Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells CHIH-KO
YEH, INDU
S. AMBUDKAR,
AND ELENI
Clinical Investigations and Patient Care Branch, National National Institutes of Health, Bethesda, Maryland 20892 Yeh, Chih-Ko, Indu S. Ambudkar, and Eleni Kousvelari. Differential expressionof early responsegenes,c-jun, c-fos, and jun B, in A5 cells. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G934-G938, 1992.-We examined the expressionof c-fos, c-jun, and jun B after activation of different
signaltransduction pathways in the A5 rat salivary epithelial cell line. Stimulation of P-adrenergicreceptorsby isoproterenol, or addition of 8bromoadenosine 3’,5’-cyclic monophosphate, inducesthe expressionof c-fos and jun B by a protein kinase A-mediated pathway. Phorbol 12.myristate 13-acetate (PMA) induces the expressionof all three genes,but with different kinetics. While c-fos andjun B mRNA levelsincreaseearly (1 h) after stimulation and transiently, those of c-jun remain higher than control even after stimulation for 8 h and return to basal levelsby 24 h. Inhibitors of protein kinaseC block the effect of PMA on c-fos, c-jun, andjun B expression,indicating that these genesare also regulatedby a protein kinase C-mediated mechanism in A5 cells. Increasesin cytosolic Ca2+ by A23187 or ionomycin induceonly the expressionof c-fos gene.This induction is abolishedwhen A5 cells are loadedwith 1,2-bis(Z-aminophenoxy)ethane-N,N,N’JV’-tetraacetic acid before treatment with the ionophores,or when serumis excluded from the incubation medium.Exclusion of serum from the medium doesnot change the effects of isoproterenol or PMA on c-fos, c-jun, or jun B. These results strongly suggestthat serum factors act synergistically with Ca2+ to induce c-fos expressionin A5 cells. The studiespresentedhere indicate that different signaltransduction pathways operate in A5 cells for the induction of c-fos, c-jun, and jun B genes. protooncogenes;signal transduction; protein kinase A; protein kinaseC; calcium ion; salivary cell line THE EXPRESSION of the c-fos protooncogene
is rapidly and transiently activated by a diverse array of stimuli, including growth factors, phorbol esters, neurotransmitters, agents that elevate cytosolic Ca2+, and adenosine 3’,5’-cyclic monophosphate (CAMP), in a variety of tissues and cell types (10, 16). The c-fos protooncogene encodes a protein that forms a stable complex in the nucleus with the c-jun product. This heterodimer has been shown to recognize the same DNA binding site (5’-ATGACTCAT-3’) as the activator protein-l (AP-1) (15, 32). Recently, it has become clear that the AP-1 complex includes different members of the c-fos (fos B, fra 1, fra 2) and c-jun (jun B, jun D) families (23,24,35). The conserved DNA binding sequences among these families allow the formation of heterodimeric complexes between them and contribute to the affinity by which they bind to DNA (15, 24). The members of both families act as transcription factors that presumably mediate the effects of various stimuli on gene transcription (12, 13). Lately, there has been an increased interest in resolving the modes of expression of the genes of the c-jun and c-fos families by different stimuli (3, 20). We are studing the characteristics of expression of the early response genes c-fos, c-jun, and jun B in A5 cells. The A5 cell line is a cloned epithelial cell line derived G934
KOUSVELARI Institute
of Dental Research,
from the rat submandibular glands (6). This cell line has been shown to possess functional cyl- and P-adrenergic receptors and it has been used as an in vitro model to study receptor-mediated regulation of processes such as water and ion transport, phosphatidylinositol turnover, and amino acid transport (18, 19). Recently, we have shown that increases in intracellular CAMP either by stimulation of ,&adrenergic receptors by isoproterenol, or directly by addition of &bromo-CAMP (8-BrcAMP), in A5 epithelial cells result in the early and transient induction of c-fos and jun B gene expression (33, 34). Their inducibility by both agents is transcriptionally regulated (33). We have also shown that c-fos and jun B may be a requirement for A5 cells to enter the cycle, since their mRNA is increased at the G1/S phase of the cell cycle. In addition, these genes can be induced throughout the A5 cell cycle by isoproterenol and/or 8-BrcAMP (33). These results have suggested that the same signal transduction pathways are involved in the regulation of c-fos and jun B gene expression, and that they are similar in both asynchronized and synchronized cells. We have undertaken the present study to examine the pattern of expression of c-fos, c-jun, and jun B, three members of the AP-1 family, after activation of different signal transduction pathways in asynchronous A5 cells. Our data show that c-fos, c-jun, and jun B gene expression is differentially regulated by the activation of different intracellular signaling pathways, CAMP/protein kinase A, protein kinase C, and Ca2+, in A5 cells. EXPERIMENTAL PROCEDURES CeZZcultures. A5 cells were grown in modified McCoy’s 5-A medium (Biofluids, Rockville, MD) supplementedwith 10%fetal bovine serum(FBS) and penicillin G and streptomycin sulfate (100 U/ml and 100pg/ml, respectively) (Biofluids) at 37°C in a 5% CO2 incubator. A5 cellswere usedbetweenpassages16 and 20, seededat a density of 0.5 x lo5 cells/cm2onto loo-mm culture dishes, and allowed to grow to near confluence. The cells were incubated with or without isoproterenol (10 PM), 8-BrcAMP (1 mM), phorbol 12-myristate 13.acetate (PMA, 50 nM), A23187 (1 PM), or ionomycin (1 MM) (Sigma, St. Louis, MO) in McCoy’s medium with only 2% FBS unlessotherwise indicated. To test the effect of protein kinaseC (PKC) or PKA inhibitors H-7, H-8, staurosporine(SS), a nonspecificinhibitor of PKC (29), or 1,2-bis(Z-aminophenoxy)ethane-N,N,N’,N’tetraacetic acid (BAPTA), a Ca2+chelator, cells were first incubated in the presenceof the inhibitor for 30 min, then the appropriate agent wasadded and incubations continued for another 60 min, or cellswere only incubated in the presenceof the inhibitors. At the end of eachexperimental condition, cellswere stored at -70°C until usedfor RNA isolation. RNA isolation and Northern blot analysis. Cellswere scraped and homogenizedas describedpreviously (34). Total RNA was isolatedby the guanidinium-cesiumchloride method (7). Equal amountsof RNA were electrophoresedin 2.2 M formaldehyde,
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PROTOONCOGENE
EXPRESSION
1.2% agarose gels. The RNA was visualized by ethidium bromide staining and transferred to nitrocellulose membranes. The cDNA probes used for hybridizations were a c-fos cDNA of 2.2 kb (11) (kindly provided by Dr. T. Curran), a 0.4-kb EcoR I-Sac I c-jun fragment, a 0.5kb EcoR I-Sac Ijun B fragment (25), and an 18s genomic DNA fragment. These probes were labeled by the random primer procedure described by Feinberg and Vogelstein (14), using [s2P]dCTP (ICN Biochemicals, Costa Mesa, CA). Blots were subjected to autoradiography at -70°C for 5 days, or for 2 h for the 185 probe, using Kodak AR-5 X-ray film (Kodak, Rochester, NY). RESULTS
