]
[in a new window]
|
Figure 5.
Immunofluorescence confocal microscopy
localization of erbB-4 in the medial basal hypothalamus-median
eminence region of peripubertal female rats. ErbB-4 was detected with a
monoclonal antibody directed against an epitope in the human erbB-4
sequence, and the reaction was developed with a Texas Red-conjugated
secondary antibody. GFAP was detected with a monoclonal antibody
directly labeled with Oregon Green 488. A,
Low-magnification view showing erbB-4-immunoreactive cells in the
arcuate nucleus (arrow) and glial cells of the median
eminence (arrowhead). B, Higher
magnification view of the arcuate nucleus showing erbB-4-positive
neuron-like cells (arrows). C,
High-magnification view of erbB-4-positive astrocyte-like cells of the
median eminence (arrowheads). D-F,
Presence of erbB-4 in astrocytes of the ventrolateral aspect of the
median eminence. Notice that not all astrocytes contain erbB-4
immunoreactivity. G-I, Presence of erbB-4 in astrocytes
adjacent to tanycytes of the third ventricle. J-L,
Presence of erbB-4 in astrocytes of the medial basal hypothalamus.
M, Higher magnification view of erbB-4-positive
astrocytes associated with blood vessels in the median basal
hypothalamus, away from the median eminence. Scale bars:
A, 100 µm; B, C, 25 µm; D-I, 40 µm; J-L, 20 µm;
M, 30 µm.
|
|
Hypothalamic levels of erbB-2 and erbB-4 mRNA increase during
juvenile and peripubertal development
To determine whether changes in the hypothalamic gene expression
of erbB-2 and erbB-4 may occur in association with female sexual
maturation, the tissue content of the encoding mRNAs in this brain
region was quantitated by RNase protection assay. The hypothalamic
levels of erbB-2 and erbB-4 mRNA increased for the first time during
late juvenile development (postnatal day 28) and then again during the
peripubertal period, reaching maximal values in the afternoon of the
first proestrous day (LP), i.e., coinciding with the time of the first
preovulatory surge of gonadotropins (Fig.
6, top and bottom),
previously shown to occur in the afternoon of this day (Ojeda and
Urbanski, 1994). The cerebral cortex, a brain region irrelevant to
neuroendocrine control, displayed low and unchanging levels of erbB-2
mRNA throughout the juvenile and peripubertal period (Fig. 6,
top). Although the cortical levels of erbB-4 mRNA were
higher than those seen in the hypothalamus during midjuvenile
development (postnatal day 24-26), they did not increase with the
advent of sexual maturity (Fig. 6, bottom).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 6.
Developmental changes in erbB-2
(top) and erbB-4 (bottom) mRNA content in
the medial basal hypothalamus (median eminence-arcuate nucleus, ME-ARC
region) during juvenile and peripubertal development of the female rat.
The increase in gene expression associated with the advent of sexual
maturation is contrasted with the lack of changes in the cerebral
cortex (CTX), a region of the brain irrelevant to
reproductive neuroendocrine control. The dotted vertical
line separates the juvenile from the peripubertal periods.
EP, Early proestrous phase of puberty; a time during
which the first morphological manifestations of puberty become evident
as an increase in uterine weight and accumulation of uterine fluid.
LP, The first proestrus; the first preovulatory
discharge of gonadotropins (arrows) takes place in the
afternoon of this day. E, The first estrus; ovulation
occurs in the early morning hours of this day. Each
point represents the mean ± SEM mRNA values
(vertical lines) of three independent observations. Each
observation derives from hypothalamic tissue pooled from three rats.
*p < 0.05, first significant increase over
early (24-d-old) juvenile values.
|
|
The juvenile increase in hypothalamic erbB-2 and erbB-4 mRNA levels is
gonad-independent, but the peripubertal increase is an ovarian
steroid-regulated event.
The juvenile increase in hypothalamic levels of erbB-2 and erbB-4 mRNA
occurs at the time when circulating levels of ovarian steroids are low
(Table 1). Thus, this initial increase in
expression appears to be a centrally driven, gonad-independent event.
