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Regulation of proto-oncogenes in rat parotid acinar cells in vitro after stimulation of β- adrenergic receptors
E.xperimentul
Cell Research
I70 (1988) 194-203
Regulation of Proto-oncogenes in Rat Parotid Acinar Cells in Vitro after Stimulation of P-Adrenergic Receptors ELENI
KOUSVELARI,*,’
JOHN M. LOUIS,+ and TOM CURRAN*
LAN-HSIANG
HUANG,*
*Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Mavland 20892, TDivision of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and *Department of Molecular Oncology. Roche Institute of Molecular Biology. Roche Research Center, Nutley, New Jersey 07110
Stimulation of /?-adrenoreceptors in rat parotid acinar cells in vitro by the /I-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by CAMP. Increased levels of ~53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-a61 was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar ceils we observed no correlation between DNA synthesis and cfos induction. 0 1988 Academic Press, ITIC.
Cellular events in rat parotid acinar cells are regulated by a varitey of cellsurface receptors. Specific stimulation of /3-adrenergic receptors in vitro increases levels of CAMP, protein production, phosphorylation, and secretion [l, 21. Recently we have also shown that B-adrenoreceptor stimulation of rat parotid acinar cells in vitro increases protein N-glycosylation by influencing specific enzymes in the N-linked oligosaccharide pathway [3,4]. During the course of our studies we observed increased synthesis of a spectrum of important parotid tissue-specific proteins rich in proline, the “so called” proline-rich proteins (PRP). All these latter processes, at least in uitro, are regulated by CAMPmediated mechanisms [5, 61. On the other hand chronic treatment of rats in uiuo with the /3-adrenergic agonist isoproterenol has been shown to cause a profound hyperplastic and hypertrophic enlargement of the parotid gland [7]. In addition to these morphological changes, isoproterenol treatment also causes extremely high induction of transcription and synthesis of the PRP [8-l 11. The proto-oncogene c-j&s, a cellular homolog of the FBJ murine osteosarcoma virus [12], is induced by a variety of stimuli including growth factors, agents that induce differentiation, and factors that interact with voltage-dependent calcium channels [ 13-171. In some cells CAMP was shown to be the second message for ’ To whom correspondence
should be addressed.
Copyright @ 1988 by Academic Press. Lnc. AU rights of reproduction in any form reserved 0014-4827/88 $03.00
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the induction of c-fos transcription [I& 191. These findings suggest the possibility that c-fos can be induced by membrane-originating stimuli such as receptor-ligand interactions. c-fos encodes a nuclear protein that exhibits DNA-binding activity in vitro and has been proposed to act as nuclear intermediary in the transduction and translation of cell-surface signals at the level of gene expression [2&22]. At present there is no information about oncogene expression in the highly regulated exocrine rat parotid acinar cell. Accordingly we have chosen to investigate the expression of the c-fos proto-oncogene in rat parotid cells in vitro after /Iadrenergic stimulation. In addition the expression of c-myc, ~53, c-abl, and c-sis proto-oncogenes, which have been shown to change during cell proliferation and differentiation [23-261, has also been studied in these cells.
