Effects of Sialagogues on Ornithine Decarboxylase Induction and Proto-oncogene Expression in Murine Parotid Gland M. KAWANO, A. UENO, Y. ASHIDA, N. MATSUMOTO', and H. INOUE2 Departments of Biochemistry and 'Removable Prosthodontics, School of Dentistry, The University of Tokushima, Kuramotocho, Tokushima 770, Japan The mechanism of a sialagogue-induced increase in ornithine decarboxylase (ODC) activity and the expressions of proto-oncogenes in murine parotid gland were investigated by use of isoproterenol (IPR), carbachol (CC), and methoxamine (MTX). The results were as follows: (1) The three sialagogues had similar effects on the parotid in vivo (mouse parotid after a single injection of IPR) and/or in vitro (rat parotid explants cultured on siliconized lens paper floating on 199 medium containing IPR, CC, or MTX), the order of their effectiveness being IPR > CC > MTX. (2) Northern/dot and Western blot analyses revealed that the sialagogues elevated the steady-state levels of ODC mRNA and ODC protein to maxima at two h and six h, respectively, after stimulation. The increases were roughly proportional to those in ODC activity, suggesting that sialagogue-dependent enzyme induction is regulated at the transcriptional level. (3) The mRNAs of four of nine proto-oncogenes examined showed sialagogue-dependent increases to maxima at 30 min (c-fos) or 60 min (c-jun, c-myc, and c-src) after the beginning of stimulation. These increases were all transient, with the levels returning to the control values (without sialagogue) within 60 min. (4) The IPR-dependent elevations of ODC activity and the mRNAs of ODC, c-fos, and c-jun were inhibited by monensin, but not by polymyxin B. On the other hand, the CC-dependent increases in these parameters were inhibited by polymyxin B but not by monensin. The IPR- and CC-induced increases in c-myc and c-src mRNAs were not inhibited by either monensin or polymyxin B, suggesting that the c-Fos and c-Jun proteins participate in this transcriptional control through the AP-1 site of the ODC gene. J Dent Res 71(12):1885-1890, December, 1992

Introduction. Our previous papers have shown that diverse sialagogues such as isoproterenol (IPR, a Pi -adrenergic agonist), carbachol (CC, a cholinergic agonist), and methoxamine (MTh, an a-adrenergic agonist) stimulate DNA synthesis of murine parotid gland in vivo and in vitro, and that this stimulation is always associated with increase in ornithine decarboxylase (ODC) activity in the pre-replicative period (Kikuchi et al., 1987). In these experiments, sialagoguedependent changes in polyamine metabolism in cultured rat parotid explants closely reflected those observed in vivo in mice (Inoue et al., 1974; Kikuchi et al., 1987). We also reported that the sialagogueinduced increase in ODC activity was preceded by increase in phosphorylation of specific proteins during the early pre-replicative period in cultured parotid explants. These stimulations were roughly proportional to the sialagogue-induced increases in amylase secretion, the order of effectiveness ofthe sialagogues being IPR > CC > MTX. Regardless of the type of sialagogue, the maximal increases of protein phosphorylation, ODC activity, and DNA synthesis were detected two, six, and 24 h, respectively, after stimulation of the parotid by the sialagogues (Ueno et al., 1991b). Experiments on the time-dependence of the effects of IPR and CC showed Received for publication March 11, 1992 Accepted for publication June 3, 1992 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 2To whom correspondence and reprint requests should be addressed

that parotid explants must be cultured with these agonists for at least four and 12 h for maximal increases in ODC activity and DNA synthesis, respectively (Inoue et al., 1985; Kikuchi et al., 1987). Although ODC-dependent increase in the putrescine level is a prerequisite for subsequent stimulation of DNA synthesis in this growth system (Inoue et al., 1975), the mechanism of increase in ODC activity by the sialagogues is unknown. For further examination of sialagogue-induced biochemical changes in the early G1 phase (Whitlock et al., 1968; Inoue et al., 1974), in this study changes were measured in the concentrations of ODC protein and expressions ofthe ODC gene and proto-oncogenes related to DNAbindingprotein (fos,jun, myc, myb), signal transduction (src, raf, Ha-ras), and growth factor (erbB, erbB2) in sialagoguestimulated murine parotid gland.