In contrast, the changes in mRNA content observed during normal puberty occur at the time when the plasma levels of E2
first, and progesterone (P) later, are elevated (Ojeda and Urbanski,
1994), suggesting that at least part of the peripubertal changes in
mRNA levels is caused by the rising circulating levels of these ovarian
steroids. Mimicking in ovariectomized juvenile rats the preovulatory
increase in plasma E2 that occurs at puberty
(Andrews et al., 1980), via implantation of
17-E2-containing capsules (Andrews et al.,
1981), resulted 2 d later in a significant increase in
hypothalamic erbB-4 mRNA levels but not in erbB-2 mRNA content (Fig.
7), thus reproducing the changes in mRNA
content observed during normal puberty before the afternoon
preovulatory discharge of gonadotropins (Fig. 6). Administration of a
single dose of P- to E2-treated animals, to produce plasma P levels similar to those found at the time of the
gonadotropin surge (Andrews et al., 1980), resulted in a marked increase in erbB-2 mRNA content but no further change in erbB-4 mRNA
levels compared with animals treated with E2
alone (Fig. 7). The changes caused by this sequential
E2 plus P treatment were again similar to those
observed in the afternoon of the first proestrus during normal puberty
(Fig. 6). P alone was ineffective. Thus, most of the increase in
hypothalamic erbB-2 and erbB-4 gene expression at the time of puberty
appears to be a gonad-dependent event.
View this table:
[in this window]
[in a new window]
|
Table 1.
Serum levels of sex steroid in juvenile female rats at the
time when hypothalamic erbB-2 and erbB-4 mRNA content first increases
before puberty
|
|
View larger version (38K):
[in this window]
[in a new window]
|
Figure 7.
Increase in steady-state levels of erbB-2 and
erbB-4 mRNA in the medial basal hypothalamus (ME-ARC region) of
juvenile ovariectomized rats induced by E2 or the
sequential combination of E2 plus P. The animals were
ovariectomized on postnatal day 22 and received 5 d later a
subcutaneous SILASTIC capsule containing 17-E2 at a
concentration that produces preovulatory levels of serum
E2. Control animals received a capsule filled with the
vehicle (V, corn oil). P was administered 50 hr later
(at 12:00 P.M.) as a single subcutaneous injection. All animals were
euthanized 4 hr after the P injection (i.e., at 4:00 P..M., 54 hr after
receiving the E2-containing capsule). Each
bar represents the mean ± SEM of three independent
observations (vertical lines). Each observation derives
from hypothalamic tissue pooled from three rats.
**p < 0.01 versus V-treated group.
CTX, Cerebral cortex.
|
|
NRG-dependent phosphorylation of erbB-2 in astroglia requires the
participation of erbB-4
The mammary tumor cell line MCF-7 that lacks erbB-1 but
contains erbB-2 receptors (Karunagaran et al., 1996) failed to respond to EGF with either erbB-1 or erbB-2 tyrosine phosphorylation (Fig. 8A, left)
but showed a strong increase in erbB-2 phosphorylation upon stimulation
with the NRG NDF-2. In contrast to MCF-7 cells, hypothalamic
astrocytes, which contain functional erbB-1 receptors (Ma et al.,
1994a, 1997a), responded to the EGF relative TGF, with tyrosine
phosphorylation of both erbB-1 and erbB-2 (Fig. 8A,
right). In addition, they showed erbB-2 and erbB-4 tyrosine phosphorylation when exposed to NDF-2 (Fig. 8A,
right). Cerebrocortical astrocytes, on the other hand,
responded to TGF with erbB-1 and erbB-2 phosphorylation (Fig.
8B, left) but showed no erbB-2
phosphorylation upon exposure to NDF-2. Because cortical astrocytes
express the erbB-2 but not the erbB-3 or erbB-4 genes (Fig. 2), we
reasoned that the inability of NDF-2 to induce erbB-2
phosphorylation was because of the lack of appropriate neuregulin
receptors in these cells. Hypothalamic astrocytes, which express the
erbB-4 gene, respond to NDF-2 with erbB-2 phosphorylation (Fig.
8B, middle). We, therefore, tested the
possibility that the lack of NRG-dependent erbB-2 phosphorylation in
cortical astrocytes was caused by the absence of erbB-4. Expression of
erbB-4 in these cells, via transient transfection with cNHER4 (Plowman
et al., 1993b), a plasmid that contains a cDNA encoding the human
erbB-4 receptor, led to an marked increase in both basal and
NDF-2-induced erbB-2 phosphorylation (Fig. 8B,
right).