MATERIALS
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METHODS
Animals and tissue preparurions. Male Wistar rats 3 months old were purchased from Harlan Sprague-Dawley. All animals were allowed water and NIH-Purina laboratory chow ad libitum. Rats were sacrificed, and parotid glands were removed and trimmed of fat and connective tissue. Enzymatically dispersed cell aggregates were prepared essentially as described earlier [2, 31. Following enzyme digestion, cell aggregates were washed three times with Ham’s F-12 medium containing 0.022% bovine serum albumin. Thereafter the cells were resuspended in fresh Ham’s F-12 medium and incubated in the presence or absence of the ,!I-adrenoreceptor agonist isoproterenol (10m5 M, Sigma) and/or [methyl-‘Hlthymidine (New England Nuclear) for various time intervals up to 4 h. For /I-adrenergic blocking experiments cell aggregates were preincubated (15 min) in the presence of 5~ 10e5 M propranolol (Sigma) prior to adding the agonist and/or [merhyL3H]thymidine and incubations were continued for another hour. When the effect of CAMP was to be studied, cells were exposed to 1 mM I-bromo-cyclic AMP (8-Br-CAMP, Sigma) for 60 min. At the appropriate time points, cells were separated from the media by centrifugation (15 s at 4Og), washed, frozen in liquid nitrogen, and stored at -70°C until use for RNA extraction. Incorporation of [methyl-‘Hlthymidine in cellular homogenates was detected by liquid-scintillation spectrophotometry and the DNA content was determined by the diphenylamine method [27]. In uivo treatmenr of ruts. The animals were anesthetized with 5 mg of sodium phenobarbital per kilogram body weight and a mini osmotic pump (Model 2002, Alza) was inserted subcutaneously in the back of the animal as per the manufacturer’s instructions. The minipumps delivered 1 mg/day of the /l-adrenergic agent isoproterenol over an I-day period. Analysis of steady-stare leuels of mRNA. Total cellular RNA was isolated from rat parotid acinar cells after incubation in either the presence or absence of isoproterenol by the guanidinium/hot phenol method [28] or by the method described in [29] with minor modifications. Equal amounts of RNA were used to select for poly(A)’ RNA using the method as described [30]. Aliquots of 8 ug of poly(A)+ RNA were denatured and separated by electrophoresis on 1.1% agarose12.2 M formaldehyde gels [28]. The mRNA was transferred and immobilized to GeneScreen (Dupont/New England Nuclear) membranes, as fust described [31], and hybridized with nick-translated probes [32]. Hybridization with the specific nick-translated probes was carried out at 42°C for 24-l8 h in 50% formamide, 0.02% polyvinylpyrolidone, 0.02 % bovine serum albumin, 0.02% Ficoll, 5x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% SDS, and 100 &ml denatured salmon sperm DNA. Filters were washed and exposed for autoradiography. Sizes (kb) of mRNA were estimated by ,UHindIII DNA or by the 28 and 18 S ribosomal RNA markers. The DNA probes employed here were as follows: a fulllength rat-specific c-fos cDNA, [44] a full-length mouse ~53 cDNA (pSV53-c clone) [33], human c-myc genomic full-length clone, v-sis and v-abl were obtained from Oncor, Inc. (Gaithersburg, MD). Immunocytochemistry. Rat parotid acinar cells from 60- and 120-min incubations either with or without isoproterenol were fixed in 4% paraformaldehyde overnight. Next, the cells were immersed in 0.1 M sodium phosphate containing 4% sucrose for 2 days and cryoprotected with 30% sucrose. The cells were mounted, quick-frozen on dry ice, and sectioned on a cryostat by standard techniques. The buffer used for immunocytochemistry consisted of 75 mM sodium phosphate, pH 7.2, and 75 mM sodium chloride. The buffer used to dilute the antibodies and/or block the sections contained 0.2%
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Fig. I. Effect of isoproterenol on c-fos expression. (A) Parotid acinar cells were incubated (in vitro) in the presence or absence of lo-’ M isoproterenol (ISO) for times indicated in the figure. Eight micrograms of poly(A)+ RNA was applied per lane and analyzed by Northern blot hybridization. Lane I, poly(A)’ RNA from an untreated parotid gland (UPC?). Lane 9, cells were first preincubated (15 min) with propranolol(5~ IO-’ M) (PROP) and then incubated for 60 min in the presence of 10e5M isoproterenol. The autoradiograph was exposed overnight for lanes 1,2, IO-15 and 6 h for lanes 3-9. (II) Poly(A)+ RNA was extracted from parotid acinar cells after 60 min incubations without (lane I) and with (lane 2) 1 rnkf Sbromo-cyclic AMP.