Materials and methods. Materials.-Plasmid pOD48 containing mouse ODC cDNA was a generous gift from Dr. Philip Coffino, University of California, San Francisco (McConlogue et al., 1984). The plasmids pfos-1, pJun, pMC3, pEB1, pKX044, pmyb, and 361 1E-H containing cDNA sequences from v-fos, v-jun, v-myc, v-erbB, c-erbB2, v-myb, and vraf, respectively, were supplied by the Japanese Cancer Research Resources Bank. Plasmid pSV-src [pCD-X (Okayama and Berg, 1983) with RSV v-src cDNA inserted at a BamH I site after removal ofthe X-region] was kindly provided by Dr. Masuo Yutsudo, Osaka University. Human P-actin cDNA probe, a-amanitin, and cycloheximide were obtained from Wako Pure Chemical Co., Osaka. Molecular-weight markers were from Pharmacia LKB Biotechnology (protein) and Bethesda Research Laboratories (RNA). Unless otherwise noted, other reagents for blotting and hybridization experiments were purchased from Boehringer-Mannheim or Takara Shuzo Co., Kyoto. [a-32P]dCTP was a product ofDuPont/NEN. Other chemicals used were of reagent grade and were obtained as reported previously (Kikuchi et al., 1987; Ueno et al., 1991a). Treatment of animals.-Male albino ddY strain mice (30-35 g) were used for in vivo experiments. They were allowed free access to water and laboratory chow. IPR (0.3 mmol/kg body weight) was dissolved in saline and injected intraperitoneally in a volume of 0.2 mL per head (Inoue et al., 1974). The same volume of saline was administered to control mice. Parotid glands were removed from mice under ether anesthesia between 14:00 and 16:00 h. Culture ofratparotid explants.-Explants (approximately 1 mg each) were prepared aseptically from the parotid glands of male Wistar-strain rats weighing from 200 to 300 g each. These explants were incubated at 370C in a CO2 incubator on siliconized lens paper floating on serum-free 199 medium containing insulin, dexamethasone sulfate, and antibiotics, as reported previously (Inoue et al., 1985; Kikuchi et al., 1987). Assay of ODC activity.-The parotid gland (15-20 mg) was homogenized in 0.2 mL of chilled 0.1 mol/L Tris-HCl buffer (pH 7.5) containing 7 mmol/L dithiothreitol, 0.2 mmolIL pyridoxal phosphate, and 0.1 mmol/L EDTA in a Polytron-type microhomogenizer M-100 (Tokai Irika Co., Tokyo). The homogenate was centrifuged at 15,000 g for 20 min at 4°C. ODC activity in the supernatant was assayed with L-[1-14C] ornithine as reported previously (Inoue et al., 1986). Protein was assayedby the method ofRoss and Schatz (1973) with bovine serum albumin (BIO-RAD) as a standard.

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1885

1886

Klll.. et al.

J Dent Res December 1992

A

B 5 _

119

22:kb

67K 941 67

M1K -61K

431f

43 K

B-actn

30

301f

-22 k5e

0 0.51

20'p

(h)

2

4

6 (h)