View larger version (46K):
[in this window]
[in a new window]
|
Figure 8.
A, Left to
right, Tyrosine phosphorylation of erbB-1 and erbB-2
receptors in MCF-7 cells induced by EGF and NDF-2, and
phosphorylation of erbB1, erbB-2, and erbB-4 receptors in hypothalamic
astrocytes (H.A.) induced by TGF or NDF-2. The
cells were exposed for 5 min to each ligand (100 ng/ml) before lysis.
The erbB receptors were immunoprecipitated (IP) with
antibodies specific to each protein, and the tyrosine phosphorylated
receptors were identified by Western blots using phosphotyrosine
antibodies. B, Left to
right, Phosphorylation of erbB-1 and
transphosphorylation of erbB-2 in cerebrocortical astrocytes
(C.A.) by TGF; inability of NDF-2 to
phosphorylate erbB-2 in cortical astrocytes; phosphorylation of erbB-2
in hypothalamic astrocytes by NDF-2; and effectiveness of NDF-2
to induce erbB-2 phosphorylation in cortical astrocytes after transient
overexpression (Transf.) of erbB-4.
|
|
NRG-dependent activation of erbB-4 in hypothalamic astrocytes
involves formation of erbB-4-erbB-2 heterodimeric complexes
To determine whether exposure of hypothalamic astrocytes to
neuregulins results in receptor heterodimerization as shown in cell
lines (Spivak-Kroizman et al., 1992; Cohen et al., 1996), astrocytic cultures were treated with NDF-2 (3 min at 37°C)
followed by cross-linking with bis (sulfosuccinimidyl) suberate (Staros and Kakkad, 1983). Immunoprecipitation of the reactive species with
monoclonal antibodies to either erbB-2 or erbB-4, followed by Western
blot analysis using antibodies to erbB-2, demonstrated in both cases
the presence of a high molecular weight complex of ~365 kDa (Fig.
9, left and
middle). To verify the presence of erbB-4 in this complex,
the cross-linked species were immunoprecipitated with antibodies to
erbB-2, and the Western blot was developed with monoclonal antibodies
to erbB-4. The results showed the presence of erbB-4 in a high
molecular weight species identical in size to that detected by the
erbB-2 antibodies (Fig. 9, right).
View larger version (70K):
[in this window]
[in a new window]
|
Figure 9.
Heterodimerization of erbB-4 with erbB-2 receptors
in hypothalamic astrocytes. After exposure to NDF-2 (500 ng/ml, 3 min), the cells were exposed to the cross-linker
BS3, lysed, immunoprecipitated (IP)
with either erbB-2 or erbB-4 antibodies, and blotted
(IB) with erbB-2 or erbB-4 antibodies. Notice the
formation of a high molecular weight complex containing both erbB-2
(left and middle) and erbB-4
(right).
|
|
Effect of NRG1 isoforms on the secretion of PGE2 from
hypothalamic astrocytes
In a previous study, we showed that TGF-dependent activation of
erbB-1 receptors in hypothalamic astrocytes results in the release of
PGE2 (Ma et al., 1997a), a prostaglandin involved
in mediating neurotransmitter-induced LHRH secretion from the
hypothalamus (Ojeda et al., 1986b). To determine whether NRG-1 exerts a
similar stimulatory effect, cultured hypothalamic astrocytes were
treated with three different isoforms of NDF, including NDF-1, 2,
and 2 (Wen et al., 1994). These isoforms were selected because of their proven ability to promote glial cell function. Thus, the 1 and
2 forms induce astrocyte maturation (Pinkas-Kramarski et al., 1994),
and the 2 form facilitates the survival and maturation of glial cell
precursors (Dong et al., 1995). Figure
10 (left) shows that
NDF-2 and NDF-2 were as effective as TGF in stimulating PGE2 release. Surprisingly, NDF-1, the
reported major neuronal NDF isoform (Wen et al., 1994), was ineffective
(Fig. 10, left).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 10.
