Activity-dependent Expression of Parathyroid Hormone-related Protein (PTHrP) in Rat Cerebellar Granule Neurons
REQUIREMENT OF PTHrP FOR THE ACTIVITY-DEPENDENT SURVIVAL OF GRANULE NEURONS*

(Received for publication, October 22, 1996, and in revised form, March 7, 1997)

Tomio Ono Dagger §, Kaoru Inokuchi , Akihiko Ogura par , Yoji Ikawa **, Yoshihisa Kudo Dagger Dagger and Seiichi Kawashima Dagger

From the Dagger  Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan, the  Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan, the par  Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan, the ** Department of Retroviral Regulation, Medical Research Division, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan, and the Dagger Dagger  Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and Life Science, School of Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

To identify genes whose expression is neuronal activity-dependent, we used an mRNA differential display technique and discovered that parathyroid hormone-related protein (PTHrP) is expressed in an activity-dependent manner in primary cultures of rat cerebellar granule neurons. PTHrP mRNA was expressed as early as 1 h by the addition of KCl to a final concentration of 25 mM to the culture medium. This expression was induced by Ca2+ influx through voltage-dependent L-type Ca2+ channels and regulated at the transcriptional step. PTHrP mRNA was persistently expressed before and after the time of commitment of granule neurons to apoptosis when they are cultured in the presence of 25 mM KCl or both 150 µM N-methyl-D-aspartic acid and 15 mM KCl, both of which promote the survival of these neurons. PTHrP was rapidly secreted into the culture medium in a depolarization-dependent manner. Parathyroid hormone/PTHrP receptor mRNA was also expressed in the primary cultures, and its expression was up-regulated by KCl and/or N-methyl-D-aspartic acid. The addition of anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. These results suggest that PTHrP is involved in an autocrine loop and required for the survival of granule neurons.


INTRODUCTION

Activity-dependent modifications of neuronal functions and differentiation are the cellular bases of both synaptic and developmental plasticity (1). Activity-dependent survival of neurons is one such form of plasticity that may be involved in neuronal network formation during development. In primary culture, several neurotransmitters and their agonists promote the survival of several types of neurons. Glutamate and (NMDA)1 promote the survival of cerebellar granule neurons (2), Purkinje cells (3), spinal cord neurons (4), and hippocampal neurons (5). A metabotropic glutamate receptor agonist, L-(+)-2-amino-4-phosphonobutyric acid (6), kainate (7), and muscarinic-cholinergic receptor agonists (8) have been reported to promote the survival of cerebellar granule neurons. Depolarizing concentrations of KCl or electrical stimulation have also been reported to promote the survival of several types of neurons in primary culture (9). Cerebellar granule neurons gradually undergo apoptosis within a few days after their inoculation into a culture medium containing 5 mM KCl. Stimulation of voltage-dependent L-type Ca2+ channels with 25 mM KCl or NMDA receptors with 150 µM NMDA inhibits this apoptosis, thus resulting in a marked increase in the survival of the granule neurons (2, 10). To identify activity-dependent genes whose expression levels are regulated during the activity-dependent survival of cerebellar granule neurons, we employed an mRNA differential display technique and found that the gene encoding parathyroid hormone-related protein (PTHrP) is one such gene.

PTHrP was originally discovered as a causative factor of humoral hypercalcemia of malignancy (11, 12). The structural similarity of PTHrP to parathyroid hormone (PTH) is confined to the amino-terminal 32 amino acid residues that interact with the PTH/PTHrP receptor and exert their functions through adenylate cyclase- and phospholipase C-mediated pathways (13). PTHrP has been suggested to act as an autocrine and/or a paracrine factor that regulates cell proliferation, differentiation, and apoptosis (14-17). PTHrP is expressed in a large variety of tissues including the central nervous system, heart, pancreas, adrenal glands, smooth muscle, lactating mammary tissue, bladder, skin, and pregnant uterus (18-20). In situ hybridization studies have revealed that in the central nervous system PTHrP mRNA and its receptor mRNA are localized in the cerebellum, cortex, hippocampus, and neuroendocrine cells (19, 21). Although its abundance and specific localization in the central nervous system may suggest that PTHrP must have some specific functions in the nervous system, a little has been elucidated about the biological functions of PTHrP and the PTH/PTHrP receptor and regulation of the genes encoding them in neurons (19, 21). Here we report on activity-dependent regulation of the expression of PTHrP and the PTH/PTHrP receptor and the involvement of PTHrP in the activity-dependent survival of cerebellar granule neurons.