AND
G935
IN A5 CELLS
A
B
DISCUSSION
We have hypothesized that stimulation of different signaling pathways may result in distinct patterns of c-fos, c-jun, and jun B gene expression. To test this hypothesis, we have investigated the effect of three main intracellular signals, cAMP/PKA, PKC, and cytosolic Ca2+, on c-fos, c-jun, and jun B gene expression. Effect of isoproterenol and CAMP on c-fos, c-jun, and jun B gene expression. A5 cells were incubated in the
presence of 10 PM isoproterenol or 1 mM 8-BrcAMP for various times (30 and 60 min and 2, 4, 8, and 24 h), and the pattern of c-fos, c-jun, and jun B expression was followed. As we have reported previously, the induction of c-fos and jun B by isoproterenol or 8-BrcAMP is high at 30 and 60 min and returns to basal levels after 4 h (31, 32). Unlike c-fos and jun B, the expression of c-jun is not activated by either isoproterenol or 8-BrcAMP at any time point examined. The differences in expression between the three genes at 1 h are demonstrated in Fig. 1. These results are conistent with a transcriptional mechanism for c-fos and jun B regulation that is modulated by CAMP, as we have shown recently (33); however, c-jun does not appear to be similarly regulated. A CAMP response element (TGACGTCA) (CRE) consensus sequence (22) has been identified in the promoter of the c-fos gene (4), and therefore c-fos expression could potentially be directly regulated by CAMP. Our data demonstrate a role for CAMP in the regulation of c-fos expression in A5 cells. Although it is not known whether the promoter of jun B contains CRE matching sequence, it has been shown that increasing levels of intracellular CAMP in BALB/c-3T3 cells induces jun B expression (18). In the same cells, stimulation of c-jun by 12-0tetradecanoylphorbol 13-myristate (TPA) is totally inhibited in the presence of CAMP. We have also shown (21) that in rat parotid salivary glands, stimulation by isoproterenol activates only the expression of c-fos and jun B but not of that of c-jun. Additionally, it has been shown that jun B competes with c-jun for the AP-1 site within the c-jun promoter, resulting in the repression of c-jun (9, 26). Presently, we have no evidence to suggest that jun B may act as a negative regulator of c-jun in salivary cells. The data in Fig. 1B support the interpretation that induction of c-fos and jun B by isoproterenol or CAMP is mediated by PKA. Preincubations of A5 cells with H-8 (100 PM), H-7 (100 PM), or SS (1 PM) abolish the effect of isoproterenol or CAMP on c-fos and jun B expression (Fig. 1B). Therefore, it appears that activation of PKA leads to different responses of three related genes, which are members of the AP-1 family and basically contain similar DNA binding domains. Differences in the expres-
c-jun -
c-fos -
jun B-
18S-
Ij
Fig. 1. Northern blot analysis of c-jun, c-fos, andiun B in A5 cells after @-adrenergic stimulation, addition of 8-BrcAMP, or addition of different protein kinase inhibitors. A: total RNA was isolated from cells treated with either isoproterenol or 8-BrcAMP. B: total RNA was isolated from cells preincubated with 100 staurosporin (SS) for 30 min, after which added for 60 min. Blots were successively jun B cDNA 32P-labeled probes or with observations of 4 experiments.
1M of H-7 or H-8 or 1 ,.rM time isoproterenol(1 FM) was hybridized with c-jun, c-fos, or the 18s probe. Data represent
sion of c-fos, c-jun, and jun B have also been observed in PC-12 cells after KCl-induced membrane depolarization or treatment with the neurotransmitter analogue nicotine (3). Effect of PKC on c-fos, c-jun, and jun B gene expression.
We have next examined the effect of PMA, a direct activator of PKC, on c-fos, c-jun, and jun B expression. A5 cells were incubated in the presence or absence of this agent for various time intervals starting at 1, 4, 8, and 24 h. The autoradiograms of the Northern blot analysis are shown in Fig. 2, A and B. The expression of all three genes is induced by PMA (50 nM), but with different kinetics. c-Fos and jun B mRNA levels are highly induced after 1 h of incubation with PMA and return to basal levels by 4 h (Fig. 2A). The levels of c-jun mRNA are high at 1 h, and even at 8 h after stimulation, and return to the basal levels after 24 h. A PMA analogue 4cr-phorbol12,13-didecanoate (PDD), which does not activate PKC, had no effect on the expression of c-fos, c-jun, and jun B. The temporal differences in the expression of c-jun may reflect differences in the activation of the c-jun promoter. It has been shown that induction of c-jun transcription in
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G936
PROTOONCOGENE
EXPRESSION
A PMA
1 hr
r-----7nnn -+-+
c-jun
pn
4 hr
8 hr -
24
+-
hr
+
-
B-
18S-
IN A5 CELLS
physiological levels of extracellular Ca2+. The increase in cytosolic Ca2+ is a result of both intracellular Ca2+ release and Ca2+ entry (data not shown). As shown in Fig. 3, both ionophores induce the expression of c-fos in these cells after 1 h, but have no effect on c-jun or jun B expression. The levels of c-fos mRNA return to basal levels after 4 h. The pathway by which increases in intracellular Ca2+ regulate c-fos expression has been studied in PC-12 cells. It has been shown in these cells that a specific DNA sequence is important for the induction of c-fos by A23187 (28). Surprisingly, in our study c-jun and jun B mRNA levels are not changed by either A23187 or ionomycin (Fig. 3). Recently, Trejo and Brown (31) have shown that increase in cytoplasmic Ca2+ is a negative modulator of c-jun expression in 1321Nl cells. They have suggested that the repression of c-jun expression probably occurs though a pathway affecting c-jun mRNA degradation. To determine whether the induction of c-fos was mediated by increases in cytosolic Ca2+, A5 cells were buffered intracellularly with the Ca2+ chelator BAPTA (50 PM). In BAPTA-loaded A5 cells, ionomycin no longer induced the levels of c-fos mRNA. These data clearly
A
B
Fig. 2. Effects of phorbol 12-myristate 13-acetate (PMA) and protein kinase inhibitors on expression of c-jun, c-fos, and jun B. A: total RNA was isolated from A5 cells treated with 50 PM PMA for various times indicated. B: total RNA was isolated from cells treated with H-7 (100 FM) or staurosporin (1 PM) for 30 min before addition or no addition of 50 &M PMA for 1 h. RNA was analyzed by hybridization to c-j,,, c-fos, jun B, and 18s probes. Data in this and next 2 figures represent observations of 3 different experiments. X-ray films in B were overexposed to demonstrate mRNA levels in control.
