Cyclic AMP Regulation of Pro-vasoactive Intestinal Polypeptide/PHM ...

Vol. 259, No. 14,Issue of July 25, pp. 9207-9211, 1984 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Cyclic AMP Regulation of Pro-vasoactive Intestinal Polypeptide/PHM-27 Synthesis in Human Neuroblastoma Cells* (Received for publication, October 18, 1983)

Yumiko Hayakawa, Ken-ichi Obata, Nobuyuki Itoh, Noboru YanaiharaS, and Hiroshi Okamotot From the Department of Biochemistry, Toyama Medicaland Pharmaceutical University School of Medicine, Toyama 930-01,Japan andthe $Laboratory of Bioorganic Chemistry,Shizuoka College of Pharmacy, Shizuokn 422, Japan

The mechanism of N’, Oz’-dibutyryl adenosine 3’:5’monophosphate (Bt2cAMP)induction of pro-vasoactive intestinal polypeptide (VIP)/PHM-27 biosynthesiswas investigated in human neuroblastoma cells in culture. When neuroblastoma cells were grownfor 48 h in the presence of 1 mM Bt2cAMP, the synthesis of pro-VIP/ PHM-27 was stimulated ll-fold. The amount of prepro-VIP/PHM-2’7mRNA determined both by hybridization with cloned prepro-VIP/PHM-27 cDNA and a reticulocytecell-freetranslationassaywasalsoincreased ll-fold in the Bt2cAMP-induced cells. Transcription of prepro-VIP/PHM-27 mRNA in isolated nuclei was observed in induced cells, but not in uninduced cells. Blot hybridization with prepro-VIP/PHM27 cDNA of total nuclear RNA isolated from neuroblastoma cells revealed an RNA species corresponding to mature prepro-VIP/PHM-27 mRNA, and the amount of the RNA was markedly increased in the induced cells. Thequantity of VIPIPHM-27 gene in the DNA of neuroblastoma cellswas analyzed afterhydrolysis with a restriction endonuclease, EcoRI. However, VIP/ PHM-27 gene was not amplified in the induced cells. These results indicate that Bt2cAMP-inducedpro-VIP/ PHM-27 synthesis is achieved by enhancing the transcription rate of prepro-VIP/PHM-27 mRNA. VIP,’ a 28-amino acid peptide, was originally isolated from porcine duodenum (1).VIP is present not only in gastrointestinal tissues but also neural tissues possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion, and release of insulin, glucagon, and several anterior pituitary hormones) (2-5). However, the biosynthetic mechanism of VIP remains to be elucidated. Recently, we reported that VIP was synthesized from a precursor (pro-VIP) in human neuroblastoma cells and that the synthesis of pro-VIP was stimulated by BtzcAMP (6). More recently, we have cloned the DNA sequence comple*This workwas supported in part by grants-in-aid for Cancer Research and for Scientific Research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom all correspondence should be addressed at Department of Biochemistry, Toyama Medical and Pharmaceutical University School of Medicine, Toyama 930-01, Japan. The abbreviations used are: VIP, vasoactive intestinal polypeptide; SDS, sodium dodecyl sulfate; bp, base pairs; kb, kilobase; 1 X SET, 10 mM Tris-C1, pH 7.5, 5 mM EDTA, 1%SDS; Hepes, 4-(2hydroxyethy1)-l-piperazineethanesulfonicacid Btp CAMP,dibutyryl cyclic AMP.

mentary to the mRNA coding for human VIP and analyzed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids, PHM-27 (7,8),which differs by only two amino acids from porcine PHI-27 (9). In order to clarify the mechanism of the induction of proVIP/PHM-27 synthesis by Bt,cAMP, we have now used a prepro-VIP/PHM-27 cDNA hybridization method to determine the amount of the specific mRNA in neuroblastoma cells and the transcriptional activity of nuclei isolated from neuroblastoma cells. Available evidence indicates that theinduction of pro-VIP/ PHM-27 synthesis by BtzcAMP is due to an increase in the rate of transcription of the VIP/PHM-27 gene. EXPERIMENTALPROCEDURES