Left, Stimulatory effect of
neuregulin isoforms (each at 50 ng/ml) on PGE2 release from
cultured hypothalamic astrocytes and comparison with the effect of
TGF (50 ng/ml). Right, Potentiation of the effect of
TGF on PGE2 release from hypothalamic astrocytes by a
dose of NDF-2 (10 ng/ml) that by itself is ineffective.
Bars or circles represent the mean of
five to nine culture wells per group. Vertical lines are
SEM. **p < 0.01 versus control
(C) group.
|
|
Neuregulins facilitate the stimulatory effect of TGF on PGE2
release from hypothalamic astrocytes
Because neuregulins bind only to erbB-3 and erbB-4 receptors but
recruit erbB-2 and erbB-1 receptors for expanded signal transduction (Beerli et al., 1995; Karunagaran et al., 1996; Lemke, 1996; Burden and
Yarden, 1997), we sought to determine whether neuregulins would be able
to potentiate the effect of low concentrations of TGF on glial
PGE2 release. Exposure of hypothalamic astrocytes to a low, ineffective dose of NDF-2 (10 ng/ml) markedly facilitated the effect of marginally effective doses of TGF (Fig. 10,
right), suggesting that the two peptides are required for
full erbB receptor-dependent activation of eicosanoid synthesis in
hypothalamic astrocytes.
Neuregulin-induced activation of glial PGE2 release
requires erbB-2 receptors
In a variety of cell types, erbB-2 is required for
neuregulin-dependent signaling via erbB-4 receptor activation (Carraway and Cantley, 1994; Beerli et al., 1995). ErbB-2 also functions, via
receptor-receptor interactions, as an auxiliary subunit in erbB-1-mediated signal transduction (Karunagaran et al., 1996). It was,
therefore, important to determine whether targeted inactivation of
erbB-2 receptors would affect NRG and/or TGF signaling capacity in
hypothalamic astrocytes, as assessed by the ability of these growth
factors to stimulate glial PGE2 release in
erbB-2-deficient cells. Because inactivation of erbB-2 receptors can be
efficiently achieved by either administration of antisense ODNs
(Colomer et al., 1994) or the intracellular expression of a recombinant
single-chain antibody (Berrli et al., 1994; Beerli et al., 1995), we
chose one of these approaches for our studies. Figure
11 (top left) demonstrates the effectiveness of an erbB-2 ODN treatment (1 µM for 16 hr) to reduce the levels of
phosphorylated erbB-2 in hypothalamic astrocytes exposed to a 5 min
NDF-2 pulse. The inhibitory effect of the erbB-2 ODN was not seen
when the cells were treated with an oligodeoxynucleotide containing the
same base composition but in a scrambled order. No effect of the erbB-2
ODN on phosphorylated erbB-4 content was observed (Fig. 11, top
right), indicating that the ODN selectively targets erbB-2.
NDF-2-induced PGE2 release from hypothalamic
astrocytes was abolished by exposing the cells to the same dose of
erbB-2 ODN for the duration (16 hr) of the NRG treatment (Fig. 11,
middle). The scrambled sequence was ineffective. The ODN
partially blocked the effect of TGF, further indicating that, as in
immortalized cell lines (Beerli et al., 1995), erbB-2 is required for
full expression of ligand-initiated, erbB-1-dependent signaling in
primary astrocytes. The specificity of the erbB-2 ODN effect was
further demonstrated by its failure to alter the stimulatory effect of
basic FGF, or the lack of effect of IGF-I, on
PGE2 release (Fig. 11, middle). Thus,
its inhibitory effect on NRG and TGF signaling is not a result of a
general inactivation of receptor tyrosine kinases in glial cells.
View larger version (43K):
[in this window]
[in a new window]
|
Figure 11.
