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Arthur M. Fishberg Research Center for Neurobiology and Henry L. Schwartz Department of Geriatrics and Adult Development, Neurobiology of Aging Laboratories, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., Neurobiology of Aging Laboratories, Box 1639, Mount Sinai School of Medicine, New York, New York 10029. E-mail: gore{at}msvax.mssm.edu
| Abstract |
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| Introduction |
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The maturation of the neuroendocrine hypothalamus is profoundly affected by neurotrophic factors, whose synthesis and release increase dramatically during the first 2 postnatal weeks of life in rodents (7, 8, 9). This period corresponds exactly with that of the most profound alterations in GnRH gene expression in rodents, between postnatal days (P) 5 and 10 (4, 5, 10), suggesting that the maturation of GnRH neurons may be a result of an increase in neurotrophic factor input. A likely candidate for this maturation is insulin-like growth factor-I (IGF-I), which is synthesized in peripheral tissues (11) and in the brain (12, 13), and whose receptors are expressed in neurons (14) and are abundant in the median eminence (15, 16), the site of GnRH neuroterminals. Furthermore, IGF-I is strongly implicated in reproductive function, as it causes a dose-dependent increase in GnRH secretion from median eminence explants (17), and plasma IGF-I levels increase during puberty (18, 19). The temporal pattern of binding of IGF-I to its receptors in the hypothalamus, peaking between P5 and P7 (8), a period of alterations in GnRH gene expression in vivo (4, 5, 10), raises the possibility that this growth factor is one of the metabolic signals important or obligatory for the maturation of the neuroendocrine axis and, ultimately, the attainment of adult reproductive function.
It is hypothesized that IGF-I would cause alterations in GnRH biosynthesis in GT17 cells, such that they would become a more representative model for an adult GnRH neuron. IGF-I receptors, as demonstrated by receptor binding, are present in GT1 cells (20). Treatment with this specific growth factor produces changes in GnRH release and GT1 morphology (21, 22). Thus, in the present study, we tested the hypothesis that treatment with IGF-I would cause the maturation of the immortalized GT17 cell line, as evaluated by alterations in biosynthetic and morphological indices. These experiments are intended to establish a cell system that is both morphologically and physiologically more representative of the adult GnRH neuron, for future studies on the regulation of GnRH gene expression. We also report the novel observation that GT17 cells themselves are IGF-I-immunopositive, suggesting the intriguing possibility of autofeedback of IGF-I onto its own receptors on GnRH cells.
| Materials and Methods |
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Experimental design
IGF-I effects on GnRH gene expression, release, and GT17 cell
morphology. Dose-response experiments were initially performed to
determine the optimal dose of IGF-I on GnRH gene expression. GT17
cells were treated with 0.1, 10, and 100 ng/ml IGF-I (Sigma, St. Louis,
MO) or vehicle (the same volume of ethanol in dH2O), after
1 h of serum deprivation. Cells were harvested after 24 h,
and GnRH RNA levels were measured by RNase protection assay. Then,
using the 10 ng/ml dose of IGF-I, which was determined to have the most
potent effect on GnRH gene expression, time-course experiments were
performed: cells were treated with IGF-I or vehicle for 0, 2, 4, 8, 24,
or 48 h. GT17 cells were harvested and RNase protection assays
performed separately on GnRH messenger RNA (mRNA) in the cytoplasm, and
GnRH primary transcript, an index of GnRH gene transcription (6), in
the nucleus. A 1-ml aliquot of medium was collected at the time of
harvest for RIA of GnRH peptide. Differences between groups were
analyzed by two-way ANOVA, followed by Fishers protected
least-significant-difference post hoc test. Significance was
set at P < 0.05.
IGF-I effects on GnRH mRNA half-life. To determine whether IGF-I affects GnRH mRNA half-life, indicating a posttranscriptional mechanism of action of IGF-I on GnRH mRNA levels, GT17 cells were serum-deprived for 1 h. Then, at t = -2.5 h, cells were treated with IGF-I or vehicle. At t = 0 h, cells were treated with the RNA polymerase II inhibitor 5,6-dichloro-1-ß-ribofuranosylbenzimidazole (DRB; 100 µg/ml final concentration, as described previously) (24). Cells were harvested at t = 0, 2, 4, 8, 16, or 24 h after DRB treatment. The GnRH mRNA half-life was determined by performing a regression analysis on the changes in GnRH mRNA levels for both control and IGF-I-treated cells.