Materials-Fetal bovine serum was obtained from Gibco Laboratories. BtzcAMP was purchased from Yamasa Shoyu Co. (Chiba, Japan); ~-[~’S]Methionine (specific activity, 1300 Ci/mmol), [a-”P] dCTP (specific activity, 3000 Ci/mmol), [a-32P]UTP(specific activity, 400 Ci/mmol), and [5,6-3H]UTP (specific activity, 40 Ci/mmol) were from Amersham; DPT paper was from Schleicher and Schuell, Inc.; methyl mercury hydroxide was from Alfa Chemicals, Danvers, MA; guanidinium thiocyanate was from Fluka; kanamycin was from Meiji Seika Kaisha, Ltd. (Tokyo, Japan); ribonuclease inhibitor (RNasin) and Escherichia coliRNA polymerase were from Biotec, Inc. All other reagents were of the highest analytic grade available. Cell Culture-A human neuroblastoma cell line (NB-1) was originally established by Miyake et al. (10). The cells were grownin RPMI 1640 (Nissui Seiyaku Co., Ltd., Tokyo, Japan) supplemented by 10% fetal bovine serum and 0.05% kanamycin in 75-cmZCorning tissue culture flasks. Two days prior to induction, the culture medium was replaced with a fresh medium. Cells were stimulated at 37 “C for 48 h in the presence of 1 mM BtzcAMP. Quantitation of Newly Synthesized Pro- VIP/PHM-27 and Cell-free Translation Assay-All experimental procedures were described in our previous report (6). Isolation of Total Cellular RNA-RNA was isolated by phenol extraction followed by LiCl precipitation (6). Isolation of Nuclear RNA-Nuclei were isolated from neuroblastoma cells (1.1 X 10’ cells) as described by Maurer ( l l ) , except that the homogenization buffer contained 2 mM DTT and 1,000 units/ml RNasin. The isolated nuclear pellet was dissolved in 9 ml of guanidinium thiocyanate solution (4 M guanidinium thiocyanate, 0.5% sodium N-lauroyl sarcosinate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol, 0.1% Antifoam A) using a Dounce homogenizer. The homogenate was layered over 6 ml of 5.7 M CsC1,O.l M EDTA, pH 7.0, and centrifuged at 26,000 rpm at 20 “C for 16 h in a Beckman SW 27.1 rotor (12). The RNAwhich pelleted through the CsCl cushion was dissolvedin sterilewater and precipitated by addition of 0.1 volume of 3 M sodium acetate, pH7.0, and 2.5 volumes of ethanol. Isolation of High Molecular Weight DNA-High molecular weight DNA was isolated from neuroblastoma cells (4.5 X lo6 cells) by the method of Blin and Stafford (13). Prepro-VIPIPHM-27 cDNAClone, pVIP-1, and Nick Translation-In our previous report, we cloned the DNA sequence comple-