response to TPA is mediated by binding of jun/AP-1 to an AP-1 binding site in the jun-promoter region (2). This direct stimulation of c-jun by its own gene product is likely to be responsible for prolonging the transient expression of c-jun in A5 cells, as has been suggested for HeLa TK- cells (1). PMA has been shown to activate the expression of c-jun, c-fos, and jun B in other cells, such as 1321Nl astrocytoma cells, BALB/c-3T3 cells, and human U-937 leukemia cells, by a PKC-mediated mechanism (17, 19, 31). We have, therefore, pretreated A5 cells with H-7 or SS, two relatively nonspecific protein kinase inhibitors when used at high concentrations. Both inhibitors blocked the effect of PMA on the expression of c-fos, c-jun, and jun B (Fig. 2B). They also decreased the basal mRNA levels of all three genes (compare lanes Con, +H-7, +SS in Fig, 2B). It should be noted however that in Fig. lB, control levels of c-fos and jun B mRNA are not clearly visualized because of lower exposure times of the X-ray film. Role of Ca2+ on expression of c-fos, c-jun, and jun B. To examine the effect of cytosolic Ca2+ on the expression of c-fos, c-jun, and jun B genes, A5 cells were incubated in the presence of the Ca2+ ionophores A23187 (1 pm) or ionomycin (1 PM). In fura-2-loaded A5 cells, we have observed that these ionophores induce immediate sustained elevations in cytosolic Ca2+ in the presence of
c-jun -
c-fos -
jun B-
18S-
18S-
Fig. 3. Role of cytosolic Ca2+ on expression of c-jun, jun B, and c-fos. A: total RNA was isolated from A5 cells incubated with A23187 (1 PM) or ionomycin (1 PM) for 1 h. B: total RNA was isolated from cells first loaded with BAPTA for 30 min and then treated with ionomycin (1 PM) for 1 h. RNA was analyzed by hybridization to c-jun, c-fos, jun B, and 18s probes.
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PROTOONCOGENE
EXPRESSION
suggest that in A5 cells, elevation in cytosolic Ca2+ is also a signal for c-fos expression. Interestingly, the effect of the Ca2+ ionophores on the expression of c-fos is influenced by the composition of the incubation medium. As shown in Fig. 4, elimination of serum in McCoy’s 5-A medium blocks the effect of ionomycin on c-fos induction. The removal of serum from the medium has little effect on the increase of c-fos mRNA levels by isoproterenol or PMA. Furthermore, there is no time dependency on the serum requirement on c-fos induction (Fig. 4, bottom panel). These results clearly indicate that the induction of c-fos by increases in cytosolic Ca 2+ depends on the presence of serum in the medium. What these findings further suggest is that a factor(s) in the serum likely acts synergistically with ionomycin to induce c-fos gene expression. Although we provide no evidence, we can suggest that activation of c-fos by Ca2+ is probably mediated by a serum response ele-
Serum
CON r---lnnr----l +-+-+-+-
IS0
‘ON
’ 1 hr.
Ion0
PMA
18S-
Ion0
Serum
4 hrs.
8 hrs. ’
-l-----Ill+-+-+-+-
G937
IN A5 CELLS
ment (SRE). Indeed, it has been shown that c-fos SRE can mediate transcriptional activation in response to thapsigargin, an agent that increases cytosolic Ca2+ without hydrolysis of inositol phospholipids and has no effect on protein kinase activity (27). Importantly, elimination of serum from the media did not alter the results of isoproterenol or PMA on jun B expression, or the effect of PMA on the expression of c-jun (data not shown). Reported evidence supports our findings that early response genes can be regulated differentially by different stimuli in the same cell (3, 18, 31), indicating that the biochemical pathways by which these genes are activated are distinct. Indeed, activation of c-fos, c-jun, and jun B can be mediated through the SRE or the phorbol ester response element (1, 25, 30). Alternatively, c-fos can be activated by an element located -60 bp upstream of the c-fos mRNA initiation site that has sequence homologies with CRE (4), and by DNA specific sequence important for the induction of this gene by A23187 (28). Taken together, our results indicate that distinct combinations of the early response genes can be activated in response to three different intracellular signals, CAMP/ PKA, PKC, and Ca2+, in A5 cells. A summary of the results is shown in Table 1. Because c-fos, c-jun, and jun B have been identified as transcription regulators, it is possible to imagine that distinct heterodimeric fos-jun, fos-jun B, jun-jun B, or homodimeric jurz-jun complexes among them will be required to link extracellular signals with events in the nucleus. The observed dissociation of c-fos, c-jun, and jun B gene expression is, therefore, important because it may result in different dimerization forms, and by doing so will determine the transcriptional activation of the target genes. For example, fos-jun heterodimers bind to DNA more tightly than do jun-jun homodimers (22). c-dun and jun B are also different in their biological properties regardless of their sequence homologies. The c-jun homodimers are more efficient transcription activators for promoters containing a single AP-1 site than those formed with jun B. Thus differential regulation of this group of early response genes may play an important role in the long-term responses of A5 cells to extracellular stimuli. A5 cells are rapidly proliferating cells. Their doubling time Is 18 h (Yeh, unpublished observations). Recently, we were able to demonstrate that c-fos and jun B expression may be important in the early phase of the A5 cell cycle (33). Therefore, it is possible that distinct combinations of c-fos, c-jun, and jun B are required to control the proliferative activity of A5 cells. Although it is clear Table 1. Differential expression of c-fos, c-jun, and jun B by various intracellular signals in A5 cells Intracellular Signals
Agents
18S\ . Fig. 4. Effect of serum in isoproterenol-, PMA-, or Ca”+-activated c-fos expression. Total RNA was isolated from A5 cells incubated in McCoy’s medium in the presence or absence of 2% serum with or without isoproterenol, ionomycin, or PMA for 1 h. Cells were also incubated for 1, 4, and 8 h with or without ionomycin in the presence or absence of 2% serum. The same blot was hybridized first with c-fos cDNA probe, stripped, and then rehybridized with 18s probe.
Isoproterenol 8-BrcAMP PMA PDD Ionomycin Ionomycin + serum A23187 + serum
c-fos
Protein
kinase
A
Protein
kinase
C
[Caz+li [Ca*+]i ICa2+lz
increase increase? increase?
8-BrcAMP, 8-bromoadenosine phorbol 12-myristate 13-acetate;
+ + + + +
c-~un
jun B
+ -
+ + + -
3’,5’-cyclic monophosphate; PMA, PDD, 4oc-phorbol 12,13-didecanoate.