201 ZJ

4

af

Fig. 2-Changes in steady-state levels of ODC mRNA in parotid Fig. I Sialagoguedependent changes in steady state levels of ODC protein i munne parotid gland. Western blotting was carried out with S0 glands of IPR-treated mice. The gland was excised at the indicated times pg of parotid protein, as described in 'Materials and methods". (A) In vwo after injection of IPR, and parotid total RNA was used for Northern and experiment. Mouse parotid gland was excised at the indicated times after dot blotting, as described in "Materials and methods" (A) Northern injection of IPR (0.3 mmollkg body weight). (B) I vitro experiments. Rat blotting with parotid RNA 10 gg and labeled cDNA probes of ODC and parotid explains were cultured for six h in the absence (I) or presence of 10 P-actin. The positions of marker RNAs are shown (in kb) on the left. (B) pmol/mL IPR (2), 10 pmollmL CC (3) or 20 molmLMIX(4). The positions Dot blotting with parotid RNA (0.2, 1, 5 pg and cDNA of ODC, of marker proteins are shown (in kDa) on the left of each panel. hybridization for four h at 42 C with fragmented and de natured Western blottng S polyacrylamide gel electrophoresis of salmon sperm DNA (200 pgqnL), the membrane was incubated at done by the method the supernatants of parotid homogenates 42 C for 16 h in hybridization buffer containing the P-labeled ofLaemmli (1970). Samples containing 10-50 pg proteinwere boiled cDNA fragment (1-1.5 x 101 cpm/mL, 1-5 x 101 cpmlpg) and 10% in Laemmli's SDS sample buffr and applied to a 10% polyacrylam- dextran sulfate. Then the membrane was washed 5 times at 42C with SDS SSC buffer. The radioactivity on the membrane was ide gel. After electruphuresis, proteins in the were transferred to Immobilon membrane (Millipore) by electroblotting at a determined by autoradiography with Hyperfilm-MP (Amersham), constant current of 1.5 mA/cm2 for 60 mn. Duplicate membranes followed by scanning with a Joyce-Loebl microdensitometer. The were prepared, one for staining with Coomassie Brilliant Blue and scanned area was calculated with a Luzex 500 image analyzer the other for immunodetection of ODC. The latter strip was (Nihon Regulator). The levels of transcripts were normalized with immersed in a solution of blocking reagent for ELISA (Boehringer) those of action transcripts. and then incubated with 50fod-diluted rabbit antiserum (Ueno et Radioactive DNAfagment ere paredbya routine method: al., 1991a) to a synthetic peptide corresponding to residues 341-350 restricted hydrolysis of plasmid, purification by agarose gel electroof'ODC (QKRPKPDEKY), which is a sequence commonto the mouse phoresis, and labeling with [i P-J dCTP (111 TBq:immo) and a (Eisenberg and Janne, 1989), rat (Steeg et at., 1988), and human random primer DNA labeling kit (Takara Shuzo Co., Kyoto) by the (Fitzgerald and Flanagan, 1989) enzymes. Then the strip was multiprime labeling method ofFeiiberg and Vogelstein (1983). The treated with horseradish-peroxidase-conjugated goat anti-rabbit cDNA fragments used in this study were as follows: a Hind IlI W (Seikagaku Kogyo Co., Tokyo, 1000-fIld-diluted), ad peroxi- fragment of ahout 0.96 kb from pOD48 (OD, a 1.3-kb Pt I dase was located by treatment with a mixture of 0.03% 4-chloro-1- fragment from pfos-1 (v[s), a 0.95kb EcoR I-BamH I fragment naphthol (BRL) and 0.02% H202in 50 mmoL Tris-HCI, pH 7.5, fom pdun (v-jn), a 1.52-b Pst I fragment from pMC3 (v-myc), a 5.9-b Sal I fragment from pmb (vomyb), a 1.45-kb Xho IBgl II containing 0.2 moliL NaCl (Hawkes et at., 1982). Color intensity was measured with a Shimadzu dual-wavelength TLC scanner fragment from 361 1E-H (vq-rf, a 1.7-kb Sal I fagment from pEBi1 IalationrofRNA4.ParotidtotalRNAwas isolatedbythe method (v-erB), a 0.44-kb BamH I fragment rom pI044 (c-erbB2), a 0.8described in Molecua Cloning (Sambrook et at., 1989. with kb BamH I fragment from pSV-arc (v-a), and a 0.44-b Hinf I slight modifications. Briefly, tissue (20-30 mg) was homogenized in fament ofthe human P-actin gene (I-actin). Sizes ofmRNAs were 0.3 mL of 5 molJL guanidinium thiocyanate containing 0.1 moi/ estimated with a 0.24-9.5-kb RNA ladder (BRL) or parotid ribosoTris-HCl buffr (pH 7.5), 1% f-mercaptoethanol, and 0.5% sodium mal RNA (2.35 and 6.33 kb). Dotblotin Vumesof200gLofMOPSbuffer(50%formamide, lauroyl sarcosinate in a Polytron-type homogenizer. Debris was removed by centrifugation (5000 g, 10 mi), and the supernatant 6 3% fomaldehyde, 20 mmol/L MOPS) containing 0.2, 1.0, or 5.0 pg was layered 0.8 mL of 5.7 mol/L CsCI-10 mmoI EDTA (pH 7.5) oide-natured parotid RNA were spotted on a Hybond-N4 membrane and centrifuged at 178,000 g for 18 h at 20C in a Hitachi CS- 100 with BIO-DOT (BIO-RAD). Subsequent procedures for determinaultracentrifuge. RNA was purified by sequential ethanol precipita- tion of hybridized radioactive cDNA on the membrane were as for Northern blotting. tions and organic extractions and stored at -200C until used. Northern blotting. -Blotting and hybridization were carried out on a Hybond-N filter (Amersham) according to the manufacture's Results. instructions, with slight modifications. Briefly, 10 pg of total 95 C for three MOPS RNA de-natured at buffr minm Efcts of sialges on the ODC content of the parotid gland.-In parotid this study, IPR was used in both in vivo and in vitro experiments, containing formaldehyde and fomamide, separated by electro 1.2% agarose gel, transferred to a Hybond-N mem- whereas CC and MIX were used oniy in in vitro experiments, phoresis brane by capillary blotting, and fixed with alkali. After prebecause CC is extremely toxic in vivo, and the increase in ODC was