EXPERIMENTAL PROCEDURES

Primary Culture of Cerebellar Granule Neurons

Cerebellar granule neurons were cultured according to the method of Levi et al. (22) with some modifications. Briefly, cerebella were dissected from 8-day-old rats and incubated in 1% trypsin (Worthington) for 20 min at 37 °C. The trypsinized tissue was triturated with a Pasteur pipette until no tissue aggregates were seen. Then the cells were washed with Eagle's basal medium (Life Technologies, Inc.) and plated on poly-D-lysine-coated plastic dishes at a density of 2.6 × 105 cells/cm2 unless otherwise stated in Eagle's basal medium containing 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At 24 h after plating, the medium was exchanged with fresh medium containing 100 µM cytosine arabinoside and various reagents as described below. In some cases 30 µM (±)-2-amino-5-phosphonopentanoic acid (AP-5) was added to the culture medium simultaneously with KCl for prevention of potential effects of glutamate, which might have been present in the fetal calf serum and/or released from the granule neurons, on the survival of granule neurons.

Cell Survival Assay

The cell survival was assayed by the MTT method as described by Mosmann (23). A one-tenth volume of 5 mg/ml MTT solution dissolved in Dulbecco's phosphate-buffered saline was added to the culture. Then the dish was returned to the incubator and incubated for a further 2-3 h. An equal volume of isopropyl alcohol containing 0.08 N HCl was then added for dissolution of the formazan dye. The absorbance at 570 nm was measured with that at 630 nm as a reference.

RNA Isolation and Northern Blotting Analysis

Total cellular RNA was isolated by the method of Chomczynski and Sacchi (24). RNA was separated on a 1% formaldehyde agarose gel and then transferred to Biodyne B membrane (Pall). The 0.24-9.5-kb RNA ladder (Life Technologies) was used as a size marker. A cDNA probe was labeled with random 9-mers (Takara) and [alpha -32P]dCTP. Hybridization was carried out at 65 °C in rapid hybridization buffer (Amersham Corp.) according to the manufacturer's instructions. After hybridization, the membrane was washed with 2 × SSC, 0.1% SDS at room temperature followed by washing with 0.1 × SSC, 0.1% SDS at 55 °C. The same filters were reprobed with the cDNA for mouse glucose-6-phosphate dehydrogenase (G6PDH) for verification of equivalent loading of RNAs (25). The expression levels of the mRNAs were normalized to that of G6PDH mRNA according to the results of densitometric analysis of the autoradiograms.

PTH/PTHrP Receptor Probe

The PTH/PTHrP receptor probe was generated by PCR with specific primers and rat kidney cDNA. The sequence of the sense primer was 5'-AGC GCA AAG CAC GAA GTG GGA-3', and that of the antisense primer was 5'-CCA TGT GCC CAG GCC AGC AG-3' (26). With these primers, a 369-base pair product, which corresponds to amino acid residues 488-610 of the rat PTH/PTHrP receptor, was generated.

Differential Display and cDNA Cloning

Differential display was carried out by the method of Liang et al. (27) and Inokuchi et al. (25) with some modifications. Total RNA was isolated from the cultured cerebellar granule neurons as described above. Six µg of total RNA was treated with 4 units of RNase-free DNase I (Life Technologies; amplification grade) in the presence of 48 units of ribonuclease inhibitor (Takara) in a total volume of 40 µl. After phenol-chloroform extraction, the RNA was precipitated with ethanol and dissolved in water. A 0.5-µg aliquot of the DNase I-treated RNA was heat-denatured at 70 °C for 5 min and quenched on ice. The reverse transcription was carried out at 35 °C for 60 min with 300 units of Super ScriptTM II RNase H- reverse transcriptase (Life Technologies), 20 µM dNTP, 40 units of ribonuclease inhibitor, and 2.5 µM of anchor primer (either T12VA, T12VG, T12VT, or T12VC (where V is a mixture of A, G, and C)) in a total volume of 20 µl. The reaction was stopped by heating at 95 °C for 5 min, and the cDNA product was stored at -80 °C until use. The polymerase chain reaction (PCR) was carried out in a siliconized tube containing a 0.5-µl aliquot of the cDNA solution, 2.0 µM dNTP, 3.1 µCi of [alpha -35S]dCTP (1,200 Ci/mmol, Amersham), 1.0 µM anchor primer, 0.5 µM arbitrary 10-mer, and 0.63 unit of Taq DNA polymerase (Toyobo) in a total volume of 5.0 µl. The cycle conditions were as follows: 40 cycles of 94 °C for 30 s, 40 °C for 2 min, and 72 °C for 30 s, followed by extension at 72 °C for 5 min. The PCR products were analyzed on 6% sequencing gels. The differentially displayed bands were extracted from the sequencing gel and reamplified as described by Liang et al. (27). The PCR products were cloned into the pCR-Script Amp SK(+) vector (Stratagene). Positive clones were selected by screening by Northern blotting analysis and sequenced with a Sequenase DNA sequencing kit (Amersham).