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G938
PROTOONCOGENE
EXPRESSION
from our data that agents such as CAMP, PMA, and Ca2+ differentially regulate the expression of the early response genes, their participation in the growth of A5 cells will have to be confirmed. We thank Dr. R. Bravo for providing the c-jun and jun B cDNA and Dr. T. Curran for providing the c-fos cDNA. We are particularly thankful to Dr. B. J. Baum for comments and encouragement during this study. Present address of C.-K. Yeh: Audie L. Murphy Memorial Veterans Hospital, 7400 Merton Minter Blvd., San Antonio, TX 78284. Address for reprint requests: E. Kousvelari, Clinical Investigations and Patient Care Branch, Bldg. 10, Rm. lA-19, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892. Received 26 February 1992; accepted in final form 30 July 1992. REFERENCES 1. Angel, P., K. Hattori, T. Smeal, and M. Karin. The jun proto-oncogene is positively autoregulated by its product, jun/ AP-1. Cell 55: 875-885, 1988. 2. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. 3. Imbra, H. J. Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. Phorbol ester inducible genes contain common cis element recognized by a TPA-modulated trans-acting factor. Cell 49: 729-739, 1987. 3. Bartel, D. P., M. Sheng, -L. F. Lau, and M. E. Greenberg. Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and .jun induction. Genes Dev. 3: 304-313, 1989. 4. Berkowitz, L. A., N. T. Riabowol, and M. 2. Gilman. Multiple sequence elements of a single function class are required for cyclic AMP responsiveness of the mouse promoter. Mol. Cell. Biol. 9: 4272-4281, 1989. 5. Brown, A. M., and B. J. Baum. System A amino acid transport in rat submandibular ductal cell line. Comp. Biochem. Physiol. A Comp. Physiol. 93: 383-389, 1988. 6. Brown, A. M., E. J. Rusnock, J. 3. Sciubba, and B. J. Baum. Establishment and characterization of an epithelial cell line from rat submandibular gland. Oral Pathol. Med. 18: 206-213, 1989. 7. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979. 8. Chiu, R., P. Angel, and M. Karin. Jun B differs in its biological properties from, and is a negative regulator of, c-jun. Cell 59: 979-986, 1989. 9. Chiu, R., W. J. Boyle, 3. Meek, T. Smeal, T. Hunter, and M. Karin. The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54: 541-552, 1988. edited by E. P. 10. Curran, T. Fos. In: The Oncogene Handbook, Reddy, A. M. Skalka, and T. Curran. Amserdam: Elsevier, 1988, p. 307-325. 11. Curran, T., M. B. Gordon, K. L. Rubino, and L. C. Sambucetti. Isolation and characterization of the c-fos (rat) cDNA and analysis of post-translational modification in vitro. Oncogene
2: 79-84,
16. Grausz, 17.
18.
19. 20.
21.
22. 23. 24. 25. 26. 27.
28.
29.
30. 31.
1987.
12. Curran, T., and P. K. Vogt. Dangerous liasons: fos and jun oncogenic transcription factors. In: Transcription Regulation, edited by K. Yamamoto and S. McKnight. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. In press. 13. Diamond, M. I., J. N. Miner, S. K. Yoshinaga, and Y. R. Yamamoto. c-&n and c-fos levels specify positive or negative glucocorticoid regulation from a composite CRE. Science Wash. DC 249: 1266-1272, 1990. 14. Feinberg, A. M., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983. 15. Gentz, R., F. Rauscher III, C. Abate, and T. Curran. Parallel association of fos and jun leucine zippers juxtapose DNA binding domains. Science Wash. DC 243: 1695-1699, 1989.
IN A5 CELLS
32. 33.
34. 35.
J. D., D. Fradelizi, R. Monier, and P. Lehn. Modulation of c-fos and c-myc mRNA levels in normal human lymphocytes by calcium ionophore A23187 and phorbol ester. Eur. J. Immunol. 16: 1217-1221, 1986. Haas, R., H.-J. Pfannkuche, S. Kharbanda, H. Gunji, G. Meyer, A. Hartman, H. Hidaka, K. Resch, D. Kufe, and M. Goppelt-Strube. Protein kinase C activation and protooncogene expression in differentiation/retrodifferentiation of human U-937 leukemia cells. Cell Growth Differ. 2: 541-548, 1991. He, X., J. Ship, X. Wu, A. M. Brown, and R. B. Wellner. ,&Adrenergic control of cell volume and choride transport in an established rat submandibular cell line. J. Cell. Physiol. 138: 527535, 1989. He, X., X. Wu, A. M. Brown, R. B. Wellner, and B. J. Baum. Characteristics of cr,-adrenergic receptors in a rat salivary cell line, RSMT-A5. Gen. Pharmacol. 20: 175-181, 1989. Metcha, F., J. Piette, S.-I. Hirai, and M. Yavin. Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes. New Biol. 1: 297-304, 1989. Mertz, P. M., P. C. Fox, A. Pluta, B. J. Baum, and E. E. Kousvelari. Effects of ionizing radiation and P-adrenergic stimulation on the expression of early response genes in rat parotid glands. Radiat. Res. 130: 104-112, 1992. Montminy, M. R., and L. M. Bilezikjian. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature Lond. 328: 175-178, 1987. Nakaabeppu, Y., K. Ryder, and D. Nathans. DNA binding activities of three murine jun proteins: stimulation by fos. Cell 55: 907-915, 1988. Ryseck, R.-P., and R. Bravo. c-Jun, jun B, and jun D differ in their binding affinities to AP-1 and CRE consensus sequences: effect of fos proteins. Oncogene 6: 533-542, 1991. Ryseck, R.-P., M. Hirai, M. Yaniv, and R. Bravo. Transcriptional activation of c-jun during Go/G1 transition in mouse fibroblasts. Nature Lond. 334: 535-537, 1988. Schutte, J., J. Viallet, M. Nau, S. Segal, J. Fedorko, and J. Mina. Jun B inhibits and c-fos stimulates the transforming and transactivating activities of c-jun. Cell 59: 987-997, 1989. Schonthal, A., J. Sgarman, J. H. Brown, M. R. Hanley, and J. R. Feramisco. Regulation of c-fos and c-jun protooncogene expression by the Ca 2+-ATPase inhibitor thapsigargin. Proc. Natl. Acad. Sci. USA 88: 7096-7100, 1991. Sheng, M., S. T. Dougan, G. McFadden, and M. E. Greenberg. Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences. 1Mol. Cell. Biol. 8: 2787-2796, 1988. Tamaoki, T., H. Nomoto, I. Takahashi, H. Kato, M. Morimoto, and F. Tomita. Staurosporine, a potent inhibitor of phospholipid/Ca 2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402, 1986. Treisman, R. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5’ element and c-fos 3’ sequences. Cell 42: 889-920, 1985. Trejo, J.-A., and J. H. Brown. ~4’0s and c-jun are induced by muscarinic receptor activation of protein kinase C but are differentially regulated by intracellular calcium. J. Biol. Chem. 266: 7876-7882, 1991. Turner, R., and R. Tjian. Leucine repeats and an adjacent DNA binding domain mediate the formation of the functional c-fos-c-jun heterodimers. Science Wash. DC 243: 1689-1694,1989. Yeh, C.-K., M. A. Chinchetru, and E. Kousvelari. Characteristics of c-fos and jun B gene expression in A5 cells after padrenoreceptor stimulation and during the cell cycle. Biochim. Biophys. Acta 1090: 173-180, 1991. Yeh, C.-K., J. M. Louis, and E. Kousvelari. /3-Adrenergic regulation of c-fos gene expression in an epithelial cell line. FEBS Lett. 240: 118-122, 1988. Zerial, M., L. Toschi, R. Ryseck, M. Schuermann, R. Muller, and R. Bravo. The product of a novel growth factor activated gene, fos B, interacts with jun proteins, enhancing their DNA binding activity. EMBO J. 8: 805-815, 1989.