Expression of IGF-I-immunoreactivity in GT17 cells. To determine whether IGF-I-like immunoreactivity is present in GT17 cells, immunocytochemistry for IGF-I was performed in GT17 cells using the rabbit polyclonal IHC-7296 antibody (Peninsula Laboratories, Belmont, CA). Cells were subcultured on sterile 22-mm tissue culture cover slips (Nunc, Naperville, IL) in six-well tissue culture plates for 23 days. Then, cells were fixed in 4% paraformaldehyde, 0.12 M sucrose for 20 min at 37 C. Cells were washed three times in PBS, then permeabilized with 0.25% Triton X-100 for 5 min at room temperature. Cells were washed three times in PBS, then pretreated with 10% BSA/PBS for 1 h at 37 C. This was removed, and the primary antibody (1:500 dilution) was applied. For control experiments, the antibody was preabsorbed with 100 ng, 500 ng, or 1 µg of the antigen (IGF-I, Sigma), or the primary antibody was omitted. GT17 cells were incubated with the primary antibody overnight at 4 C, then washed three times in PBS. The biotinylated secondary antibody (goat antirabbit IgG; Vector, Burlingame, CA), at 1:200, was applied and then incubated for 1 h at 37 C. Cells were washed three times in PBS, then treated with a Vector ABC kit according to the directions; the DAB reaction was allowed to proceed for 10 min. Cells were washed three times in PBS, then dehydrated in graded ethanol series and mounted with Gurrs Fluoromount (BDH Laboratories, Poole, England).
RNA extraction
GT17 nuclear RNA, harvested above, was treated with
deoxyribonuclease (DNase) I (40 U) for 5 min at 37 C. Both cytoplasmic
and nuclear fractions were treated with proteinase K at 45 C, extracted
with phenol:chloroform:isoamyl alcohol (25:24:1) and then with
chloroform:isoamyl alcohol (24:1), and precipitated with 2.5 vol
ethanol (nuclear) or 1.5 vol isopropanol (cytoplasmic) at -20 C (23, 24). Both fractions were centrifuged and washed with 70% ethanol, and
the pellets were resuspended in diethylpyrocarbonate-water. An aliquot
from each cytoplasmic fraction was used to determine the total RNA
content using an optical density reading at 260 nm. Aliquots of 0.5
µg were resuspended in 20 µl hybridization solution (0.1
M EDTA, 4 M guanidine thiocyanate) for RNase
protection assay of GnRH mRNA. The nuclear fraction underwent a DNase I
treatment for 30 min at 37 C with 60 U DNase I, extracted as above, and
precipitated with ethanol. It was then centrifuged and washed with 70%
ethanol, and the pellets were resuspended in 20 µl hybridization
solution for RNase protection assay of GnRH nuclear RNA
transcripts.
RNase protection assay
Cytoplasmic and nuclear RNA samples were incubated overnight at
30 C with 5 µl of probe (1 ng) labeled to low (600,000 cpm/ng: GnRH
cDNA and cyclophilin) or high (1,300,000 cpm/ng: GnRH A2B) specific
activity with [
-32P]uridine 5'-triphosphate. The probe
was also incubated with increasing concentrations of sense RNA to
produce a standard curve. Samples and standards were allowed to
hybridize with the probe for 1618 h at 30 C. The remainder of the
RNase protection assay was performed exactly as described previously
(23, 24). Samples were electrophoresed through 6% nondenaturing
polyacrylamide gels, which were exposed to x-ray film for 1848 h to
produce an autoradiogram, then exposed to a phosphorimaging screen
(Molecular Dynamics, Sunnyvale, CA) for 24 h for quantitation. The
amount of radioactivity in each sample, as determined by the
phosphorimager, was compared with the amount of reference RNA, as
calculated by regression analysis, and used to calculate the amount of
RNA in each sample.