Immunocytochemistry

For immunostaining of primary cultured granule neurons, the granule neurons were cultured on eight-well slides precoated with poly-D-lysine. The slides were washed with Dulbecco's phosphate-buffered saline twice, after which the cells on the slides were fixed overnight at 4 °C in 0.1 M sodium phosphate (pH 7.2) containing 4% paraformaldehyde. Then after the cells were permeabilized for 15 min with phosphate-buffered saline containing 0.1% Triton X-100 and 5% goat serum, they were incubated with anti-PTHrP antibody (10 µg/ml IgG, Oncogene PC09) for 2 h at room temperature. Then the slides were washed with phosphate-buffered saline three times followed by repermeabilization of the cells for 15 min and incubation of the repermeabilized cells with anti-rabbit IgG conjugated with fluorescein isothiocyanate. Preabsorbed antibody was used for staining negative control preparations.

Radioimmunometric Assay of PTHrP

The PTHrP concentration in the culture medium was determined by use of a PTHrP radioimmunometric assay kit (Mitsubishi Kagaku Co., Japan) with human PTHrP-(1-137) as a standard, according to the manufacturer's instructions. This kit contains a monoclonal anti-human PTHrP-(1-34) antibody conjugated to polystyrene beads and a polyclonal anti-human PTHrP-(50-83) antibody.

Other Methods and Reagents

Protein was determined by the method of Bradford (28) by use of the Bio-Rad protein assay kit with bovine gamma -globulin as a standard. Synthetic rat PTHrP-(1-34) amide was purchased from Peptide Institute Inc. (Osaka, Japan), and rabbit anti-rat PTHrP-(1-34) antiserum was purchased from Yanaihara Institute Inc. (Sizuoka, Japan). All other chemicals were of reagent grade and were purchased from local pharmaceutical companies.


RESULTS

Determination of the Commitment Time to Apoptosis

Cerebellar granule neurons undergo apoptosis during culture in the presence of 5 mM KCl. In the presence of 25 mM KCl or both 150 µM NMDA and 15 mM KCl, this apoptosis of the granule neurons is inhibited. To characterize this apoptosis, we determined the commitment time to apoptosis of the granule neurons. The granule neurons were cultured in the presence of 5 mM KCl, 30 µM AP-5, or 15 mM KCl. At 24-h intervals, KCl or NMDA was added to a final concentration of 25 mM or 150 µM, respectively, for rescue of the cells, and the cell survival was assayed at 5.0 days in vitro (DIV). As shown in Fig. 1, 50% protection of the apoptosis was observed at 3.0 DIV under both sets of conditions, and no significant difference in the time of commitment to apoptosis was observed between these two sets of conditions. Thus, the commitment time to apoptosis under these conditions was 3.0 DIV.


Fig. 1. Determination of timing of commitment to apoptosis. The granule cells were seeded in Eagle's basal medium containing 10% fetal calf serum as described under "Experimental Procedures." At 1.0 DIV, the culture medium was exchanged with a medium containing 5 mM KCl, 30 µM AP-5 (bullet ------bullet ) or 15 mM KCl (open circle ------open circle ). At the times indicated, KCl or NMDA was added to a final concentration of 25 mM or 150 µM, respectively. Cell survival was assayed at 5.0 DIV as described under "Experimental Procedures." Each circle and bar represents the mean ± S.D. of triplicate determinations in three independent experiments.
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Identification of the PTHrP Gene as an Activity-dependent Gene

To identify activity-dependent genes whose expression is up-regulated at around the time of commitment to apoptosis, we used an mRNA differential display technique. At 2.5 and 3.5 DIV, i.e. just before and after the time of commitment to apoptosis, total cellular RNA was harvested from the granule neurons cultured under nondepolarizing conditions (culture medium containing 5 mM KCl and 30 µM AP-5) and depolarizing conditions (culture medium containing 15 mM KCl and 150 µM NMDA). These RNA samples were used as templates for PCR after they were reverse-transcribed to cDNA. We used various combinations of 10 arbitrary primers and 4 anchor primers in the PCR and detected 21 differentially displayed bands on the sequencing gels (data not shown). Among these bands, one of the most strongly displayed was chosen for further analysis. As shown in Fig. 2, the bands indicated with an arrow were detected in the lanes that were loaded with the PCR products derived from cells cultured under the depolarizing conditions (lanes B) but not in those loaded with the PCR products derived from the cells cultured under the nondepolarizing conditions (lanes A). One of these bands was cut out from the gel, and the cDNA fragment was extracted and reamplified. The product of this amplification was cloned into the pCR-Script Amp SK(+) vector and sequenced in its entirety. It was found to consist of 324 base pairs, and its sequence was found to be identical to a part of the sequence that had been published for rat PTHrP cDNA sequence (29). A full-length PTHrP cDNA was obtained by screening of a lambda  ZapII library that had been constructed from the RNA extracted from the granule neurons cultured for 5.0 DIV in the medium containing 15 mM KCl and 150 µM NMDA. The cDNA sequence of this clone was found to be 100% identical to that of the PTHrP cDNA reported previously (29). Differential expression of the PTHrP mRNA under the nondepolarizing conditions and depolarizing conditions was confirmed by Northern blotting analysis (Fig. 3).