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YEH, INDU
S. AMBUDKAR,
AND ELENI
Clinical Investigations and Patient Care Branch, National National Institutes of Health, Bethesda, Maryland 20892 Yeh, Chih-Ko, Indu S. Ambudkar, and Eleni Kousvelari. Differential expressionof early responsegenes,c-jun, c-fos, and jun B, in A5 cells. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G934-G938, 1992.-We examined the expressionof c-fos, c-jun, and jun B after activation of different
signaltransduction pathways in the A5 rat salivary epithelial cell line. Stimulation of P-adrenergicreceptorsby isoproterenol, or addition of 8bromoadenosine 3’,5’-cyclic monophosphate, inducesthe expressionof c-fos and jun B by a protein kinase A-mediated pathway. Phorbol 12.myristate 13-acetate (PMA) induces the expressionof all three genes,but with different kinetics. While c-fos andjun B mRNA levelsincreaseearly (1 h) after stimulation and transiently, those of c-jun remain higher than control even after stimulation for 8 h and return to basal levelsby 24 h. Inhibitors of protein kinaseC block the effect of PMA on c-fos, c-jun, andjun B expression,indicating that these genesare also regulatedby a protein kinase C-mediated mechanism in A5 cells. Increasesin cytosolic Ca2+ by A23187 or ionomycin induceonly the expressionof c-fos gene.This induction is abolishedwhen A5 cells are loadedwith 1,2-bis(Z-aminophenoxy)ethane-N,N,N’JV’-tetraacetic acid before treatment with the ionophores,or when serumis excluded from the incubation medium.Exclusion of serum from the medium doesnot change the effects of isoproterenol or PMA on c-fos, c-jun, or jun B. These results strongly suggestthat serum factors act synergistically with Ca2+ to induce c-fos expressionin A5 cells. The studiespresentedhere indicate that different signaltransduction pathways operate in A5 cells for the induction of c-fos, c-jun, and jun B genes. protooncogenes;signal transduction; protein kinase A; protein kinaseC; calcium ion; salivary cell line THE EXPRESSION of the c-fos protooncogene
is rapidly and transiently activated by a diverse array of stimuli, including growth factors, phorbol esters, neurotransmitters, agents that elevate cytosolic Ca2+, and adenosine 3’,5’-cyclic monophosphate (CAMP), in a variety of tissues and cell types (10, 16). The c-fos protooncogene encodes a protein that forms a stable complex in the nucleus with the c-jun product. This heterodimer has been shown to recognize the same DNA binding site (5’-ATGACTCAT-3’) as the activator protein-l (AP-1) (15, 32). Recently, it has become clear that the AP-1 complex includes different members of the c-fos (fos B, fra 1, fra 2) and c-jun (jun B, jun D) families (23,24,35). The conserved DNA binding sequences among these families allow the formation of heterodimeric complexes between them and contribute to the affinity by which they bind to DNA (15, 24). The members of both families act as transcription factors that presumably mediate the effects of various stimuli on gene transcription (12, 13). Lately, there has been an increased interest in resolving the modes of expression of the genes of the c-jun and c-fos families by different stimuli (3, 20). We are studing the characteristics of expression of the early response genes c-fos, c-jun, and jun B in A5 cells. The A5 cell line is a cloned epithelial cell line derived G934
KOUSVELARI Institute
of Dental Research,
from the rat submandibular glands (6). This cell line has been shown to possess functional cyl- and P-adrenergic receptors and it has been used as an in vitro model to study receptor-mediated regulation of processes such as water and ion transport, phosphatidylinositol turnover, and amino acid transport (18, 19). Recently, we have shown that increases in intracellular CAMP either by stimulation of ,&adrenergic receptors by isoproterenol, or directly by addition of &bromo-CAMP (8-BrcAMP), in A5 epithelial cells result in the early and transient induction of c-fos and jun B gene expression (33, 34). Their inducibility by both agents is transcriptionally regulated (33). We have also shown that c-fos and jun B may be a requirement for A5 cells to enter the cycle, since their mRNA is increased at the G1/S phase of the cell cycle. In addition, these genes can be induced throughout the A5 cell cycle by isoproterenol and/or 8-BrcAMP (33). These results have suggested that the same signal transduction pathways are involved in the regulation of c-fos and jun B gene expression, and that they are similar in both asynchronized and synchronized cells. We have undertaken the present study to examine the pattern of expression of c-fos, c-jun, and jun B, three members of the AP-1 family, after activation of different signal transduction pathways in asynchronous A5 cells. Our data show that c-fos, c-jun, and jun B gene expression is differentially regulated by the activation of different intracellular signaling pathways, CAMP/protein kinase A, protein kinase C, and Ca2+, in A5 cells. EXPERIMENTAL PROCEDURES CeZZcultures. A5 cells were grown in modified McCoy’s 5-A medium (Biofluids, Rockville, MD) supplementedwith 10%fetal bovine serum(FBS) and penicillin G and streptomycin sulfate (100 U/ml and 100pg/ml, respectively) (Biofluids) at 37°C in a 5% CO2 incubator. A5 cellswere usedbetweenpassages16 and 20, seededat a density of 0.5 x lo5 cells/cm2onto loo-mm culture dishes, and allowed to grow to near confluence. The cells were incubated with or without isoproterenol (10 PM), 8-BrcAMP (1 mM), phorbol 12-myristate 13.acetate (PMA, 50 nM), A23187 (1 PM), or ionomycin (1 MM) (Sigma, St. Louis, MO) in McCoy’s medium with only 2% FBS unlessotherwise indicated. To test the effect of protein kinaseC (PKC) or PKA inhibitors H-7, H-8, staurosporine(SS), a nonspecificinhibitor of PKC (29), or 1,2-bis(Z-aminophenoxy)ethane-N,N,N’,N’tetraacetic acid (BAPTA), a Ca2+chelator, cells were first incubated in the presenceof the inhibitor for 30 min, then the appropriate agent wasadded and incubations continued for another 60 min, or cellswere only incubated in the presenceof the inhibitors. At the end of eachexperimental condition, cellswere stored at -70°C until usedfor RNA isolation. RNA isolation and Northern blot analysis. Cellswere scraped and homogenizedas describedpreviously (34). Total RNA was isolatedby the guanidinium-cesiumchloride method (7). Equal amountsof RNA were electrophoresedin 2.2 M formaldehyde,
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PROTOONCOGENE
EXPRESSION
1.2% agarose gels. The RNA was visualized by ethidium bromide staining and transferred to nitrocellulose membranes. The cDNA probes used for hybridizations were a c-fos cDNA of 2.2 kb (11) (kindly provided by Dr. T. Curran), a 0.4-kb EcoR I-Sac I c-jun fragment, a 0.5kb EcoR I-Sac Ijun B fragment (25), and an 18s genomic DNA fragment. These probes were labeled by the random primer procedure described by Feinberg and Vogelstein (14), using [s2P]dCTP (ICN Biochemicals, Costa Mesa, CA). Blots were subjected to autoradiography at -70°C for 5 days, or for 2 h for the 185 probe, using Kodak AR-5 X-ray film (Kodak, Rochester, NY). RESULTS