9207

Transcriptional Control

9208

of Pro- VIPIPHM-27 Synthesis

mentary to the mRNA encoding for human VIP and designated pVIP-Bt2cAMP were incubated a t 37 "C for 2 h with ["S]methio1 (8).The restriction map is shown in Fig. 1. The size of the insert nine. After incubation, the cell extract was immunoprecipifor pVIP-1 is estimated to be 1227 nucleotides (nucleotide residues analyzed by SDS-polyacryl53-1279). Nick translation was performed using the400-bp and 700- tated with antiserum to VIP and bp Sau3AI/EcoRI restriction fragments (see Fig. 1) of the insert by amide gel electrophoresis followed by autoradiofluorography. the method of Weinstock et al. (14). Probe specific activities were 1- The results are shown in Fig. 2. We have already shown that 2 X 10' cpm/pg of DNA. the M, = 17,500 product is pro-VIP/PHM-27(6-8). Although RNA Gel Blots and DNA Gel Blots-RNA was denatured with 10 several products labeled with [:"S]methionine were detected, mM CH,HgOH, electrophoresed on a 1.5% agarose gel containing 5 (15).The paper was pro-VIP/PHM-27 was effectively eliminated by an excess of mM CH,HgOH, and transferred onto DPT paper unlabeled VIP during the immunoprecipitation reaction (Fig. hybridized to the nick-translated 400-bp Sau3AI/EcoRI restriction fragment and subjected to autoradiography. High molecular weight 2, lanes b and d). In the presence of Bt,cAMP, the synthesis DNA was digested completely with EcoRI, electrophoresed on a 0.7% of pro-VIPIPHM-27 was markedly induced (Fig. 2, lane a). (15).The paper was hybridagarose gel, and blotted onto DPT paper Quantitation by counting the band corresponding to pro-VIP/ ized to "P-labeled probes of 400-bp and 700-bp Sau3AI/EcoRI rePHM-27 showed that the synthesisof pro-VIP/PHM-27 instriction fragments and subjected to autoradiography. creased 11-fold in the induced cells. Nuclear RNA Synthesis in Vitro-Nuclei were isolated from neuroblastoma cells as described by Maurer (11).The nuclear pellet was Quantitation of Prepro- VIPIPHM-27mRNA in Neuroblassuspended in 20 mM Tris-CI, pH 8.0, 5 mM MgC12, 0.1 mM EDTA, 2 toma Cells-In order toclarify the mechanismof induction of mM DTT, and 40% glycerol. For transcription, 50 pl of the nuclear pro-VIPIPHM-27synthesis by Bt2cAMP, we performed suspension were incubated in a 150-pl reaction mixture containing quantitation of prepro-VIP/PHM-27 mRNA by cDNA hy15% glycerol, 20 mM Tris-CI, pH 8.0, 5 mM MgCl2, 150 mM KC1, 2 mM DTT, 0.4 mM ATP, GTP, and CTP, and125 pCi of [a-"PIUTP bridizationassay.RNA isolatedfrom neuroblastoma cells at 26 "C for 30 min (16). Then 50 pg of yeast RNA and 10X SET (to prepared for thepro-VIPIPHM-27synthesisstudies, was final concentration of 1 X SET), 100 pg/ml proteinase K were added separated on an agarose gel containing CH,HgOH, blotted at 45 "C for 30 min. The proteinaseKand the sample was incubated allowed to hybridize with the"P-labeled digested sample was combined with 2.0 ml of guanidinium thiocyanate onto DPT paper, and 400-bp Sau3AIIEcoRI restriction fragment of prepro-VIP/ solution and incubated a t 60 "C for several minutes until the chromatin dissolved (11). The sample was layered over 1.5 ml of 5.7 M PHM-27 cDNA(Fig. 1). Autoradiography of thepaperis CsCI, 0.1 M EDTA, pH 7.0, and centrifuged at 35,000 rpm a t 20 'C shown in Fig. 3. The mRNA hybridized to prepro-VIP/PHMfor 16 h in a Beckman SW 60 Ti rotor. After centrifugation, the 27 cDNA was single and the size of the mRNA was roughly solution above the CsCl cushion was removed, the sides of the tube were rinsed with sterile water, and the remaining CsCl solution was estimated tobe 1600 nucleotides long. In neuroblastoma cells treated by Bt2cAMP, the mRNA hybridized to prepro-VIP/ decanted. The RNA pellet was then dissolved in sterile water and precipitated by addition of 0.1 volume of 3 M sodium acetate, pH7.0, PHM-27 cDNA was markedly increased, indicating that the and 2.5 volumes of ethanol (11). amount of prepro-VIPIPHM-27mRNAsequencewasinFilter Hybridization-PstI-digested plasmid pVIP-1was coupled to D P T paper (15 pg of plasmid/cm2 filter) as described by Goldberg et creased by Bt2cAMP (Fig. 3). Furthermore, we examined the al. (17). Hybridization mixtures of 120 pl contained 0.5 M NaCI, 20 cell-free translation activity of prepro-VIP/PHM-27 mRNA. mM Hepes, pH 7.4,33% deionized formamide, 0.4% SDS, 2 mM RNA was translated in a reticulocyte lysate system suppleEDTA, 100pg/ml yeast tRNA, 1000 cpm of prepro-VIP/PHM-27 mented with ["Slmethionine. The translation products were [''HIcRNA, andtwo filters containingimmobilized pBR322 DNA and analyzed by immunoprecipitationandSDS-polyacrylamide pVIP-1 DNA. Prepro-VIP/PHM-27 ['HIcRNA was included to determine the efficiency of hybridization and was synthesized by the gel electrophoresis followed by autoradiofluorography (6). Quantitation by counting the band corresponding to prepromethods of Jungmann et al. (16) using prepro-VIP/PHM-27 cDNA as template. Hybridizations were carried out for 3 days a t 45 "C. VIPIPHM-27 mRNA in Fig. 3 and the band corresponding Conditions of filter washing and elution of bound RNA from the to prepro-VIP/PHM-27 in thecell-free translation assay was filters were described in Ref. 16. summarized in Table I. When synthesis of pro-VIPIPHM-27 RESULTS