Fig. 2. Identification of genes whose expression is up-regulated by depolarization. Total RNA was isolated at 2.5 and 3.5 DIV from the granule cells cultured in the presence of either 5 mM KCl, 30 µM AP-5 (lanes A) or 150 µM NMDA, 15 mM KCl (lanes B). cDNA was generated with T12VC used as an anchor primer. PCR was carried out using 5'-CGT ACT TGT G-3' and T12VC. Aliquots of two independent PCR mixtures after amplification were loaded onto a 6% sequencing gel. The arrow indicates the position of the PTHrP cDNA fragment.
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Fig. 3. Expression of PTHrP mRNA is associated with activity-dependent survival of cerebellar granule neurons. A, Northern blotting analysis of PTHrP mRNA expression in granule neurons. Either 20 µg (lanes labeled 2.5 DIV and 4.5 DIV) or 10 µg (lanes labeled 5.5 DIV and 6.5 DIV) of total RNA extracted from the granule neurons cultured in the presence of 5 mM KCl, 30 µM AP-5; 15 mM KCl, 30 µM AP-5; 25 mM KCl, 30 µM AP-5; or 15 mM KCl, 150 µM NMDA was subjected to Northern blotting analysis. The SacII/XbaI fragment of the full-length PTHrP cDNA was used as a probe. At 5.5 DIV, 300 µM AP-5 (asterisk) was added to the cultures in the presence of 150 µM NMDA, 15 mM KCl for induction of apoptosis. Twenty-four hours later, total RNA was extracted and subjected to Northern blotting analysis (lane labeled 6.5 DIV, AP-5, +*). The same volume of water was added to the control culture (lane labeled 6.5 DIV, AP-5(-)). The bottom panel shows the expression levels of G6PDH mRNA. B, down-regulation of PTHrP mRNA expression by blocking of NMDA receptor. The granule neurons were cultured for 4.5 days in the medium containing 15 mM KCl, 150 µM NMDA, and then NMDA receptor was blocked by the addition of AP-5 to a final concentration of 300 µM. Total RNA was extracted at the indicated times, and 10-µg aliquots of it were subjected to Northern blotting analysis. The numbers above the lanes indicate the number of hours after the addition of AP-5. The bottom part shows the expression levels of G6PDH mRNA.
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Up-regulation and Down-regulation of PTHrP mRNA Expression Are Strongly Associated with the Activity-dependent Survival of the Granule Neurons

In the presence of 25 mM KCl, 30 µM AP-5 or both of 150 µM NMDA and 15 mM KCl cerebellar granule neurons in primary culture survive at high rates, whereas in the presence of 5 mM KCl, 30 µM AP-5 or 15 mM KCl, 30 µM AP-5, granule neurons undergo apoptosis at high rates. To clarify the relationship between the survival rate of the granule neurons and the expression of PTHrP mRNA, we carried out Northern blotting analysis. As shown in Fig. 3A, the expression of the 1.4-kb PTHrP mRNA could already be detected at 2.5 DIV in the case of cultures under the survival-promoting conditions (25 mM KCl, 30 µM AP-5 or 150 µM NMDA, 15 mM KCl). Its level in both the survival-promoting conditions increased to 4.0 times that at 2.5 DIV by 4.5 DIV. An additional minor transcript of 3.0 kb was also expressed under these survival-promoting conditions. This expression of the PTHrP mRNAs was confined mainly to the survival-promoting conditions; little of it was detected in the case of culture under the apoptosis-inducing conditions (5 mM KCl, 30 µM AP-5 or 15 mM KCl, 30 µM AP-5). Thus, before the time of commitment to apoptosis and thereafter, the expression of PTHrP mRNA was up-regulated, when the cells were cultured under the survival-promoting conditions, while little PTHrP mRNA expression was detected in the case of culture under apoptosis-inducing conditions. The addition of AP-5 to a final concentration of 300 µM to the NMDA-supported culture at 5.5 DIV resulted in induction of apoptosis within 12 h, and approximately 50% of the cells died within 24 h.2 Within these 24 h the expression of PTHrP was down-regulated to approximately 13% of the control level (Fig. 3A, lane labeled 6.5 DIV and AP-5(-)) by inducing the apoptosis. The down-regulation of PTHrP mRNA expression was induced at between 2 and 5 h after the addition of AP-5 (Fig. 3B), and the level of PTHrP mRNA was reduced to 8.9% of the initial level within 10 h after the addition of AP-5. The expression of PTHrP mRNA was also down-regulated within 24 h after the culture medium containing 25 mM KCl, 30 µM AP-5 was exchanged with a fresh one containing 5 mM KCl (data not shown). To confirm the expression of PTHrP in the granule neurons, we carried out immunofluorescence staining with anti-PTHrP antibody (Fig. 4). Abundant PTHrP immunoreactivity was detected in the granule neurons cultured under the survival-promoting conditions (25 mM KCl, 30 µM AP-5 or 15 mM KCl, 150 µM NMDA). The immunoreactivity was localized in the somata and neurites. In the somata both the perinuclear region and the nucleus were stained, although the former was stained more strongly than the latter. The PTHrP immunofluorescence in the cells cultured under the apoptosis-inducing conditions (5 mM KCl, 30 µM AP-5 or 15 mM KCl, 30 µM AP-5) was extremely weak, which is in good agreement with the expression pattern of PTHrP mRNA in the cells cultured under these conditions. No staining was obtained in negative control preparations (data not shown). Thus, both PTHrP mRNA and its protein product were expressed in an activity-dependent manner in the granule neurons.