AND
G935
IN A5 CELLS
A
B
DISCUSSION
We have hypothesized that stimulation of different signaling pathways may result in distinct patterns of c-fos, c-jun, and jun B gene expression. To test this hypothesis, we have investigated the effect of three main intracellular signals, cAMP/PKA, PKC, and cytosolic Ca2+, on c-fos, c-jun, and jun B gene expression. Effect of isoproterenol and CAMP on c-fos, c-jun, and jun B gene expression. A5 cells were incubated in the
presence of 10 PM isoproterenol or 1 mM 8-BrcAMP for various times (30 and 60 min and 2, 4, 8, and 24 h), and the pattern of c-fos, c-jun, and jun B expression was followed. As we have reported previously, the induction of c-fos and jun B by isoproterenol or 8-BrcAMP is high at 30 and 60 min and returns to basal levels after 4 h (31, 32). Unlike c-fos and jun B, the expression of c-jun is not activated by either isoproterenol or 8-BrcAMP at any time point examined. The differences in expression between the three genes at 1 h are demonstrated in Fig. 1. These results are conistent with a transcriptional mechanism for c-fos and jun B regulation that is modulated by CAMP, as we have shown recently (33); however, c-jun does not appear to be similarly regulated. A CAMP response element (TGACGTCA) (CRE) consensus sequence (22) has been identified in the promoter of the c-fos gene (4), and therefore c-fos expression could potentially be directly regulated by CAMP. Our data demonstrate a role for CAMP in the regulation of c-fos expression in A5 cells. Although it is not known whether the promoter of jun B contains CRE matching sequence, it has been shown that increasing levels of intracellular CAMP in BALB/c-3T3 cells induces jun B expression (18). In the same cells, stimulation of c-jun by 12-0tetradecanoylphorbol 13-myristate (TPA) is totally inhibited in the presence of CAMP. We have also shown (21) that in rat parotid salivary glands, stimulation by isoproterenol activates only the expression of c-fos and jun B but not of that of c-jun. Additionally, it has been shown that jun B competes with c-jun for the AP-1 site within the c-jun promoter, resulting in the repression of c-jun (9, 26). Presently, we have no evidence to suggest that jun B may act as a negative regulator of c-jun in salivary cells. The data in Fig. 1B support the interpretation that induction of c-fos and jun B by isoproterenol or CAMP is mediated by PKA. Preincubations of A5 cells with H-8 (100 PM), H-7 (100 PM), or SS (1 PM) abolish the effect of isoproterenol or CAMP on c-fos and jun B expression (Fig. 1B). Therefore, it appears that activation of PKA leads to different responses of three related genes, which are members of the AP-1 family and basically contain similar DNA binding domains. Differences in the expres-
c-jun -
c-fos -
jun B-
18S-
Ij
Fig. 1. Northern blot analysis of c-jun, c-fos, andiun B in A5 cells after @-adrenergic stimulation, addition of 8-BrcAMP, or addition of different protein kinase inhibitors. A: total RNA was isolated from cells treated with either isoproterenol or 8-BrcAMP. B: total RNA was isolated from cells preincubated with 100 staurosporin (SS) for 30 min, after which added for 60 min. Blots were successively jun B cDNA 32P-labeled probes or with observations of 4 experiments.
1M of H-7 or H-8 or 1 ,.rM time isoproterenol(1 FM) was hybridized with c-jun, c-fos, or the 18s probe. Data represent
sion of c-fos, c-jun, and jun B have also been observed in PC-12 cells after KCl-induced membrane depolarization or treatment with the neurotransmitter analogue nicotine (3). Effect of PKC on c-fos, c-jun, and jun B gene expression.
We have next examined the effect of PMA, a direct activator of PKC, on c-fos, c-jun, and jun B expression. A5 cells were incubated in the presence or absence of this agent for various time intervals starting at 1, 4, 8, and 24 h. The autoradiograms of the Northern blot analysis are shown in Fig. 2, A and B. The expression of all three genes is induced by PMA (50 nM), but with different kinetics. c-Fos and jun B mRNA levels are highly induced after 1 h of incubation with PMA and return to basal levels by 4 h (Fig. 2A). The levels of c-jun mRNA are high at 1 h, and even at 8 h after stimulation, and return to the basal levels after 24 h. A PMA analogue 4cr-phorbol12,13-didecanoate (PDD), which does not activate PKC, had no effect on the expression of c-fos, c-jun, and jun B. The temporal differences in the expression of c-jun may reflect differences in the activation of the c-jun promoter. It has been shown that induction of c-jun transcription in
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G936
PROTOONCOGENE
EXPRESSION
A PMA
1 hr
r-----7nnn -+-+
c-jun
pn
4 hr
8 hr -
24
+-
hr
+
-
B-
18S-
IN A5 CELLS
physiological levels of extracellular Ca2+. The increase in cytosolic Ca2+ is a result of both intracellular Ca2+ release and Ca2+ entry (data not shown). As shown in Fig. 3, both ionophores induce the expression of c-fos in these cells after 1 h, but have no effect on c-jun or jun B expression. The levels of c-fos mRNA return to basal levels after 4 h. The pathway by which increases in intracellular Ca2+ regulate c-fos expression has been studied in PC-12 cells. It has been shown in these cells that a specific DNA sequence is important for the induction of c-fos by A23187 (28). Surprisingly, in our study c-jun and jun B mRNA levels are not changed by either A23187 or ionomycin (Fig. 3). Recently, Trejo and Brown (31) have shown that increase in cytoplasmic Ca2+ is a negative modulator of c-jun expression in 1321Nl cells. They have suggested that the repression of c-jun expression probably occurs though a pathway affecting c-jun mRNA degradation. To determine whether the induction of c-fos was mediated by increases in cytosolic Ca2+, A5 cells were buffered intracellularly with the Ca2+ chelator BAPTA (50 PM). In BAPTA-loaded A5 cells, ionomycin no longer induced the levels of c-fos mRNA. These data clearly
A
B
Fig. 2. Effects of phorbol 12-myristate 13-acetate (PMA) and protein kinase inhibitors on expression of c-jun, c-fos, and jun B. A: total RNA was isolated from A5 cells treated with 50 PM PMA for various times indicated. B: total RNA was isolated from cells treated with H-7 (100 FM) or staurosporin (1 PM) for 30 min before addition or no addition of 50 &M PMA for 1 h. RNA was analyzed by hybridization to c-j,,, c-fos, jun B, and 18s probes. Data in this and next 2 figures represent observations of 3 different experiments. X-ray films in B were overexposed to demonstrate mRNA levels in control.