Probes
The following probes were used: for GnRH mRNA, a 443-bp mouse
GnRH cDNA clone spanning the EcoO109I and XbaI
restriction sites and subcloned into a Bluescript SK(+) vector (23);
for cyclophilin, a 111-bp cDNA clone spanning the PstI and
XmnI restriction sites and subcloned into a Bluescript KS(+)
vector (25); and for GnRH primary transcript, a mouse GnRH clone,
complementary to the intron A-exon 2-intron B (A2B) region of the
proGnRH gene, spanning the SpeI and HindIII
restriction sites and subcloned into a Bluescript SK(+) vector
(24).
RIA
GnRH in the medium was measured in 150-µl aliquots by RIA
in a single assay using antiserum LR-10, kindly provided by Dr. R.
Guillemin. Synthetic GnRH (Richelieu Laboratory, Montreal, Canada) was
used for the radiolabeled antigen and the reference standard. The
antigen-antibody complex was precipitated with a goat antirabbit
-globulin (Calbiochem, La Jolla, CA). The sensitivity of the assay
was 0.5 pg/tube in a final vol of 500 µl without secondary antibody.
The intraassay coefficient of variation was 4%.
| Results |
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| Discussion |
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The timing of events caused by treatment with IGF-I, with an initial increase in GnRH primary transcript and a subsequent increase in GnRH mRNA levels, suggests that the elevation in GnRH mRNA is a consequence of stimulated GnRH gene transcription. This is consistent with a report in a human hypothalamic NLT cell line in which GnRH gene transcription, as measured by luciferase reporter activity, was stimulated by IGF-I (27). Such a transcriptional mechanism for the increase in GnRH mRNA levels is supported by our observation that the GnRH mRNA half-life did not change substantially after IGF-I treatment. Studies on the effects of second-messenger activators on GnRH gene expression also have indicated significant changes in GnRH gene transcription (28, 29, 30), suggesting that this is an important mechanism for the regulation of GnRH mRNA levels in hypothalamic NCLs. This does not seem to be the case in the animal, however, in which most of the regulation of GnRH mRNA levels seems to occur at a posttranscriptional level, such as mRNA stability (25, 31; reviewed in Ref. 4).
To our knowledge, this is the first report of a substance stimulating GnRH gene expression in GT17 cells. Stimulation of the protein kinase A (PKA) or C (PKC) second-messenger systems has been reported to cause rapid and substantial decreases in GnRH gene expression (23, 24, 28, 30, 32, 33). In contrast, it has been reported that neurotransmitters (such as glutamate) stimulate GnRH gene expression in the animal (25, 34, 35) but not in GT17 cells (A. C. Gore and J. L. Roberts, unpublished observation). It is possible that IGF-I represents an input to GnRH neurons that is obligatory for a stimulatory effect on the GnRH gene to occur.
Although IGF-I is stimulatory to GnRH gene expression in GT17 cells,
it was found to have an overall inhibitory effect on levels of GnRH
decapeptide in the medium. IGF-I treatment caused an initial
stimulation of GnRH peptide levels at the 2-h time point. However,
beginning at 4 h and for the rest of the 48-h time-course, GnRH
peptide levels were significantly suppressed, compared with control
peptide levels. This result differs from that of another laboratory,
which reported no effect of 2 h of exposure to IGF-I on GnRH
secretion (20). Furthermore, preliminary reports from another group
indicated a stimulatory effect of 12-h exposure to IGF-I on GnRH
release in the GT11 cell line (22). Differences between laboratories
are attributed to different cell lines (GT11 vs. GT17)
and possible so-called drift in the phenotype of the GT1 cells between
laboratories with repeated passaging. It was reported that GT1 cells
can alter the expression of certain molecules over time, and in fact,
these cells even can express glial markers, depending on the passaging
technique (36). However, it should be noted that preliminary studies in
our laboratory indicated no expression of glial fibrillary acidic
protein, an astroglial marker, in our GT17 cell cultures (K. M.
Longo and A. C. Gore, unpublished observations). Differences
between our and other studies may also involve the effects of serum
deprivation, because in our study, GnRH peptide levels increased and
then decreased over time after serum deprivation (Table 1
), whereas
other studies used different cell culture and serum starvation
conditions.