Fig. 4. Immunofluorescence staining of PTHrP in primary culture of granule neurons. The granule neurons were cultured for 5.0 days and stained with anti-PTHrP antibody and fluorescein-labeled anti-rabbit IgG. Left column, phase-contrast microscopic images. Right column, immunofluorescence staining. Upper row, granule neurons cultured in the presence of 150 µM NMDA, 15 mM KCl; second row, granule neurons cultured in the presence of 25 mM KCl, 30 µM AP-5; third row, granule neurons cultured in the presence of 15 mM KCl, 30 µM AP-5; bottom row, granule neurons cultured in the presence of 5 mM KCl, 30 µM AP-5.
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Regulation of PTHrP mRNA Expression

In an attempt to elucidate the expression mechanism of PTHrP mRNA in an activity-dependent manner, we studied the time course of PTHrP mRNA expression in the granule neurons. Stimulation with 25 mM KCl at 1.0 DIV resulted in no significant up-regulation of PTHrP mRNA expression within 5 h, suggesting that the expression is dependent on the maturation of the granule neurons. Therefore, we decided to use more mature neurons. The granule neurons were cultured in the presence of 25 mM KCl for 4 days, and then the medium was exchanged with fresh medium containing 5 mM KCl. The culture was continued for 24 h for down-regulation of PTHrP mRNA expression. Then the cells were stimulated with the reagents as shown in Fig. 5A. PTHrP mRNA was expressed as early as 1 h after the start of stimulation with 25 mM KCl or 150 µM NMDA, 15 mM KCl. An approximately 10-fold increase in the level of the expression compared with the expression level at 1 h had occurred within 5 h after the start of the stimulation with 25 mM KCl or 150 µM NMDA, 15 mM KCl. Hardly any PTHrP mRNA expression was detectable in both the cells cultured in the presence of 5 mM KCl, 30 µM AP-5 and those cultured in the presence of 15 mM KCl, 30 µM AP-5. Fig. 5B shows the effects of nifedipine, actinomycin D, and cycloheximide on the expression of PTHrP mRNA. The addition of nifedipine 30 min before the addition of 25 mM KCl resulted in complete inhibition of the expression of PTHrP mRNA. This result suggests that Ca2+ influx through voltage-dependent L-type Ca2+ channels was required for the up-regulation of the PTHrP mRNA expression in these cells. Actinomycin D inhibited completely the up-regulation of PTHrP mRNA expression induced by 25 mM KCl, suggesting that the expression of PTHrP mRNA is regulated at the transcriptional step. The addition of cycloheximide alone to the 5 mM KCl-containing culture induced no PTHrP mRNA expression. Moreover, cycloheximide did not inhibit the expression of PTHrP mRNA induced by 25 mM KCl, indicating that the expression of PTHrP mRNA was independent of translation. These results suggest that the Ca2+ influx through voltage-dependent L-type Ca2+ channels positively regulates PTHrP mRNA transcription.


Fig. 5. Expression of PTHrP mRNA upon depolarization. Cerebellar granule neurons were cultured in a medium containing 25 mM KCl for 4 days as described under "Experimental Procedures." Then the medium was exchanged with one containing 5 mM KCl, and the culture was continued for 24 h, after which the cells were stimulated with various reagents as described below. The bottom of each panel shows the levels of expression of G6PDH mRNA. A, time course of PTHrP expression. The granule neurons were stimulated with 5 mM KCl, 30 µM AP-5; 15 mM KCl, 30 µM AP-5; 25 mM KCl, 30 µM AP-5; or 150 µM NMDA, 15 mM KCl. Total RNA was harvested at 1 or 5 h after the start of stimulation, and 10-µg aliquots were subjected to Northern blotting analysis. B, effect of various inhibitors on PTHrP mRNA expression. Inhibitors indicated in the figure were added 30 min before stimulation with KCl. Total RNA was harvested at 5 h after the start of stimulation, and 10-µg aliquots of it were subjected to Northern blotting analysis.
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Activity-dependent Secretion of PTHrP into Culture Medium

It has been suggested that PTHrP is secreted from non-neuronal cells and endocrine cells and functions as an autocrine and/or a paracrine factor in those cells (30). In the present study, we found that PTHrP was secreted from the cerebellar granule neurons into the culture medium only when they are cultured under the survival-promoting conditions (Fig. 6A). This secretion was first detected after 2.0 DIV, and its level increased linearly thereafter, which is in good agreement with the PTHrP mRNA expression pattern (Fig. 3A). To see if this secretion is depolarization-dependent, we stimulated the granule neurons with 55 mM KCl after exchanging the culture medium with the one containing 5 mM KCl. We used 55 mM KCl rather than 25 mM to ensure complete depolarization of the neurons. Secretion of PTHrP was induced within 30 s after stimulation with KCl, and the PTHrP concentration in the culture medium became almost constant within 2 min (Fig. 6B). Thus, PTHrP is secreted via a depolarization-dependently regulated pathway. Since in the radioimmunometric assay for quantitation of the secreted PTHrP we used an antibody that recognizes the N-terminal region (1-34) and one that recognizes a middle region (50-83) of PTHrP, the secreted form must include at least the N-terminal and middle portions of PTHrP.