Induction of Pro- VIPIPHM-27 Synthesk by Bt2cAMPAfter treatment of neuroblastoma cells with 1 mM Bt,cAMP for 48 h, neuroblastoma cells expressed differentiated features such as elongation of neurites and increase in size of both soma and nucleus asdescribed previously (18-20). Neuroblastoma cells grown for 48 h in the presence or absenceof 1 mM 1

2W

-400-bp

Pstl SWW

400

fragmmt

600

:

EmRl

8W

I"

700-bp fragment

was stimulated 11-fold by Bt,cAMP, boththeamount of prepro-VIP/PHM-27mRNAand its translationalactivity increased 11-fold. However, total protein synthesis and the Mr

mw--

C49.2k C36.9k

1200

C24.6k C12.3k

PStI

FIG. 1. Restriction map of the cDNA insert in the plasmid pVIP-1. The cDNA insertwas cloned into the PstI of site the plasmid pBR322 by the oligo(dG)-oligo(dC) tailing method. Nucleotide residues of the cDNA insert are numbered in the directionfrom 5' to 3' in the coding strand. The cDNA insert (nucleotideresidues 53-1279) did not contain the entire protein-coding sequence (8).The shadow box, the open box, and the lines represent the coding region, the 3'noncoding region, andtheoligo(dG)-oligo(dC)tails of thecDNA insert, respectively.

a

b

c

d

FIG. 2. Induction of pro-VIP/PHM-27 synthesis by BtzCAMP. Cells grown for 48 h in the presence (a and b) or absence (c and d ) of 1 mM BWAMP were pulse-labeled with [35S]rnethionine at 37 "Cfor 2h. Immunoprecipitates were prepared from thecell extracts with antiserum to VIP in the presence (b and d ) or absence ( a and c) of unlabeled VIP and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiofluorography.


ofVIPIPHM-27 Pro- Synthesis

B

A

bases

~2,096 cprepro-VIP/PHM-27 mRNA ~1,327

c 678 a

b

a

b

FIG. 3. Hybridization of prepro-VIPfPHM-27 cDNAto sizefractionated mRNA from neuroblastoma cells. RNA isolated from cells induced by 1 mM BtsAMP (a, 10.8 pgof RNA) and uninduced cells (b, 7.2pg of RNA) were denatured by 10 mM CHaHgOH, subjected to electrophoresis in a 1.5% agarose gel containing 5mM CHaHgOH, followedby blotting onto DPTpaper, which was later hybridized with "P-labeled nick-translatedprepro-VIP/ PHM-27 cDNA restriction fragment (400-bp Sau3AI/EcoRI fragment). The amount of RNA was adjusted for each cell. A, ethidium bromide stain of the agarose gel; E , autoradiogram of the DPTpaper. The arrows are the size in nucleotides determined from a PstI-RsaI digest of pBR322 run in parallel.

9209

prepro-VIP/PHM-27 mRNA synthesis in neuroblastoma cells were estimated by in uitro transcription assay with isolated nuclei. Nuclei isolated frominduced and uninducedcells were transcribed in uitro, and the relative rates of prepro-VIP/ PHM-27 mRNA synthesiswere determined by hybridization of the nuclear RNA products to immobilized pVIP-1. The results are shown in Table 11. Although the transcriptional activity was not detectable in nuclei isolated from uninduced cells, transcription of prepro-VIP/PHM-27 mRNA was actually observed in isolated nuclei from induced cells. Nuclear RNA isolated from induced and uninduced cells was electrophoresed on a 1.5% agarose gel containing 5 mM CHsHgOH, transferred to DPT paper, andhybridized to the "'P-labeled 400-bp Sau3AIIEcoRI restriction fragment of prepro-VIP/PHM-27 cDNA. As shown inFig. 4, a n RNA species corresponding to mature prepro-VIP/PHM-27 mRNA was detected in nuclear RNA and the amount of the RNA was markedly increased in Bt'cAMP-induced cells. RNA species of molecular weight higher than mature prepro-VIP/PHM27 mRNA was found faintly at the position of about 4 kb. Trace amountsof RNA smaller than mature size prepro-VIP/ PHM-27 mRNA hybridized with prepro-VIP/PHM-27 cDNA. These results from Table I1 and Fig. 4 support the concept that Bt'cAMP increases the production of preproVIPIPHM-27 mRNA by increasing the rateof transcription. Quantitation of VIPIPHM-27 Gene in Neuroblastoma Cells-The experimental results presented so far indicate that the control pointfor the induction of pro-VIP/PHM-27 synthesis by Bt'cAMP is the transcriptional level. T o gain further evidence, quantitation of the VIPIPHM-27 gene was made. High molecular weight DNA was isolated from neuroblastoma cells grown inthepresenceorabsence of Bt'cAMP and completely digested withEcoRI. The resulting fragmentswere subjected to electrophoresis in a 0.7% agarose gel and transferred onto DPTpaper. As shown in Fig. 5, the 2.0- and 3.0kilobase pair fragments hybridized to the 400-bp Sau3AI/ EcoRI restrictionfragment of prepro-VIP/PHM-27 cDNA and the 5.5-kilobase pair fragment hybridized to the 700-bp Sau3AI/EcoRI restriction fragment of prepro-VIP/PHM-27 cDNA. The amounts of the DNA fragments hybridized were almostequalbetweeninducedanduninduced cells. From these results, we concluded that the induction of pro-VIP/ PHM-27 synthesisby Bt'cAMP was due to an increase in the transcriptional activityof the VIP/PHM-27 gene.