response to TPA is mediated by binding of jun/AP-1 to an AP-1 binding site in the jun-promoter region (2). This direct stimulation of c-jun by its own gene product is likely to be responsible for prolonging the transient expression of c-jun in A5 cells, as has been suggested for HeLa TK- cells (1). PMA has been shown to activate the expression of c-jun, c-fos, and jun B in other cells, such as 1321Nl astrocytoma cells, BALB/c-3T3 cells, and human U-937 leukemia cells, by a PKC-mediated mechanism (17, 19, 31). We have, therefore, pretreated A5 cells with H-7 or SS, two relatively nonspecific protein kinase inhibitors when used at high concentrations. Both inhibitors blocked the effect of PMA on the expression of c-fos, c-jun, and jun B (Fig. 2B). They also decreased the basal mRNA levels of all three genes (compare lanes Con, +H-7, +SS in Fig, 2B). It should be noted however that in Fig. lB, control levels of c-fos and jun B mRNA are not clearly visualized because of lower exposure times of the X-ray film. Role of Ca2+ on expression of c-fos, c-jun, and jun B. To examine the effect of cytosolic Ca2+ on the expression of c-fos, c-jun, and jun B genes, A5 cells were incubated in the presence of the Ca2+ ionophores A23187 (1 pm) or ionomycin (1 PM). In fura-2-loaded A5 cells, we have observed that these ionophores induce immediate sustained elevations in cytosolic Ca2+ in the presence of
c-jun -
c-fos -
jun B-
18S-
18S-
Fig. 3. Role of cytosolic Ca2+ on expression of c-jun, jun B, and c-fos. A: total RNA was isolated from A5 cells incubated with A23187 (1 PM) or ionomycin (1 PM) for 1 h. B: total RNA was isolated from cells first loaded with BAPTA for 30 min and then treated with ionomycin (1 PM) for 1 h. RNA was analyzed by hybridization to c-jun, c-fos, jun B, and 18s probes.
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PROTOONCOGENE
EXPRESSION
suggest that in A5 cells, elevation in cytosolic Ca2+ is also a signal for c-fos expression. Interestingly, the effect of the Ca2+ ionophores on the expression of c-fos is influenced by the composition of the incubation medium. As shown in Fig. 4, elimination of serum in McCoy’s 5-A medium blocks the effect of ionomycin on c-fos induction. The removal of serum from the medium has little effect on the increase of c-fos mRNA levels by isoproterenol or PMA. Furthermore, there is no time dependency on the serum requirement on c-fos induction (Fig. 4, bottom panel). These results clearly indicate that the induction of c-fos by increases in cytosolic Ca 2+ depends on the presence of serum in the medium. What these findings further suggest is that a factor(s) in the serum likely acts synergistically with ionomycin to induce c-fos gene expression. Although we provide no evidence, we can suggest that activation of c-fos by Ca2+ is probably mediated by a serum response ele-
Serum
CON r---lnnr----l +-+-+-+-
IS0
‘ON
’ 1 hr.
Ion0
PMA
18S-
Ion0
Serum
4 hrs.
8 hrs. ’
-l-----Ill+-+-+-+-
G937
IN A5 CELLS
ment (SRE). Indeed, it has been shown that c-fos SRE can mediate transcriptional activation in response to thapsigargin, an agent that increases cytosolic Ca2+ without hydrolysis of inositol phospholipids and has no effect on protein kinase activity (27). Importantly, elimination of serum from the media did not alter the results of isoproterenol or PMA on jun B expression, or the effect of PMA on the expression of c-jun (data not shown). Reported evidence supports our findings that early response genes can be regulated differentially by different stimuli in the same cell (3, 18, 31), indicating that the biochemical pathways by which these genes are activated are distinct. Indeed, activation of c-fos, c-jun, and jun B can be mediated through the SRE or the phorbol ester response element (1, 25, 30). Alternatively, c-fos can be activated by an element located -60 bp upstream of the c-fos mRNA initiation site that has sequence homologies with CRE (4), and by DNA specific sequence important for the induction of this gene by A23187 (28). Taken together, our results indicate that distinct combinations of the early response genes can be activated in response to three different intracellular signals, CAMP/ PKA, PKC, and Ca2+, in A5 cells. A summary of the results is shown in Table 1. Because c-fos, c-jun, and jun B have been identified as transcription regulators, it is possible to imagine that distinct heterodimeric fos-jun, fos-jun B, jun-jun B, or homodimeric jurz-jun complexes among them will be required to link extracellular signals with events in the nucleus. The observed dissociation of c-fos, c-jun, and jun B gene expression is, therefore, important because it may result in different dimerization forms, and by doing so will determine the transcriptional activation of the target genes. For example, fos-jun heterodimers bind to DNA more tightly than do jun-jun homodimers (22). c-dun and jun B are also different in their biological properties regardless of their sequence homologies. The c-jun homodimers are more efficient transcription activators for promoters containing a single AP-1 site than those formed with jun B. Thus differential regulation of this group of early response genes may play an important role in the long-term responses of A5 cells to extracellular stimuli. A5 cells are rapidly proliferating cells. Their doubling time Is 18 h (Yeh, unpublished observations). Recently, we were able to demonstrate that c-fos and jun B expression may be important in the early phase of the A5 cell cycle (33). Therefore, it is possible that distinct combinations of c-fos, c-jun, and jun B are required to control the proliferative activity of A5 cells. Although it is clear Table 1. Differential expression of c-fos, c-jun, and jun B by various intracellular signals in A5 cells Intracellular Signals
Agents
18S\ . Fig. 4. Effect of serum in isoproterenol-, PMA-, or Ca”+-activated c-fos expression. Total RNA was isolated from A5 cells incubated in McCoy’s medium in the presence or absence of 2% serum with or without isoproterenol, ionomycin, or PMA for 1 h. Cells were also incubated for 1, 4, and 8 h with or without ionomycin in the presence or absence of 2% serum. The same blot was hybridized first with c-fos cDNA probe, stripped, and then rehybridized with 18s probe.
Isoproterenol 8-BrcAMP PMA PDD Ionomycin Ionomycin + serum A23187 + serum
c-fos
Protein
kinase
A
Protein
kinase
C
[Caz+li [Ca*+]i ICa2+lz
increase increase? increase?