Nonidet-P40 and 20% normal goat serum. ARinity-purified primary antibody, specific for a synthetic peptide corresponding to amino acids 127 to 152 of the c-fos sequence [20], was used at a 1: 20 dilution for 1 h. For controls, sections were incubated with normal rabbit serum. Detection of antigen-antibody complexes was accomplished with the Zymed streptavidin-biotin system (Zymed Laboratories). This system utilizes a horseradish peroxidase-streptavidin conjugate as biotinylated secondary antibody and a substrate
RESULTS Expression of c-fos mRNA at Various Time intervals after /3-Adrenergic Receptor Stimulation
The steady-state level of c-fos mRNA was examined in rat parotid acinar cells in vitro after incubation for various time periods in either the absence or presence of 10m5 M isoproterenol, and also in cells from parotid glands before and after Equal amounts (8 kg) of chronic (8 days) in vivo treatment with isoproterenol. poly(A)+ RNA from each experimental point (0 to 240 min) were analyzed by Northern blot hybridization to 32P-labeled rat-specific c-fos full-length cDNA. A representative autoradiograph of the Northern blotting analysis is shown in Fig. 1 A. In freshly excised parotid glands, c-fos transcripts were barely detectable (Fig. IA, lane I). However, at 0 min, which is after mincing and treatment of parotid cells with collagenase and hyaluronidase, the c-fos mRNA was higher in comparison with the levels seen in freshly excised parotid glands (Fig. 1A, lane 2). This result is not surprising since it has been shown, in amnion cells [32], that
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Fig. 2. Effect of isoproterenol on ~53, c-myc, c-&t, and c&s mRNA levels. Parotid acinar cells were incubated in vitro in the presence or absence of 1O-5 M isoproterenol for the times indicated in the figure. Eight micrograms of poly(A)+ RNA was applied to each lane and analyzed by Northern blot hybridization with different probes: (A) ~53, (B) c-myc, (C) c-abl, and (0) c-k. For (C) lane 9 poly(A)+ RNA was from rats treated chronically (8 days) with isoproterenol.
c-fos proto-oncogene expression is under the regulation of many external factors. The expression of c-fos decreased to the basal levels after 15 min incubation. A dramatic increase (20- to 30-fold) was observed at 60 min following incubation of parotid acinar cells with isoproterenol (Fig. 1 A, lane 8). Following this induction, steady-state levels of mRNA gradually declined and showed a second induction at 240 min (Fig. 1A, lane 15). This induction was 5- to lo-fold higher than the control 240-min sample (Fig. 1 A, lane IS). Pretreatment of rat parotid acinar cells with the fi-adrenergic antagonist propranolol (5x 10m5 M) blocked the effect of isoproterenol on c-fos induction (Fig. 1A, lane 9). c-fos mRNA was undetectable in parotid cells from chronically treated animals. Since the effect of isoproterenol on c-fos induction was inhibited by proprano101, we hypothesized that cyclic AMP might be the second message involved in cfos gene induction, The results are shown in Fig. 1B. Addition of 1 miI4 %bromoCAMP for 60 min increased c-fos mRNA levels above basal levels. However, cfos expression was not elevated to a level equivalent to that of isoproterenol. This result suggests a weaker potency of this CAMP analog in c-fos induction. by Isoproterenol on Effect of /3-Adrenergic Receptor Stimulation c-myc, ~53, c-abl, and c-sis Gene Expression To determine whether there was any degree of specificity in the induction of cfos expression after /?-adrenergic receptor stimulation in rat parotid acinar cells, we examined ~53, c-myc, c-abl, and c-sis mRNA levels following treatment of cells with isoproterenol from 0 to 240 min. ~53, which has also been implied to play a role in normal cellular processes [24, 321, showed increased levels (5- to lofold) of mRNA only after 60 min of incubation of rat parotid acinar cells with isoproterenol (Fig. 2A, lane 2). ~53 transcripts were not detected at any other time points tested. The untreated (control) 60-min cells reproducibly exhibited a