amount of total RNA were only increased by a very small amount. Based on these results, it is clear that the induction of proVIP/PHM-27 synthesis by Bt'cAMP in neuroblastoma cells is primarily the consequence of a n increased amountof translatable prepro-VIP/PHM-27 mRNA. Effect of Bt'cAMP on Nuclear Transcription of Prepro- VIP/ PHM-27 mRNA-The finding that Bt'cAMP caused a n increase in the actual amount of prepro-VIP/PHM-27 mRNA DISCUSSION implies that synthesis or degradation of prepro-VIP/PHM-27 mRNA sequences in the nucleus is being altered. As an initial BtZcAMP is known to induce some neural specific enzymes, approach to examining these possibilities, the ratesof nuclear such as tyrosine hydroxylase, choline acetyltransferase, and TABLE I Effect of BtSAMP on pro- VIPIPHM-27 synthesis, prepro- VIPIPHM-27mRNA level, and translational activity in human neuroblastoma cells Total protein synthesis was determined by radioactivity of trichloroacetic acid-insoluble materials of aliquots of cell extracts from neuroblastoma cells incubated a t 37 "C for 2 h with [?3]methionine in the presence or absence of 1 mM BbcAMP. The band corresponding to pro-VIP/PHM-27 in Fig. 2 was excised from the gels to determine the radioactivity of pro-VIP/PHM-27. The amountof prepro-VIP/PHM-27 mRNA was determined by measuring the radioactivity of the band corresponding to prepro-VIP/PHM-27 mRNA in Fig. 3. Translational activity was determined by measuring the radioactivity of the band corresponding to prepro-VIP/PHM-27 (6). The numbers in parentheses were related to that uninduced by BtsAMP. ~~

Protein Treatment TotalTotal cpm/4 X

~-

None 1 mM-~ BtzcAMP

3.4 x lo6 (1.0) 6.2 X lo6 (1.8)

RNA Pro-VIP/ PHM-27 synthesis

IO" cells

440 (1.0) 4810 (10.9)

Prepro-VIP/ PHM-27 mRNA pg/2 x I@ cells

18 (1.0) 27 (1.5)

Translational activity

cpm/2 X 10s cells

105 (1.0) 1210 (11.5)

148 (1.0) 1570 (10.6)

Transcriptional Control of Pro- VIPIPHM-27 Synthesis

9210

TABLE I1 Transcription of prepro- VIP/PHM-27 mRNA in isolated nuclei Nuclei were isolated from 1 mM BWAMP-induced cells and from uninduced cells. Isolated nuclei were allowed to incorporate [“PI UTP. The [32P]RNAwas isolated, purified, and hybridized to DPT filters carrying immobilized pVIP-1 DNA or pBR322 DNA. Hybridized radioactivity was eluted from the filters and determined by liquid scintillation counting. For experimental details, see “Experimental Procedures.” The efficiency of hybridization was assessed by determination of the recovery of added [3H]cRNAstandard. Nonspecific hybridization to pBR322 filtershas been subtracted from counts hvbridized to DVIP-1 filters. 13*P]RNAhybridized to

