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• W2044961980 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 mmKCl or both 150 μm N-methyl-d-aspartic acid and 15 mmKCl, 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/orN-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. 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 mmKCl or both 150 μm N-methyl-d-aspartic acid and 15 mmKCl, 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/orN-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. Activity-dependent modifications of neuronal functions and differentiation are the cellular bases of both synaptic and developmental plasticity (1Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2762) Google Scholar). 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) 1The abbreviations used are:NMDAN-methyl-d-aspartic acid;MTT3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;PTHparathyroid hormone;PTHrPparathyroid hormone-related protein;AP-5(±)-2-amino-5-phosphonopentanoic acid;kbkilobase pair(s);G6PDHglucose-6-phosphate dehydrogenase;PCRpolymerase chain reaction;DIVdays in vitro. promote the survival of cerebellar granule neurons (2Balázs R. Jørgensen O.S. Hack N. Neuroscience. 1988; 27: 437-451Crossref PubMed Scopus (428) Google Scholar), Purkinje cells (3Yuzaki M. Forrest D. Verselis L.M. Sun S.C. Curran T. Conner J.A. J. Neurosci. 1996; 16: 4651-4661Crossref PubMed Google Scholar), spinal cord neurons (4Brenneman D.E. Forsythe I.D. Nicol T. Nelson P.G. Dev. Brain. Res. 1990; 51: 63-68Crossref PubMed Scopus (63) Google Scholar), and hippocampal neurons (5Bambrick L.L. Yarowsky P.J. Krueger B.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9692-9696Crossref PubMed Scopus (76) Google Scholar). A metabotropic glutamate receptor agonist,L-(+)-2-amino-4-phosphonobutyric acid (6Graham M.E. Burgoyne R.D. Eur. J. Pharmacol. 1994; 288: 115-123Crossref PubMed Scopus (32) Google Scholar), kainate (7Balázs R. Hack N. Jørgensen O.S. Neuroscience. 1990; 37: 251-258Crossref PubMed Scopus (65) Google Scholar), and muscarinic-cholinergic receptor agonists (8Yan G.-M. Lin S.-Z. Irwin R.P. Paul S.M. Mol. Pharmacol. 1995; 47: 248-257PubMed Google Scholar) 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 (9Franklin J.L. Johnson Jr., E.J. Trends Neurosci. 1992; 15: 501-508Abstract Full Text PDF PubMed Scopus (326) Google Scholar). 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 (2Balázs R. Jørgensen O.S. Hack N. Neuroscience. 1988; 27: 437-451Crossref PubMed Scopus (428) Google Scholar, 10Gallo V. Kingsbury A. Balázs R. Jørgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). 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. N-methyl-d-aspartic acid; 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; parathyroid hormone; parathyroid hormone-related protein; (±)-2-amino-5-phosphonopentanoic acid; kilobase pair(s); glucose-6-phosphate dehydrogenase; polymerase chain reaction; days in vitro. PTHrP was originally discovered as a causative factor of humoral hypercalcemia of malignancy (11Gillespie M.T. Martin T.J. Mol. Cell. Endocrinol. 1994; 100: 143-147Crossref PubMed Scopus (49) Google Scholar, 12Harvey S. Fraser R.A. J. Endocrinol. 1993; 139: 353-361Crossref PubMed Scopus (26) Google Scholar). 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 (13Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.-F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Crossref PubMed Scopus (1007) Google Scholar). PTHrP has been suggested to act as an autocrine and/or a paracrine factor that regulates cell proliferation, differentiation, and apoptosis (14Kaiser S.M. Laneuville P. Bernier S.M. Rhim J.S. Kremer R. Goltzman D. J. Biol. Chem. 1992; 267: 13623-13628Abstract Full Text PDF PubMed Google Scholar, 15Birch M.A. Carron J.A. Scott M. Fraser W.D. Gallagher J.A. Brit. J. Cancer. 1995; 72: 90-95Crossref PubMed Scopus (62) Google Scholar, 16Stolpe A. Karperien M. Löwik C.W.G.M. Jüppner H. Segre G. Abou-Samra A.-B. de Laat S.W. Defize L.H.K. J. Cell Biol. 1993; 120: 235-243Crossref PubMed Scopus (113) Google Scholar, 17Henderson J.E. Amizuma N. Warshawsky H. Biasotto D. Lanske B.M.K. Goltzman D. Karaplis A.C. Mol. Cell. Biol. 1995; 15: 4064-4075Crossref PubMed Scopus (307) Google Scholar). 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 (18Daifotis A.G. Weir E.C. Dreyer B.E. Broadus A.E. J. Biol. Chem. 1992; 267: 23455-23458Abstract Full Text PDF PubMed Google Scholar, 19Weir E.C. Brines M.L. Ikeda K. Burtis W. Broadus A.E. Robbins R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 108-112Crossref PubMed Scopus (119) Google Scholar, 20Yamamoto M. Harm S.C. Grasser W.A. Thiede M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5326-5330Crossref PubMed Scopus (135) Google Scholar). 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 (19Weir E.C. Brines M.L. Ikeda K. Burtis W. Broadus A.E. Robbins R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 108-112Crossref PubMed Scopus (119) Google Scholar, 21Weaver D.R. Deeds J.D. Lee K. Segre G.V. Mol. Brain Res. 1995; 28: 296-310Crossref PubMed Scopus (74) Google Scholar). 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 (19Weir E.C. Brines M.L. Ikeda K. Burtis W. Broadus A.E. Robbins R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 108-112Crossref PubMed Scopus (119) Google Scholar, 21Weaver D.R. Deeds J.D. Lee K. Segre G.V. Mol. Brain Res. 1995; 28: 296-310Crossref PubMed Scopus (74) Google Scholar). 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. Cerebellar granule neurons were cultured according to the method of Levi et al. (22Levi G. Aloisi F. Ciotti M.T. Thangnipon W. Kingsbury A. Balázs R. Shahar A. Vellis J. Habu B.A. Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, Inc., New York1989: 211-214Google Scholar) 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 × 105cells/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. The cell survival was assayed by the MTT method as described by Mosmann (23Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46809) Google Scholar). 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. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63223) Google Scholar). 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 [α-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 (25Inokuchi K. Kato A. Hiraia K. Hishinuma F. Inoue M. Ozawa F. FEBS Lett. 1996; 382: 48-52Crossref PubMed Scopus (75) Google Scholar). The expression levels of the mRNAs were normalized to that of G6PDH mRNA according to the results of densitometric analysis of the autoradiograms. 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′ (26Ureña P. Kong X.-F. Abou-Samra A.-B. Jüppner H. Kronenberg H.M. Potts Jr., J.T. Segre G.V. Endocrinology. 1993; 133: 617-623Crossref PubMed Scopus (197) Google Scholar). 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 was carried out by the method of Liang et al. (27Liang L. Averboukh L. Pardee B. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (885) Google Scholar) and Inokuchi et al. (25Inokuchi K. Kato A. Hiraia K. Hishinuma F. Inoue M. Ozawa F. FEBS Lett. 1996; 382: 48-52Crossref PubMed Scopus (75) Google Scholar) 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 [α-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. (27Liang L. Averboukh L. Pardee B. Nucleic Acids Res. 1993; 21: 3269-3275Crossref PubMed Scopus (885) Google Scholar). 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). 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. 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. Protein was determined by the method of Bradford (28Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217316) Google Scholar) by use of the Bio-Rad protein assay kit with bovine γ-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. 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. 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 mmKCl 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 (29Yasuda T. Banville D.L. Rabbani S.A. Hendy G.N. Goltzman D. Mol. Endocrinol. 1989; 3: 518-525Crossref PubMed Scopus (127) Google Scholar). A full-length PTHrP cDNA was obtained by screening of a λ 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 (29Yasuda T. Banville D.L. Rabbani S.A. Hendy G.N. Goltzman D. Mol. Endocrinol. 1989; 3: 518-525Crossref PubMed Scopus (127) Google Scholar). Differential expression of the PTHrP mRNA under the nondepolarizing conditions and depolarizing conditions was confirmed by Northern blotting analysis (Fig. 3).Figure 3Expression 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 labeled2.5 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 Sac II/Xba I 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. Thebottom part shows the expression levels of G6PDH mRNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 μmAP-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. 3 A, 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 mmKCl, 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. 2T. Ono and S. Kawashime, unpublished data. Within these 24 h the expression of PTHrP was down-regulated to approximately 13% of the control level (Fig. 3 A, lane labeled6.5 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. 3 B), 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 μmAP-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. 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.5 A. 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.5 B 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. 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 (30Plawner L.L. Philbrick W.M. Burtis W.J. Broadus A.E. Stewart A.F. J. Biol. Chem. 1995; 270: 14078-14084Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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. 6 A). 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. 3 A). 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. 6 B). 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 theN-terminal region (1Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2762) Google Scholar, 2Balázs R. Jørgensen O.S. Hack N. Neuroscience. 1988; 27: 437-451Crossref PubMed Scopus (428) Google Scholar, 3Yuzaki M. Forrest D. Verselis L.M. Sun S.C. Curran T. Conner J.A. J. Neurosci. 1996; 16: 4651-4661Crossref PubMed Google Scholar, 4Brenneman D.E. Forsythe I.D. Nicol T. Nelson P.G. Dev. Brain. Res. 1990; 51: 63-68Crossref PubMed Scopus (63) Google Scholar, 5Bambrick L.L. Yarowsky P.J. Krueger B.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9692-9696Crossref PubMed Scopus (76) Google Scholar, 6Graham M.E. Burgoyne R.D. Eur. J. Pharmacol. 1994; 288: 115-123Crossref PubMed Scopus (32) Google Scholar, 7Balázs R. Hack N. Jørgensen O.S. Neuroscience. 1990; 37: 251-258Crossref PubMed Scopus (65) Google Scholar, 8Yan G.-M. Lin S.-Z. Irwin R.P. Paul S.M. Mol. Pharmacol. 1995; 47: 248-257PubMed Google Scholar, 9Franklin J.L. Johnson Jr., E.J. Trends Neurosci. 1992; 15: 501-508Abstract Full Text PDF PubMed Scopus (326) Google Scholar, 10Gallo V. Kingsbury A. Balázs R. Jørgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar, 11Gillespie M.T. Martin T.J. Mol. Cell. Endocrinol. 1994; 10" @default.
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