Fig. 6. Secretion of PTHrP into the culture medium. A. Secretion of PTHrP. The granule cells were cultured in the presence of 5 mM KCl, 30 µM AP-5 (square ------square ); 15 mM KCl, 30 µM AP-5 (diamond ------diamond ); 25 mM KCl, 30 µM AP-5 (bullet ------bullet ); or 15 mM KCl, 150 µM NMDA (black-triangle------black-triangle). Aliquots of culture medium were removed at 24-h intervals and subjected to radioimmunometric assay. The data shown are representative results of two independent experiments. Each value is the mean ± S.D. of triplicate determinations. All of the S.D. values were smaller than the range of the symbols. B, depolarization-dependent secretion of PTHrP. The granule neurons were cultured for 5.5 days in the medium containing 25 mM KCl on 3 × 3 cm coverslips at a density of 2.8 × 106 cells/cm2. Then the coverslips were transferred to a medium containing 5 mM KCl and 10% fetal calf serum, and the culture was continued for another 2 h. After exchanging the medium for fresh medium, KCl was added to a final concentration of 55 mM (bullet ------bullet ). For a negative control, NaCl was added to a final concentration of 50 mM (open circle ------open circle ). Aliquots of the culture medium were removed at timed intervals and subjected to radioimmunometric assay. The data shown are representative results of two independent experiments. Each value is the mean ± S.D. of triplicate determinations.
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PTH/PTHrP Receptor mRNA Expression and Its Regulation in Primary Cultures of the Granule Neurons

Both PTH and PTHrP interact with the PTH/PTHrP receptor, which recognizes the N-terminal sequence of each of these ligands. If PTHrP functions as an autocrine factor, its receptor would be expected to be expressed in granule neurons. To investigate this issue, we analyzed the expression pattern of the PTH/PTHrP receptor mRNA in the rat cerebellar granule neurons by Northern blotting analysis. As shown in Fig. 7, PTH/PTHrP receptor mRNA was expressed in the 2.5-DIV cultures under both the apoptosis-inducing and the survival-promoting conditions. The major transcript was 2.4 kb, which is similar to the length of a PTH/PTHrP receptor mRNA expressed in the rat kidney (26). An additional minor transcript of 4.3 kb was also expressed. The expression of these receptor mRNAs was up-regulated to an extent that depended on the concentration of KCl and/or NMDA in the culture medium. At 4.5 DIV, the expression levels of the major transcript in the cells cultured in the presence of 5 mM KCl, 30 µM AP-5 and 15 mM KCl, 30 µM AP-5 were 3.2 and 53% of that in the cells cultured in the presence of 25 mM KCl, 30 µM AP-5, respectively. Similar to the expression pattern of PTHrP mRNA in granule neurons cultured in the presence of NMDA and AP-5 (Fig. 3A, lanes labeled 6.5 DIV), the receptor mRNA expression was down-regulated, to 8.6% of the control level, within 24 h after the addition of 300 µM AP-5 to the culture medium when cultured in NMDA-containing medium (Fig. 7, lanes labeled 5.5 DIV). These results indicate that the PTH/PTHrP receptor was expressed in the primary cultures of the granule neurons and that the expression was regulated by KCl and/or NMDA. The expression and secretion of PTHrP and the expression of the PTH/PTHrP receptor mRNA in the same primary culture suggest that the secreted PTHrP could function as an autocrine factor for the granule neurons.


Fig. 7. PTH/PTHrP receptor mRNA expression and its regulation in primary cultures of cerebellar granule neurons. Total RNA was extracted from the granule neurons cultured for 2.5-4.5 days in the medium containing 5 mM KCl, 30 µM AP-5; 15 mM KCl, 30 µM AP-5; 25 mM KCl, 30 µM AP-5; or 15 mM KCl, 150 µM NMDA, and 10-µg aliquots of this RNA were subjected to Northern blotting analysis (lanes labeled 2.5-4.5 DIV). At 4.5 DIV, 300 µM AP-5 (asterisk) was added to the 15 mM KCl, 150 µM NMDA-containing culture for induction of apoptosis. Twenty-four hours later, total RNA was harvested, and 10-µg aliquots of it were subjected to Northern blotting analysis (lane labeled 5.5 DIV, AP-5, +*). The same volume of water was added to the control culture (lane labeled 5.5 DIV, AP-5(-)). Bottom panel shows the levels of expression of G6PDH mRNA.
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Effect of Anti-PTHrP-(1-34) Antiserum on the Activity-dependent Survival of the Granule Neurons