Input Cell treatment

of 1 ~ 2 ~ 1

Hybridi-

zyll

PreproVIP/ PHM-27 synthesis mRNA

5% input

5%

RNApVIp-1pBR322cRNA x 10-7 cpm

Experiment 1 Control BWAMP Experiment 2 Control Bt2cAMP Experiment 3 Control BWAMP Experiment 4 Control BbcAMP

0.63 0.62

44 110

52 38

27 28

O.oo00

1.02 1.05

56 232

53 112

29 27

O.oo00

1.22 1.27

66 219

56 91

27 29

1.32 1.49

58 240

60 80

28 29

0.0041 0.0042 O.oo00

0.0035

O.oo00 0.0037

A

B

C

bP

C23.3k C 6.4k C 4.2k

2.2k

a b

a b

a b

FIG. 5. Southern blot analysis of VIP/PHM-27 gene. High molecular weight genomic DNAwas isolated from neuroblastoma cells grown in the presence or absence of 1 mM BWAMP andcleaved with EcoRI. Twenty-eight pg of EcoRI-digested DNA from induced cells (a) and from uninduced cells ( b )were subjected to electrophoresis in lanes a and b. Experimental details were described under “Experimental Procedures.” A, ethidium bromide stain of the agarose gel. E, autoradiogram of the DPT paper hybridized with “P-labeled 400-bp Sau3AIIEcoRI restriction fragment of prepro-VIP/PHM-27 cDNA. C, autoradiogram of the same paper as in B rehybridized with ‘*Plabeled 700-bp SauSAI/EcoRI restriction fragment of prepro-VIP/ PHM-27 cDNA after removal of the first probe.

bases of prepro-VIP/PHM-27 mRNA (Table I1 and Fig. 4), but the abundance of VIP/PHM-27 gene was not affected by BbcAMP (Fig. 5). From these results, it is reasonable to assume that the induction of pro-VIP/PHM-27 synthesis by Bt2cAMP is attributed to the newly synthesized prepro-VIP/ 4 2,096 PHM-27 mRNA andthat pro-VIP/PHM-27 synthesis in human neuroblastoma cells is regulated by Bt,cAMP at the -1,327 transcriptional level. Trace amounts of nuclear RNA species were noted in BtcAMP-induced cells(Fig. 4). They maybe a possible 678 precursor (4 kb) of prepro-VIP/PHM-27 mRNA and a series of smaller, breakdown products. There were twofragments of EcoRI-digested DNA hybridized to the400-bp Sau3AI/EcoRI restriction fragment of prepro-VIP/PHM-27 cDNA (Fig.5B). This suggested that human VIP/PHM-27 gene consisted of a b FIG. 4. Analysis of nuclearprepro-VIP/PHM-27 mRNA. at least two exons, since there was no recognition sequence Nuclear RNA wasisolated from cells as described under “Experimen- for EcoRI within the 400-bp Sau3AI/EcoRI restriction fragtal Procedures.” Fifteen pgof nuclear RNA from 1 mM BWAMP- ment of prepro-VIP/PHM-27 cDNA. Alternatively, one of induced cells (a) and from uninduced cells ( b ) were denatured, elec- the two EcoRI fragments may contain an unexpressed pseutrophoresed, blotted, and hybridized to nick-translated 32P-labeled dogene or a duplicate copy of the human VIP/PHM-27 gene. prepro-VIP/PHM-27 cDNA restriction fragment (400-bp Sau3AI/ EcoRI fragment) as described in Fig. 3. The large closed arrow marks Nucleotide sequence analysis of the VIP/PHM-27 gene is in progress in our laboratory. the mature prepro-VIP/PHM-27 mRNA. The open arrow (about 4 From the present experiments, it may be concluded that kb) marks a possible precursor of prepro-VIP/PHM-27 mRNA. cAMP exerts itseffect on the rate of transcription of preproacetylcholine esterase, as well as the morphological differen- VIPIPHM-27 mRNA. The intracellular cAMP level may be increased by some hormones or neurotransmitters via the tiation in mammalian neuroblastoma cells (21). VIP-like im- receptor-adenylate cyclase system. If such transmitters are munoreactive materials in human neuroblastoma cells were demonstrated not only in vitrobut also in vivo, then they may also increased by BbcAMP (20). In the present experiment, be the naturalregulator of the precursor protein. Furthermore, Bt2cAMP was shown to induce the synthesis of pro-VIP/ the control mechanism of the processing step of the VIP/ PHM-27 in neuroblastoma cells (Fig. 2). Evidence that the PHM-27 precursor is of great importance in understanding induction of pro-VIP/PHM-Z7 synthesis is the consequence the biosynthesis of VIP and PHM-27. Although VIP is preof an increased amount of prepro-VIP/PHM-27 mRNA was ceded by a pairof basic amino acids, Lys-Arg, inthe precursor obtained from quantitation of prepro-VIP/PHM-27 mRNA protein, PHM-27 is preceded by only one basic amino acid, both by cDNA hybridization and cell-free translation assay Arg (8).Furthermore, both VIP and PHM-27 contain a pair (Fig. 3 and Table I). The increased amount of prepro-VIP/ of internal basic amino acid residues, such as Lys-Lys and PHM-27 mRNA was caused by increasing the transcription Arg-Lys (8).These could reflect a differential tissue-specific