The observation that GnRH levels in the medium were decreased under those conditions in which increases in GnRH gene expression were observed suggests an uncoupling of synthesis-secretion events. Such an uncoupling was observed when GT1 cells were treated with phorbol ester, which causes decreases in GnRH gene expression concomitantly with increases in GnRH release (24, 28, 30, 32, 33). In the case of IGF-I treatment, it seems that there is a shift in favor of biosynthesis, perhaps at the expense of translational, posttranslational events and/or release. Moreover, it is unknown whether the overall decrease in GnRH decapeptide levels is caused by a decrease in release of GnRH peptide, or by an increase in the degradation of GnRH peptide.
Concomitantly with the effects of IGF-I on GnRH gene expression, a qualitative change in the morphology of GT17 cells also was observed, similar to the finding by other laboratories (22, 26). Interestingly, the most profound changes induced by IGF-I on GT17 cell morphology occurred at the same dose (10 ng/ml) that caused the greatest increases in GnRH gene expression. When IGF-I was added, the cells formed longer and more extended processes. It previously has been reported that GT17 cells form gap junctions with one another (37); it is therefore possible that IGF-I affected the number of gap junctions, because the number of contacts between GT17 cells seemed to increase. However, it is necessary to perform quantitative analyses of such changes in future studies to prove this hypothesis. Interestingly, PKC activators, which have opposite effects to IGF-I on GnRH gene expression and peptide release, also have opposite morphological effects to IGF-I on GT17 cell morphology: PKC caused the cell body to become more condensed and processes to retract (30), whereas IGF-I caused the cell body to become more flattened, with an appearance of an enlarged surface area, and an extension of processes. It also may be relevant that during postnatal development, GnRH neurons undergo morphological changes that make them become more irregular in shape (38), suggesting an increase in cell surface area, with the potential for increased synaptic input. A similar effect seems to be induced by IGF-I treatment in GT17 cells, suggesting a recapitulation of events occurring during the development of GnRH neurons in vivo.
Immunocytochemical studies indicated the novel observation that GT17 cells are immunoreactive for IGF-I. This is not the only growth factor synthesized in GT17 cells, which were reported to produce basic fibroblast growth factor as well (39). It is possible that these cells are under a tonic autofeedback loop from the endogenous IGF-I that they are producing. This is consistent with the report that in hypothalamic neuronal and glial cultures, IGF-I can down-regulate its own receptors (40). However, our observation that IGF-I stimulates GnRH gene expression in GT17 cells suggests that the amount of IGF-I released into the medium may not be sufficient to down-regulate the IGF-I receptors on these cells. Furthermore, changing the GT17 cells medium before IGF-I treatment may resensitize them to IGF-I. In future studies, we will measure IGF-I levels in the medium to determine the exposure of GT17 cells to endogenous IGF-I under basal conditions. The expression of IGF-I in GT17 cells also suggests the intriguing possibility that GnRH neurons in vivo may synthesize their own IGF-I. The report that embryonic day-17 rat neuronal primary cultures express IGF-I mRNA (41) supports the idea that neurons are capable of synthesizing this neurotrophic factor. Studies are currently underway in our laboratory to determine whether GnRH neurons in vivo are IGF-I immunopositive.
The present findings suggest that IGF-I may be directly involved in alterations in the gene expression and morphology of the immortalized GT17 cell line. These studies are the first crucial steps in producing a more representative model of the adult mammalian GnRH neuron. Regarding in vivo models, it was reported that IGF-I knockout mice have a smaller central nervous system size and do not undergo reproductive development (42). It is not known whether this latter effect is caused by a primary central nervous system, pituitary, or gonadal deficiency. However, the preliminary observation in our laboratory that GnRH mRNA levels are substantially lower in IGF-I knockout mice (A. C. Gore, unpublished observation) suggests that the lack of exposure of developing GnRH neurons to IGF-I results in a more immature phenotype, in fact, similar to that observed in GT17 cells. Future studies will examine changes in IGF-I input to the GnRH system of developing mice.
| Acknowledgments |
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| Footnotes |
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Received September 24, 1997.
| References |
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