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smear in the high-molecular-weight region in the autoradiograph of the Northern blots (Fig. 2A, lane 1). The c-fos and c-myc genes have been shown to be a set of coordinately regulated genes which cooperatively contribute to the mitogenic response in lymphocytes [ 131. Moreover c-fos expression has been shown to precede that of c-myc in these cells. In our studies c-myc gene expression was induced at the same time (60 min) as that of c-fos (Fig. 2B, lanes 1 and 2). The pattern of induction of both c-abl (Fig. 2 0 and c-sis (Fig. 20) oncogenes was different from that of c-fos at all time points studied (0, 15, 30, 60, 90, 120, 240 min and 8 days). c-abl and c-sis transcripts were undetectable at all time points tested (Fig. 2C, lanes l-4 and Fig. 20, lanes I, 2) in cells from control incubations. The c-sis transcripts were present at later (120 and 240 min) but not at earlier time points (0, 15, 30, 60, 90 min) following isoproterenol treatment (Fig. 20, lanes 3,4). Low c-abl mRNA levels were detectable at 60, 90, 120, and 240 min (Fig. 2 C, lanes 5, 6, 7, 8). Surprisingly, high levels of c-abl were present in cells from parotid glands of animals chronically (8 days) treated with isoproterenol (Fig. 2 C, lane 9). The transcripts were detected as two bands of 1.5 and 1.3 kb. The 1.3-kb transcript was not present in the in vitro incubated cells. In our study we have observed the transcripts of c-abl and c-sis to be of smaller sizes. However, low molecular sizes (2.6 and 1.3) for c-sis have been reported [26] in HUT 102 cells and were attributed to differences in methods adopted for extraction. However, it is not clear at this point if the low molecular sizes observed are tissue specific. The effect of /?-adrenergic receptor stimulation on the above oncogenes (~53, c-myc, c-abl, and c-sis) was blocked by the /I-adrenergic antagonist, propranolol (data not shown). Immunological Studies of the c-fos Protein in Rat Parotid Acinar Cells To determine if the induction of c-fos mRNA is accompanied by an increase in the expression of its gene product we used rat parotid acinar cells following 60and 120-min incubations with and without isoproterenol. A comparison of immunoperoxidase staining for c-fos protein in control and isoproterenol-treated cells is shown in Fig. 4. In both control samples (60 and 120 min; Fig. 4A and B) most of the staining reaction was observed in the nucleus of cells of the striated and intercalated ducts. Weakly stained nuclei were occasionally observed in some acinar cells. The cytoplasm of these cells was not stained at all, as in the case of the 60-min sample, or exhibited a faint, diffuse staining which is likely to be associated with endogenous peroxidase activity known to occur in the parotid gland and is also present in the specimens treated with normal rabbit serum (Fig. 3A). A dramatic induction of c-fos protein is observed in isoproterenol-treated cells after 60 and 120 min (Fig. 4C and D). Strong and homogenous immunoperoxidase staining was associated with the nuclei of the acinar cells at 120 min, while at 60 min some acinar cell nuclei had a pale appearance. The cytoplasm, in cells from both time points, displayed peroxidase staining mainly localized in the basal portion of the acinar cells. The intensity of the cytoplasmic staining was slightly reduced at 120 min as compared to that of cells treated for 60 min with
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Fig. 3. Light micrographs of rat parotid acinar cells treated with normal rabbit serum. Cells after treatment with or without isoproterenol reacted with normal rabbit serum. Faint but not specific immunoperoxidase staining is present in the cytoplasm of the cells. The nuclei of ductal (arrow) or acinar cells (arrowhead) are not stained. Magnifications: (A) x350; (B) x350.
isoproterenol. It is likely that some of the cytoplasmic staining observed is due to endogenous peroxidase activity since it is also present in the tissue specimens treated only with normal rabbit serum (Fig. 329. Also, the antiserum to c-fos synthetic peptide may recognize products of antigenically related cellular genes. Isoproterenol caused very little change in the immunoperoxidase staining of parotid striated and intercalated ductal cells. DISCUSSION The results presented in this study strongly indicate that stimulation of rat parotid acinar cells at the level of /I-adrenergic receptors by isoproterenol has a profound effect on c-fos mRNA transcription and c-fos protein levels in these cells. Elevation in the steady-state levels of c-fos mRNA was dramatic at 60 min followed by a decrease in expression at 2 h with a second peak at 4 h. Induction of c-fos appears to be specific since increased levels of ~53 and c-myc were detected only at one time point (60 min), while c-abl and c-sis were induced by isoproterenol treatment in a pattern quite different from that seen with c-fos. c-abl was the only proto-oncogene in rat parotid gland which Furthermore, showed increased expression after animals were treated chronically with isoproterenol. Similar results on c-fos expression have been reported by others in NIH 3T3 cells following serum stimulation [13, 361. These workers observed a strong correlation of c-fos expression with mitogenic responses of cells. In rat parotid cells we observed no correlation between DNA synthesis and c-fos induction. Incorporation of [methyL3H]thymidine/ug DNA was studied in two separate experiments for O-240 min. The results were variable but detectable differences were not observed between treated and untreated cells (data not shown). Although the induction of c-fos is coincident with the onset of mitogenesis in many