3 I,o r

-

-


of Pro- VIPIPHM-27 Synthesis

processing pathway, yielding either VIP or PHM-27, or both. REFERENCES 1. Mutt, V., and Said,

S.I. (1974) Eur. J . Biochem. 42,581-589

2. Said, S.I. (ed) (1982) Vasoactive IntestinalPeptide, Advances in Peptide Hormone Research Series, Raven Press, New York 3. Kato, Y., Iwasaki, Y., Iwasaki, J., Abe,H., Yanaihara, N., and Imura, H. (1978) Endocrinology 1 0 3 , 554-558 4. Vijayan, E., Samson, W. K., Said, S. I., and McCann, S.M. (1979) Endocrinology 104, 53-57 5. Ottesen, B., Andersen, A. N., Gerstenberg, T., Ulrichsen, H., Manthorpe, T., and Fahrenkrug, J. (1981) Lancet 2,696 6. Obata, K., Itoh, N., Okamoto, H., Yanaihara, C., Yanaihara, N., and Suzuki, T. (1981) FEBS Lett. 136,123-126 7. Okamoto, H., Itoh, N., and Obata, K. (1983) Regul. Peptides 6, 320 8. Itoh, N., Obata, K., Yanaihara, N., and Okamoto, H. (1983)

Nature (Lond.)3 0 4 , 547-549 9. Tatemoto, K., and Mutt, V. (1981) Proc. Natl. Acad. Sci. U. S. A. Yanaihara, 78,6603-6607 10. Miyake, S., Shimo, T., Kitamura, Y., Nojyo, T., Nakamura, s., Imashuku, S.,and Abe, T. (1973) Autonomic Nervous System 10,115-120 (in Japanese)

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11. Maurer, R. A. (1981) Nature (Lond.) 2 9 4 , 9 4 4 7 12. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 13. Blin, N., and Stafford, D. W. (1976) Nucleic Acids Res. 3, 23032308 14.Weinstock, R., Sweet, R., Weiss, M., Cedar, H., and Axel, R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1299-1303 15. Barinaga, M., Franco, R., Meinkoth, J., Ong, E., and Wahl, G. M. (1981) Methods for the Transfer of DNA, RNA and Protein to Nitrocellulose and Diazotized Paper Solid Supports, Schleicher & Schuell, Inc, Keene, NH 16. Jungmann, R. A., Kelley, D. C., Miles, M. F., and Milkowski, D. M. (1983) J. Biol. Chem. 258,5312-5318 17. Goldberg, M. K. L., Lifton, R. P., Stark, G. R., and Williams, J . G . (1979) Methods Enzymol. 68, 206-220 18. Shimo, Y., Miyake, S., and Kitamura, T. (1972) in Proceedings of the Japanese Cancer Association, the 31st Annual Meeting (Akazaki, K., ed) p. 70, Nagoya, Japan 19. Ishikawa, S. (1977) Acta Pathol. Jpn. 27, 697-711 20. Yanaihara, N., Suzuki, T., Sato, H., Hoshino, M., Okaru, Y., and C. (1981) Biomed. Res. 2, 728-734 21. Parasad, K. N. (1977) in Cell, Tissue and Organ Cultures in Neurobiology (Fedoroff, S., and Hertz, L., eds) pp. 447-483, Academic Press, New York