an

on

was

on

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SIALAGOGUES AND ORNITHINE DECARBOXYLASE

Vol. 71 Noi. 12 A

B

Non

PR

cc

1887

MT

(h5 pg

KO §1

.2551 022kbI

a

MITX

c-jun

C

01

2b**

02

z 6S 5s E

4

Fig. 3-Sialagogue dependent in

/

creases in levels of ODC mRNA in cul rat parotid explaits A Northern

\atured blot analysis oftotalRNA (10 g) isaed from parotid explats cultured with 10

3gmol/lmL JPR for 0 h (left) and

two h

1

right). The sizes (inkb) ofODC mRNAs 2 the p sitios of marker calculated RNAs (datafrem not shown) are hown on tie riht (B) Dot-blot analysis of RNA iso0 late ftom parotid explains cultured for in2 4 6 the idicated times in the ab ence o Tie h presence of10gpmollmLIPR, 10 fnnoI/mL CC, o 20 pmol/L MX (C) Changes in relative ODC mRNA levels. Te experimental condit ios were the same as

4Akb-

0

metry of autoradiograms for dot blotting. Values separate experiments

are

4*-

40

in B The mRNA levels were calculated fron he data obtained by deristom

means for the

activity is detectable only after repeated inrctions of CC (Kikuchi

Fig. 4ialagogue-dependert increases in mRNA levels of pinto. oncognes in cultured rat parotid explants. The experimental conditions were the same as for Fig. 3 except for the culture times. Northem blotting was done with RNA isolated from IPR-treated explants.

fbld the control activity (without sialagogui) in the presence of IPR, CC, and MTX, respectively (Kikiwhi et al. 1987). The sialagugue dependent increases in parotid ODC activities in vit o (Inoue et al. 1985) and in vwo (Inoue et al., 1974) are inhibited by yloheximide, suggesting that sialagogues stimulate de novo synthesis of ODC protein. To confirm this suggestion we quantified ODC protein in parotid extracts by Western blotting Fig. 1 shows that, like ODC in other mammalian tissues (Gupta and Coftino, 1985; Wen et al., 1989), parotid ODC consisted of subunit proteins with molecular weights of about 51,000 and that the steady-state levels of these proteins were increased in vivo and in vitro by IPR, CC and MTX (Fig. 1). lime-course experiments showed that the level ofODC protein in parotid extracts reached a maximum after six h on treatment with IPR rinvvo (Fig. 1 A) and in vitro (data not shown), and that the color intensity ofODC protein was roughly proportional to the ODC activity in the parotid extract used for Western blotting. Effects of sialagcgues on the ODC mRNA content in parotid gland.- To test whether sialagogue-stimulated ODC induct ion was preceded by an increase in expression of the ODC gere, we measured the steady-state levels of ODC mnRNA in sialagogue-stimulated parotid gland by Northern and dot blotting. Fig. 2 shows the effect of IPR in vivo. Murne parotid gland contained two sizes (2.2 and 2.7 kb) of ODC mRNA the latter being predominant These two sizes of ODC mRNA have also been found in other murine tissues (McConlogue et ali. 1984, Kahana and Nathans, 1985). When mouse parotid gland was stimulated by a single injection of IPR, the ODC mRNA level began to increase after one h, reached a maximum after two h of 15-fod over the unstimulated level and then decreased gradually However, after six h, it