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cell types, it has been reported that the expression of c-fos can be induced even in the absence of DNA synthesis and cell proliferation [18, 37, 38, 391. Moreover, the hypertrophy and hyperplasia of the parotid gland produced by chronic administration of isoproterenol were not correlated with an elevation of c-fos mRNA. Our results are consistent with those reported previously [38] for submandibular gland of 5- and 14-day-old and adult mice. Incorporation of [methyl3Hlthymidine was not significant but variable and was not correlated with c-fos induction. The low levels of constitutively expressed c-fos detected in parotid cells from control incubations, and in glands from untreated rats, may indicate a physiologic regulation of this gene. Interestingly, we observed slightly elevated levels of c-fos mRNA at 0 time and this increase could be attributed to a number of external stimuli [35]. For example, expression of c-fos was increased in amnion cells at early times after plating, but when cells were transferred into normal cell culture medium, c-fos expression was barely detectable. In rat parotid glands, P-adrenoreceptor stimulation elicits effects on a spectrum of processes including protein biosynthesis, phosphorylation, glycosylation, and exocytosis, all of which are mediated by cyclic AMP [l-3, 5, 6, 8, 9, 391. Our present results suggest that induction of c-fos gene expression in these cells is also mediated by cyclic AMP. It has been reported by Bravo et al. [18] that cyclic AMP induces c-fos gene transcription in macrophages, and that the cyclic AMP induction pathway may be cell type specific. The gene product, c-fos protein, has been shown by immunocytochemical techniques to occur throughout the nucleus in a variety of cells [ 13,2 1,22, 381. In our studies with control preparations, the c-fos protein was primarily localized in the nuclei of duct cells. After incubations (60 and 120 min) with isoproterenol, the levels and distribution of this protein increased dramatically. As shown in Figs. 4 C and D there was an increase in the number and intensity of stained acinar cell nuclei between 60 and 120 min. c-fos protein has also been shown by Barka et al. to be localized in the nuclei of duct cells in mouse submandibular gland and in the nuclei of acinar cells after 30 and 60 min of in vivo administration of isoproterenol 1371. Degradation of c-fos mRNA occurs very rapidly [ 13, 211. The half-life of the induced c-fos protein in NIH 3T3 cells has been shown [ 131 to be about 2 h. In rat parotid acinar cells the levels of c-fos protein appeared stable at 120 min poststimulation. The c-fos, ~53, and c-myc genes are members of the “nuclear” class of oncogenes. They all encode proteins which are located in the nucleus, phosphorylated, and metabolically unstable. All three genes are found to be induced at the same time (60 min) after /3-adrenergic stimulation. In our studies the induction of all of these genes occurs in the absence of DNA synthesis and cell proliferation. In general, our results support the notion [17,40] that these genes are involved in the intranuclear transduction of extracellular signals by participating in the regulation of other genes [40-42]. We have also shown that c-abf is the only oncogene whose expression seems to correlate with the hypertrophy and hyperplasia of the salivary gland after chronic isoproterenol treatment of the rat. Northern blot analysis showed that the size of the transcripts observed under these experimen-
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Fig. 4. Immunocytochemical localization of c-fos protein in rat parotid acinar cells. A comparison of immunoperoxidase staining of c-fos in control [A (60 min) and B (120 min)] and isoproterenoltreated cells [C (60 min) and D (120 min)] in vitro. Immunoreactivity in controls at 60 min (A) and 120 min (B) is present in the nuclei of the duct cells (arrowhead) while the nuclei of the acinar cells exhibited no (A, arrow) or sporadic (B) staining. The amount of immunoreactivity is greatly increased in the nuclei (arrow) and to some extent in the cytoplasm of the acinar cells after 60-min (C) or 120min (D) incubations with isoproterenol. Magnifications: (A) x440; (B) x300; (C and D) x350.