The depolarization-dependent secretion of PTHrP and the expression of the PTH/PTHrP receptor mRNA in the same primary culture led us to speculate that PTHrP might promote the survival of the granule neurons. To test this hypothesis, we added PTHrP-(1-34) to a final concentration of 1 nM to 1 µM to the culture medium containing 5 mM KCl, 30 µM AP-5 at 1.0 DIV, and the cell survival was assayed at 5.0 DIV. However, we observed no survival-promoting effects of PTHrP-(1-34) on the granule neurons cultured in this supplemented medium (data not shown). Next we added anti-PTHrP-(1-34) antiserum to the cultures containing 25 mM KCl, 30 µM AP-5 to block the activity of secreted PTHrP into the culture medium. As shown in Fig. 8 this antibody inhibited the activity-dependent survival of the granule neurons. The addition of PTHrP-(1-34) to a final concentration of 1 µM resulted in partial blockage of this inhibitory effect, showing the specificity of the antibody. We used this concentration of PTHrP to rule out the possibility of a toxic effect of excess PTHrP in the culture medium on the granule neurons. We found that the addition of PTHrP-(1-34) alone to a final concentration of up to 1 µM to the cultures containing 25 mM KCl, 30 µM AP-5 had no effect on the survival rate of the granule neurons. However, the addition of more PTHrP-(1-34) alone to a final concentration of such as 10 µM to the cultures containing in 25 mM KCl, 30 µM AP-5 resulted in a decrease in the survival rate of the granule neurons, to 64% of the survival rate of the cultures in the presence of no PTHrP-(1-34). Therefore, we used 1 µM PTHrP to block the effect of the antiserum. These results show that PTHrP is not sufficient to promoting the survival of granule neurons under the apoptosis-inducing conditions but is required for promoting the activity-dependent survival of the granule neurons.


Fig. 8. Effect of anti-PTHrP-(1-34) antiserum on the activity-dependent survival of the granule neurons. The granule neurons were cultured in a 96-well plate at a density of 2.5 × 105 cells/well containing 200 µl of the culture medium in each well. At 1.0 DIV, the culture medium was exchanged with fresh medium containing 25 mM KCl, 30 µM AP-5 and 2% anti-PTHrP-(1-34) antiserum with or without PTHrP-(1-34). The mixture of PTHrP-(1-34) and the antiserum was preincubated at 37 °C for 60 min prior to its addition to the culture medium. Two percent nonimmune rabbit serum was used as a negative control. The cell survival at 5.0 DIV was assayed by the MTT method as described under "Experimental Procedures." The data shown are representative results of two independent experiments. Each value is the mean ± S.D. of duplicate determinations. Statistical analysis was carried out using Student's t test.
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DISCUSSION

Activity-dependent signal transductions play crucial roles in developmental plasticity, synaptic plasticity, neuronal differentiation, cell survival, and excitotoxicity. In an attempt to identify the target genes of depolarization-dependent signals, we used an mRNA differential display technique and found that one such gene encodes PTHrP. To date, a wide variety of positive and negative regulators of PTHrP expression have been identified (11); however, the mechanisms of regulation of PTHrP gene expression in the nervous system have not been fully elucidated. Our findings demonstrate that depolarization is a positive regulator for the induction of PTHrP mRNA in rat cerebellar granule neurons and that this induction is regulated at the transcriptional step. Although we have not identified the precise mechanism(s) of PTHrP induction downstream of Ca2+ influx induced by depolarization in granule neurons, regulatory pathways for other Ca2+-responsive genes have been reported. Ca2+/calmodulin-dependent protein kinases have been reported to be involved in some of these pathways. The influx of Ca2+ results in activation of Ca2+/calmodulin-dependent protein kinases II and IV, the latter of which is abundant in cerebellar granule neurons (31). These enzymes then catalyze the phosphorylation of the cyclic AMP response element-binding protein and/or serum response factor and mediate stimulation of Ca2+-dependent transcription of immediate early genes (32, 33). Since cyclic AMP is a positive regulator of PTHrP (34, 35) and since multiple copies of a cyclic AMP response element-like motif, which is a common element with the Ca2+-response element (32), are present in the upstream 5'-flanking region and exons of the PTHrP gene (34, 35), phosphorylation of cyclic AMP response element-binding protein by Ca2+/calmodulin-dependent protein kinases could be part of the mechanism of induction of PTHrP mRNA. Other potential pathways for stimulation of Ca2+-dependent gene transcription involve Ras and/or Src (36). It has been reported that Ca2+ influx through voltage-dependent Ca2+ channels results in activation of Ras and a downstream mitogen-activated protein kinase (37). Rusanescu et al. (38) reported that Ca2+ influx through voltage-dependent Ca2+ channels results in Src kinase activation in PC12 cells. Li and Drucker (39) demonstrated that overexpression of Ha-ras and v-src results in enhancement of PTHrP mRNA transcription in NKR 49F cells and RCB 2.2 cells, indicating that the PTHrP gene is a downstream target of Ras and Src. Therefore, Ras- and/or Src-mediated pathways could be other routes to stimulate depolarization-dependent PTHrP mRNA expression.