50% of the maximum level (Fig. 2 A). These results confirmed by dot blot analyses (Fig. 2, B). This change preceded the increase in ODC activity, which began after three h and was maximal after six h. No change in expression of the parotid I-actin gene was detected in the six-hour observation period (Fig. 2).

et 1l.1987) or MTX unpublished observations) which disturb timecourse experiments. Previous reports showed that the ODC activity in cultured parotid explants begins to increase three h after additions of sialagogues, reaching maxima six h later of about 19- six- and two-

was still

The

were

sialagogue-dependent changes

of ODC mRNA in cultured

parotid explants are shown in Fig. 3. The three sialagogues induced increases in the ODC mRNA content in proportion to their capaci ties for ODC induction, their order of effctiveness being IPR > CC > MIX The time courses of these increases were similar to those induced in vivo, except that the maximal levels induced by IPR and MIX at two h did not decrease within six h. Eects of sinaigogues on proto oncogene expressions in parotid giand.-There are reports that IPR and CC enhance the expression of immediate early genes in murine salivary gland (Barka et al., 1986; Kousvelari et al., 1988; Mirels et al., 1989). To examine the biochemical changes that precede ODC induction in the parotid,

we

examined the

expressions ofthe proto-oncogenes that are

related to growth factors, protein kinases, and sequence-specific DNA binding proteins. Four of nine proto-oncogenes tested-fos. jun, 7y, and src-showed sialagogue-dependent increases in their mRNA levels (Fig. 4), whereas the other five genes-erbB, erbB2, nmyb, raf and Ha-ms-showed no change in mRNA level by six h after stimulation, when the sialagogue-induced ODC activity was maximal (data not shown). The time courses of increase in the mRNA levels of the four proto-oncogenes induced by the three

sialagogues were similar showing maxima 30 min (c-s) and 60

min (cjun, c-nyc, and c-sre) after stimulation. In all cases, the

increases were transient, and the levels returned to the control values (without sialagogue) within 60 min. No second increase

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1888

KAWANO et al.