Top left, Selective decrease of
phosphorylated erbB-2 in hypothalamic astrocytes treated with an
antisense oligodeoxynucleotide to erbB-2 (erbB-2 ODN). The cells were
treated with the ODN (A, 1 µM) or a
scrambled sequence (S) for 16 hr. Then, some
dishes were left untreated (C) and others were
exposed to NDF-2 (N, 50 ng/ml) for 5 min before
immunoprecipitation and electrophoretic separation of phosphorylated
erbB receptors. The phosphorylated receptors were detected with an
antiphosphotyrosine monoclonal antibody. Top right,
Failure of erbB-2 ODN to alter NDF-2-induced erbB-4 receptor
phosphorylation. Middle, Blockade of the stimulatory
effect of NDF-2 and partial inhibition of the effect of TGF on
PGE2 release from cultured hypothalamic astrocytes by
ODN-mediated inhibition of erbB-2 synthesis. Each growth factor was
tested at 50 ng/ml; the erbB-2 ODN was used at a 1 µM
concentration. bFGF, Basic fibroblast growth factor, 50 ng/ml; IGF-I, insulin-like growth factor I, 50 ng/ml;
C, untreated controls; A, erbB-2 ODN;
S, scrambled erbB-2 ODN sequence. Bottom,
Stimulatory effect of a culture medium conditioned by exposure of
hypothalamic astrocytes to NDF-2 or TGF on LHRH release from the
GT1-1 LHRH-producing cells and blockade of this effect by treating the
astrocytes with an erbB-2 ODN. N, GT1-1 cells treated
directly with NDF-2 (50 ng/ml); CM, cells treated
with the conditioned medium from astrocytes cultured in the absence of
added growth factors; N-CM, astrocyte culture medium
con- ditioned by treating the astrocytes with NDF-2 (50 ng/ml) for 16 hr; T-CM, astrocyte culture medium
conditioned by TGF treatment (50 ng/ml, 16 hr); S-CM,
culture medium conditioned by treating the astrocytes with the
scrambled ODN sequence. Bars are mean of six wells per
group; vertical lines are SEM.
|
|
Neuregulins induce LHRH release via a glial intermediacy
Direct exposure of the LHRH-producing GT1-1 cells to NDF-2
failed to stimulate LHRH release (Fig. 11, bottom). In
contrast, the culture medium of hypothalamic astrocytes treated with
NDF-2 (N-CM) increased the release of LHRH more than threefold over control values. The changes observed were similar to those induced by
the culture medium of astrocytes treated with TGF (T-CM) (Fig. 11,
bottom), a conditioned medium previously shown to stimulate LHRH secretion via its PGE2 content (Ma et al.,
1997a). Concomitant treatment of the astrocytes with either NDF-2 or
TGF and the erbB-2 ODN abolished the effect of the
NDF-2-conditioned medium on LHRH release and partially suppressed
that of the TGF-conditioned medium (Fig. 11, bottom).
Culture medium from astrocytes treated with either NDF-2 or TGF
in the presence of the scrambled erbB-2 ODN sequence was as effective
in stimulating LHRH release as culture medium from astrocytes treated
with the growth factors alone. Direct application of culture medium
from astrocytes treated with the scrambled sequence to GT1-1 cells
also failed to affect basal LHRH release (Fig. 11, bottom).
Thus, neuregulins appear to stimulate the secretory activity of
LHRH-producing neurons via an astroglial-dependent, erbB-mediated
activation of PGE2 synthesis.
In vivo disruption of erbB-2 receptor synthesis by
central administration of an erbB-2 ODN delays the onset of female
puberty
To determine the physiological importance of hypothalamic erbB-2
receptor signaling in the central control of sexual maturation, juvenile female rats were treated with the same erbB-2 ODN used in the
in vitro studies. The ODN was administered into the third ventricle of the brain via a cannula connected to a subcutaneously implanted osmotic minipump, delivering its content at a rate of 2.5 µg/hr. The ODN infusion was initiated on postnatal day 25, i.e.,
before the first increase in hypothalamic erbB-2 mRNA expression that
occurs between postnatal day 26 and 28. Control animals received an
infusion of the scrambled ODN or were left intact.
Both control groups reached puberty at a very similar age (Fig.
12), so that by postnatal day 37 all of
them had ovulated (mean age at first ovulation, 35.5 ± 0.27 and
36.0 ± 0.30 d for intact and scrambled ODN-treated groups,
respectively). In striking contrast, the animals infused with the
erbB-2 ODN did not ovulate until after the content of the pump was
exhausted (i.e., after 14 d of infusion) (Fig. 12). Ovulation
occurred within 1-4 d after termination of the infusion, so that the
mean age at first ovulation was 41.6 ± 0.43 d
(p < 0.01 vs both control groups). Although
these results indicate that erb |
TOP
ABSTRACT
INTRODUCTION