8-BrcAMP, 8-bromoadenosine phorbol 12-myristate 13-acetate;
+ + + + +
c-~un
jun B
+ -
+ + + -
3’,5’-cyclic monophosphate; PMA, PDD, 4oc-phorbol 12,13-didecanoate.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
G938
PROTOONCOGENE
EXPRESSION
from our data that agents such as CAMP, PMA, and Ca2+ differentially regulate the expression of the early response genes, their participation in the growth of A5 cells will have to be confirmed. We thank Dr. R. Bravo for providing the c-jun and jun B cDNA and Dr. T. Curran for providing the c-fos cDNA. We are particularly thankful to Dr. B. J. Baum for comments and encouragement during this study. Present address of C.-K. Yeh: Audie L. Murphy Memorial Veterans Hospital, 7400 Merton Minter Blvd., San Antonio, TX 78284. Address for reprint requests: E. Kousvelari, Clinical Investigations and Patient Care Branch, Bldg. 10, Rm. lA-19, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892. Received 26 February 1992; accepted in final form 30 July 1992. REFERENCES 1. Angel, P., K. Hattori, T. Smeal, and M. Karin. The jun proto-oncogene is positively autoregulated by its product, jun/ AP-1. Cell 55: 875-885, 1988. 2. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. 3. Imbra, H. J. Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. Phorbol ester inducible genes contain common cis element recognized by a TPA-modulated trans-acting factor. Cell 49: 729-739, 1987. 3. Bartel, D. P., M. Sheng, -L. F. Lau, and M. E. Greenberg. Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and .jun induction. Genes Dev. 3: 304-313, 1989. 4. Berkowitz, L. A., N. T. Riabowol, and M. 2. Gilman. Multiple sequence elements of a single function class are required for cyclic AMP responsiveness of the mouse promoter. Mol. Cell. Biol. 9: 4272-4281, 1989. 5. Brown, A. M., and B. J. Baum. System A amino acid transport in rat submandibular ductal cell line. Comp. Biochem. Physiol. A Comp. Physiol. 93: 383-389, 1988. 6. Brown, A. M., E. J. Rusnock, J. 3. Sciubba, and B. J. Baum. Establishment and characterization of an epithelial cell line from rat submandibular gland. Oral Pathol. Med. 18: 206-213, 1989. 7. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979. 8. Chiu, R., P. Angel, and M. Karin. Jun B differs in its biological properties from, and is a negative regulator of, c-jun. Cell 59: 979-986, 1989. 9. Chiu, R., W. J. Boyle, 3. Meek, T. Smeal, T. Hunter, and M. Karin. The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54: 541-552, 1988. edited by E. P. 10. Curran, T. Fos. In: The Oncogene Handbook, Reddy, A. M. Skalka, and T. Curran. Amserdam: Elsevier, 1988, p. 307-325. 11. Curran, T., M. B. Gordon, K. L. Rubino, and L. C. Sambucetti. Isolation and characterization of the c-fos (rat) cDNA and analysis of post-translational modification in vitro. Oncogene
2: 79-84,
16. Grausz, 17.
18.
19. 20.
21.
22. 23. 24. 25. 26. 27.
28.
29.
30. 31.
1987.
12. Curran, T., and P. K. Vogt. Dangerous liasons: fos and jun oncogenic transcription factors. In: Transcription Regulation, edited by K. Yamamoto and S. McKnight. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. In press. 13. Diamond, M. I., J. N. Miner, S. K. Yoshinaga, and Y. R. Yamamoto. c-&n and c-fos levels specify positive or negative glucocorticoid regulation from a composite CRE. Science Wash. DC 249: 1266-1272, 1990. 14. Feinberg, A. M., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983. 15. Gentz, R., F. Rauscher III, C. Abate, and T. Curran. Parallel association of fos and jun leucine zippers juxtapose DNA binding domains. Science Wash. DC 243: 1695-1699, 1989.
IN A5 CELLS
32. 33.
34. 35.
J. D., D. Fradelizi, R. Monier, and P. Lehn. Modulation of c-fos and c-myc mRNA levels in normal human lymphocytes by calcium ionophore A23187 and phorbol ester. Eur. J. Immunol. 16: 1217-1221, 1986. Haas, R., H.-J. Pfannkuche, S. Kharbanda, H. Gunji, G. Meyer, A. Hartman, H. Hidaka, K. Resch, D. Kufe, and M. Goppelt-Strube. Protein kinase C activation and protooncogene expression in differentiation/retrodifferentiation of human U-937 leukemia cells. Cell Growth Differ. 2: 541-548, 1991. He, X., J. Ship, X. Wu, A. M. Brown, and R. B. Wellner. ,&Adrenergic control of cell volume and choride transport in an established rat submandibular cell line. J. Cell. Physiol. 138: 527535, 1989. He, X., X. Wu, A. M. Brown, R. B. Wellner, and B. J. Baum. Characteristics of cr,-adrenergic receptors in a rat salivary cell line, RSMT-A5. Gen. Pharmacol. 20: 175-181, 1989. Metcha, F., J. Piette, S.-I. Hirai, and M. Yavin. Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes. New Biol. 1: 297-304, 1989. Mertz, P. M., P. C. Fox, A. Pluta, B. J. Baum, and E. E. Kousvelari. Effects of ionizing radiation and P-adrenergic stimulation on the expression of early response genes in rat parotid glands. Radiat. Res. 130: 104-112, 1992. Montminy, M. R., and L. M. Bilezikjian. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature Lond. 328: 175-178, 1987. Nakaabeppu, Y., K. Ryder, and D. Nathans. DNA binding activities of three murine jun proteins: stimulation by fos. Cell 55: 907-915, 1988. Ryseck, R.-P., and R. Bravo. c-Jun, jun B, and jun D differ in their binding affinities to AP-1 and CRE consensus sequences: effect of fos proteins. Oncogene 6: 533-542, 1991. Ryseck, R.-P., M. Hirai, M. Yaniv, and R. Bravo. Transcriptional activation of c-jun during Go/G1 transition in mouse fibroblasts. Nature Lond. 334: 535-537, 1988. Schutte, J., J. Viallet, M. Nau, S. Segal, J. Fedorko, and J. Mina. Jun B inhibits and c-fos stimulates the transforming and transactivating activities of c-jun. Cell 59: 987-997, 1989. Schonthal, A., J. Sgarman, J. H. Brown, M. R. Hanley, and J. R. Feramisco. Regulation of c-fos and c-jun protooncogene expression by the Ca 2+-ATPase inhibitor thapsigargin. Proc. Natl. Acad. Sci. USA 88: 7096-7100, 1991. Sheng, M., S. T. Dougan, G. McFadden, and M. E. Greenberg. Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences. 1Mol. Cell. Biol. 8: 2787-2796, 1988. Tamaoki, T., H. Nomoto, I. Takahashi, H. Kato, M. Morimoto, and F. Tomita. Staurosporine, a potent inhibitor of phospholipid/Ca 2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135: 397-402, 1986. Treisman, R. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5’ element and c-fos 3’ sequences. Cell 42: 889-920, 1985. Trejo, J.-A., and J. H. Brown. ~4’0s and c-jun are induced by muscarinic receptor activation of protein kinase C but are differentially regulated by intracellular calcium. J. Biol. Chem. 266: 7876-7882, 1991. Turner, R., and R. Tjian. Leucine repeats and an adjacent DNA binding domain mediate the formation of the functional c-fos-c-jun heterodimers. Science Wash. DC 243: 1689-1694,1989. Yeh, C.-K., M. A. Chinchetru, and E. Kousvelari. Characteristics of c-fos and jun B gene expression in A5 cells after padrenoreceptor stimulation and during the cell cycle. Biochim. Biophys. Acta 1090: 173-180, 1991. Yeh, C.-K., J. M. Louis, and E. Kousvelari. /3-Adrenergic regulation of c-fos gene expression in an epithelial cell line. FEBS Lett. 240: 118-122, 1988. Zerial, M., L. Toschi, R. Ryseck, M. Schuermann, R. Muller, and R. Bravo. The product of a novel growth factor activated gene, fos B, interacts with jun proteins, enhancing their DNA binding activity. EMBO J. 8: 805-815, 1989.
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