J Dent Res December 1992

was detected within six h after stimulation. IPR was the most Discussion. effective of the sialagogues, the order of effectiveness (IPR > CC It has been reported that ODC activity, which changes rapidly and > MTX) for ODC mRNA induction (Fig. 3) being the same as that markedly in response to exogenous stimuli, is delicately regulated for proto-oncogene expressions (Table 1). The effects of IPR on by a variety of cell type-specific mechanisms, including transcripthese proto-oncogenes in vivo (data not shown) were the same as tional, post-transcriptional, translational, and post-translational those observed in vitro in IPR-stimulated parotid explants. For example, Abrahamsen and Morris (1990) showed that controls. Effects of a-amanitin and cycloheximide on IPR-dependent ODC mRNA level is regulated transcriptionally in serumincreases of mRNA levels and ODC activity.-When parotid ex- the plants were cultured with a-amanitin or cycloheximide, IPR- activated Swiss 3T3 cells but post-transcriptionally in lectin-stimuinduced accumulation of c-fos mRNA was completely inhibited by lated T lymphocytes, which attain a high level of ODC mRNA o-amanitin but not by cycloheximide. In fact, cycloheximide without increase in the transcriptional activity. ODC expression been suggested to be regulated at the translational level in caused a marked increase in c-fos mRNA, in both the absence has mouse testis (Alcivar et al., 1989), Xenopus laevis oocytes (Bassez et (613% of the control value) and presence (1194% of the control 1990), Ehrlich ascites tumor cells (Wallon et al., 1990), and value) of IPR, suggesting that a short-lived protein may contrib- al., murine 3T3 fibroblasts (Levine et al., 1990). There are also reports ute to negative control of c-fos expression. Similar cycloheximideinduced superinduction of c-fos mRNA was observed in growth- that ODC activity is post-translationally inactivated in polyaminecultured cells (Flamigni et al., 1988) and regenerating liver factor-activated cultured cells (Greenberg et al., 1986). On the treated ethanol-fed rats (Diehl et al., 1990) and activated in murine other hand, the IPR-dependent increase in the ODC mRNA level of was almost completely inhibited by both a-amanitin and cyclo- colony-stimulatingfactor-dependent cells (Farrar and Harel-Bellan, and injured gastric mucosa of aged rats (Edgerton et al., 1991). heximide (data not shown). These results suggest that sialagogue- 1989) The present results indicate that three different sialagogues induced increases in mRNAlevels are due to stimulation ofmRNA that increase ODC activity elevated the steady-state levels synthesis and that, in contrast with c-fos mRNA, sialagogue- ofODC proteinparotid and the mRNAs of ODC, c-fos, c-jun, c-myc, and c-src enhanced synthesis of ODC mRNA is dependent upon protein rat parotid gland in vitro. The IPR-induced increases in their synthesis. The dependency of ODC gene expression on protein in synthesis in the cultured cells has also been reported (Shurtleff levels and the time courses of their changes in cultured rat parotid explants were quite similar to those observed in the parotid gland et al., 1988; Gilmour and O'Brien, 1989). Effects of monensin and polymyxin B on IPR- and CC-induced of IPR-treated mice, suggesting that the results of in vitro experiincreases in mRNA levels.-We next investigated the relationship ments closely reflect the in vivo effects of sialagogues in the murine gland. between elevations of proto-oncogene mRNA levels and ODC induc- parotid The sialagogue-dependent increases in the steady-state levels of tion using monensin and polymyxin B, which are specific inhibitors 1, B) and its mRNA (Fig. 3) were roughly ofIPR- and CC-dependent ODC induction, respectively (Ueno et al., ODC proteinto(Fig. the sialagogue-induced increases in ODC activity. 1991b). We have reported that neither monensin nor polymyxin B proportional inhibits sialagogue-dependent amylase secretion or the incorpora- Moreover, these sialagogue-induced increases were completely intion of phosphate into nucleic acids and total cellular proteins in hibited by a-amanitin or cycloheximide. These results strongly that, in this growth system, the initial increase in ODC cultured parotid explants. However, IPR-dependent ODC induc- suggest was mainly controlled by its de novo synthesis, which is tion was depressed by monensin but not by polymyxin B, whereas activity CC-dependent ODC induction was inhibited by polymyxin B but not by monensin. Table 3 shows that, in parotid explants stimulated by TABLE 2 the sialagogues, the IPR- and CC-induced increases in mRNA levels of c-fos, c-jun, and ODC were selectively inhibited by monensin and EFFECTS OF MONENSIN AND POLYMYXIN B polymyxin B, respectively. On the other hand, the IPR- and CCON SIALAGOGUE-DEPENDENT INCREASES induced mRNA inductions of c-myc and c-src were not inhibited by IN mRNA LEVELS OF ODC AND PROTOmonensin or polymyxin B. ONCOGENES IN RAT PAROTID EXPLANTS TABLE 1 SIALAGOGUE-DEPENDENT INCREASES IN mRNA LEVELS OF PROTO-ONCOGENES IN CULTURED RAT PAROTID EXPLANTS

Addition

ODC

fos

jun

myc

src

IPR (10 pmol/mL)

100

100

100

100

100

IPR + MS (0.11 pmol/mL) 25*

35*

18*

95

92

Addition

fos

jun

myc

src

IPR + PMB (200 U/mL)

119

93

108

102

84

None (control)

100

100

100

100

CC (10 pmol/mL)

100

100

100

100

100

IPR (10 pmol/mL)

680*

308*

728*

285*

CC + MS (0.1pmol/mL)

98

112

91

92

95

CC (10 pimolmL)

253*

182t

287*

208t

CC + PMB (200 U/mL) 47t 39* 31* 87 88 Parotid explants were pre-cultured with monensin (MS) or polymyxin B (PMB) for 30 min and then stimulated by IPR or CC. Parotid RNA was isolated 30 min (fos), one h (jun, myc, src), or two h (ODC) later for dot-blot analysis. Relative mRNA levels were calculated from the data obtained by densitometry of autoradiograms. Values are means for three separate experiments, shown as percentages of the control values (sialagogue alone). Data were analyzed by Student's t test. *p < 0.01, t p < 0.05 (for difference from values with IPR or CC alone).