Although PTHrP has been suggested to be a neurotransmitter (21), no evidence of depolarization-dependent secretion of PTHrP from neurons has been reported. Previous studies on non-neuronal cells have revealed that PTHrP is secreted by means of a regulatory and/or constitutive pathway from such cells, depending on cell type (30). Our findings are the first reported evidence that PTHrP is secreted from neurons by means of a depolarization-dependently regulated pathway. Although some of the mature secretory forms of PTHrP have been elucidated in various tissues and cell types (40), no mature secretory form of PTHrP from neurons has yet been characterized. We have shown here that one form of PTHrP secreted from cerebellar granule neurons has at least an N-terminal and a middle portion of PTHrP. The N-terminal portion of PTHrP binds to the PTH/PTHrP receptor and exerts its function(s) through cAMP and/or phospholipase C-mediated pathway(s). In situ hybridization studies have revealed that the PTH/PTHrP receptor is expressed on both cerebellar granule neurons and Purkinje cells, the latter of which are the postsynaptic cells of cerebellar granule neurons (21). The depolarization-dependent secretion of PTHrP from granule neurons and the presence of the PTH/PTHrP receptor on both Purkinje cells and granule neurons suggest that PTHrP may act as a neurotransmitter and/or an activity-dependent autocrine factor. Although the biological function(s) of PTHrP in neurons have not yet been fully elucidated, PTHrP secreted from granule neurons may regulate the Ca2+ concentration in the granule neurons and Purkinje cells. Fukayama et al. (41) reported that both N-terminal (1-34) and C-terminal (107-139) PTHrP peptides induce increases in the intracellular Ca2+ concentration of hippocampal neurons in primary culture. PTH has been reported to either induce or block increases in the intracellular Ca2+ concentration via regulation of Ca2+ channel activity (13, 42, 43). In snail neurons, PTH induces an increase in the intracellular Ca2+ concentration via modulation of N-type Ca2+ channel activity (44, 45). In mouse neuroblastoma cells (N1E-115), PTH is an inhibitor of L-type voltage-dependent Ca2+ channel activity (46). Although we have not investigated the effect of PTHrP on the intracellular Ca2+ concentration in neurons, we expect that similar regulation occurs in the granule neurons and Purkinje cells. If in fact it does, the PTHrP released from granule neurons could mediate changes in the concentration of intracellular Ca2+ in Purkinje cells as well as in granule neurons and might act as a neurotransmitter or neuromodulator.

We have demonstrated that PTH/PTHrP receptor mRNAs are expressed in primary cultures of rat cerebellar granule neurons and that their expression is up-regulated by KCl and/or NMDA. To date, there have been only a few reports concerning regulation of PTH/PTHrP receptor expression. High intracellular Ca2+ concentrations, dexamethasone, PTH, cyclic AMP, and forskolin are down-regulators of expression of this receptor (47-49). Although the molecular mechanism is not yet known, our findings constitute the first reported evidence that the expression of the PTH/PTHrP receptor mRNA is up-regulated by KCl and/or NMDA. The expression of both PTHrP and its receptor in primary culture of cerebellar granule neurons suggests that PTHrP may be involved in an autocrine stimulatory loop in such primary culture. Indeed, our data showed that the addition of the anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. PTHrP and PTH have already been reported to promote the survival of several cell types other than cerebellar granule neurons. Hayashi and Sato reported that PTH promotes the survival of GH3 cells (50). Vitry et al. (51) reported that PTH is essential for the survival of mouse nervous cell line F7. Recently Henderson et al. (17) reported that overexpression of PTHrP promotes the survival of chrondrocytes under apoptosis-inducing conditions. Although supplemented PTHrP-(1-34) did not promote the survival of granule neurons under apoptosis-inducing conditions, our results of the experiments performed using the specific antiserum suggest that PTHrP is required for the activity-dependent survival of the granule neurons.


FOOTNOTES

*   This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan (to T. O. and A. O.) and a research grant from The Tokyo Metropolitan Institute of Gerontology (to T. O. and S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101 (ext. 5248); Fax: 81-3-5685-6609; E-mail: ono{at}rinshoken.or.jp.
1   The abbreviations used are: NMDA, N-methyl-D-aspartic acid; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; AP-5, (±)-2-amino-5-phosphonopentanoic acid; kb, kilobase pair(s); G6PDH, glucose-6-phosphate dehydrogenase; PCR, polymerase chain reaction; DIV, days in vitro.
2   T. Ono and S. Kawashime, unpublished data.

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