MTX (20 pmol/mL) 207* 137t 207t 156t Parotid RNA was isolated for dot blotting 30 min (fos) or one h (Jun, myc, src) after the additions of sialagogues. Relative mRNA levels were calculated from the data obtained by scanning densitometry of autoradiograms. Values are means for three separate experiments, shown as percentages of the control values (without sialagogue). Data were analyzed by Student's t test. *p < 0.01, t p < 0.05, compared with the corresponding control.

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Vol. 71 No. 12

SIALAGOGUES AND ORNITHINE DECARBOXYLASE

1889

transcriptionally regulated through the level of ODC mRNA. However, the rates of syntheses and degradations of ODC protein and its

REFERENCES

mRNA must be determined before a final conclusion can be made. A nuclear run-off assay and a stability test of ODC message are in progress. In contrast with this early stage of ODC induction, the marked decrease in ODC activity at a later stage may not be due to reduction ofthe ODC mRNA, because 60% of the IPR-induced ODC activity was lost between six and nine h after stimulation (Inoue et al., 1985), but the decrease of mRNA during this period was only 10% (data not shown). These results suggest that ODC expression in the late stage is controlled translationally and/or posttranslationally. A similar shift of ODC regulation has been observed in regenerating rat liver (Hirvonen, 1989). Our findings are consistent with a report by Barka et al. (1986) that c-fos gene expression is transiently stimulated by IPR in vivo in mouse submandibular gland. Sialagogue-dependent increases in the mRNA levels of c-fos and c-myc have also been observed in rat parotid cells in vitro (Kousvelari et al., 1988; Mirels et al., 1989). In those experiments, pre-treatment with the P3-adrenergic antagonist propranolol blocks the effect of IPR on c-fos induction (Barka et al., 1986; Kousvelarietal., 1988), and addition of cAMP derivatives also increases c-fos mRNA levels above basal levels (Kousvelari et al., 1988). The time courses ofthese changes in proto-oncogene mRNAs are quite similar to those observed in this study. In all of these reports, however, stimulation of c-fos gene expression was concluded not to be correlated with IPR-induced cell proliferation. As shown in Fig. 4, the degrees of sialagogue-enhanced expressions of four proto-oncogenes were roughly proportional to the subsequent increases in ODC induction and DNA synthesis (Kikuchi et al., 1987), the effectiveness ofthe sialagogues being in the order IPR > CC > MTX. Studies on the relationship between proto-oncogene expression and ODC induction with use of the sialagogue-specific inhibitors of ODC induction, monensin and polymyxin B (Ueno et al., 1991b), showed that depression of ODC gene expression was preceded by decreases in c-fos and c-jun transcripts (Table 2). IPRstimulated ODC gene expression was completely abolished by cycloheximide, which must inhibit the syntheses of Fos and Jun proteins. These results strongly suggest that the accumulations of Fos and Jun proteins are prerequisites for ODC gene expression in the sialagogue-stimulated parotid gland. There are reports that the products of c-fos and c-jun form a heterodimer with a leucine-zipper motif (Landschulz et al., 1988), which functions as a transcriptional regulator through its binding to the AP-1 site of the gene (Angel et al., 1987; Chiu et al., 1988), and that the ODC gene contains an AP1 site in its 5'-flanking sequence and/or first intron (Steeg et al., 1988; Eisenberg and Janne, 1989). Therefore, we presume that the c-Fos and c-Jun proteins participate in sialagogue-induced cell proliferation as stimulatory regulators of ODC gene expression in the murine parotid gland. The roles of stimulated expressions ofthe c-myc and c-src genes in sialagogue-dependent ODC induction are unknown. In this connection, it is noteworthy that c-Myc protein was recently found to form a specific DNA-binding complex with c-Max or c-Myn (the murine homolog of c-Max), and this heterodimer was shown to recognize a palindromic core sequence (GACCACGTGGTC) for gene regulation (Blackwood and Eisenman, 1991; Prendergast et al., 1991). But this specific binding site has not been detected in the ODC gene, suggesting the absence of direct participation of cMyc in sialagogue-induced ODC gene expression. On the other hand, c-Src protein, a non-receptor-type protein tyrosine kinase, may function in ODC induction, because a preliminary experiment showed that genistein, an inhibitor of protein tyrosine kinase, depressed sialagogue-dependent ODC induction. Studies on the exact role of c-Src in sialagogue-dependent ODC induction are in progress.

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