tal condition was of a lower molecular size than those observed with cells from in vitro experiments. This could suggest a differential splicing or translocation of the c-abl gene after in uiuo treatment of rats with isoproterenol. This gene (c-ab0 has a tyrosine kinase activity and on the basis of its remarkable conservation throughout evolution has been suggested to play an essential role in cellular metabolism 1431. The c-h oncogene, which encodes for a PDGF-like molecule, in our studies was induced at 2 and 4 h after /3-adrenergic receptor stimulation. This finding may
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indicate a coordination between c-sis induction and the second peak of induction observed for c-fos. Thus, the second peak of c-fos induction at 240 min may be in part attributed to the presence of PDGF-like factors (such as c-k) after isoproterenol treatment, The role of c-fos protein in rat parotid acinar cells is not clear. As noted c-fos protein is a nuclear protein that at least in one study has been shown to complex with trans-acting factors which act as negative regulators of a P2 gene activity in preadipocytes [39]. It is possible that c-fos protein acts with putative regulatory complexes of other parotid proteins. Such interactions by c-fos with other nuclear proteins may be important in the differentiation of the parotid gland. In this context a particularly important consideration follows the suggestion 191 that cyclic AMP may be the key second messenger modulating PRP gene expression in rat parotid acinar cells. The product of the c-fos gene, the c-fos protein. has been proposed to act as a nuclear intermediary in the transduction and translation of cell-surface signals at the level of gene expression [2&22]. Our results permit the speculation that the c-fos protein may play the key linking role as suggested [9], mediating fi-adrenoreceptor-cyclic AMP coupled events with gene expression in rat parotid acinar cells. We are grateful to Dr. Bruce J. Baum for his encouragement and helpful discussions. We also thank Dr. Connie Oliver for helping us with the immunocytochemistry and Dr. Peter T. Mora for discussions.
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38. Barka, T., Cubits, R. M., and van der Noen, H. M. (1986) Mol. Cell. Biol. 6, 2984-2989. 39. Distel, R. J., Ro, H. S., Rosen, B. S., Groves, D. L., and Spiegelman, B. M. (1987) Cell 49, 835844. 40. Morgan, J. T., and Curran, T. (1986) Nature (London) 322, 552-555. 41. Muller, R. (1986) Biochim. Biophys. Acfa 823, 207-225. 42. Jenuwein, T., and Muller, R. (1987) Cell 48, 647-657. 43. Ben-Neriah, Y., Bemards, A., Paskind, M., Daley, G. Q., and Baltimore, D. (1986) Cell 44, 577586. 44. Curran, T., Gordon, M. B., Rubino, K. L., and Sambucetti, L. C. (1987) Oncogene 2, 79-84. Received February 10, 1988 Revised version received May 2, 1988
Printed
in Sweden
Cell Research
I70 (1988) 194-203
Regulation of Proto-oncogenes in Rat Parotid Acinar Cells in Vitro after Stimulation of P-Adrenergic Receptors ELENI
KOUSVELARI,*,’
JOHN M. LOUIS,+ and TOM CURRAN*
LAN-HSIANG
HUANG,*
*Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Mavland 20892, TDivision of Cancer Biology and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and *Department of Molecular Oncology. Roche Institute of Molecular Biology. Roche Research Center, Nutley, New Jersey 07110
Stimulation of /?-adrenoreceptors in rat parotid acinar cells in vitro by the /I-adrenergic agonist isoproterenol induces steady-state levels of c-fos mRNA and c-fos protein in these cells. A dramatic increase in the steady-state levels of c-fos mRNA was observed at 60 min, followed by a decrease at 2 h with a second peak at 4 h. c-fos induction in rat parotid acinar cells in vitro seems to be mediated by CAMP. Increased levels of ~53 and c-myc mRNA were detected only at 60 min. c-abl and c-sis were also induced by isoproterenol but in a pattern different from that seen with c-fos. c-a61 was the only oncogene in rat parotid gland which showed increased expression after chronic isoproterenol treatment of rats. In rat parotid acinar ceils we observed no correlation between DNA synthesis and cfos induction. 0 1988 Academic Press, ITIC.
Cellular events in rat parotid acinar cells are regulated by a varitey of cellsurface receptors. Specific stimulation of /3-adrenergic receptors in vitro increases levels of CAMP, protein production, phosphorylation, and secretion [l, 21. Recently we have also shown that B-adrenoreceptor stimulation of rat parotid acinar cells in vitro increases protein N-glycosylation by influencing specific enzymes in the N-linked oligosaccharide pathway [3,4]. During the course of our studies we observed increased synthesis of a spectrum of important parotid tissue-specific proteins rich in proline, the “so called” proline-rich proteins (PRP). All these latter processes, at least in uitro, are regulated by CAMPmediated mechanisms [5, 61. On the other hand chronic treatment of rats in uiuo with the /3-adrenergic agonist isoproterenol has been shown to cause a profound hyperplastic and hypertrophic enlargement of the parotid gland [7]. In addition to these morphological changes, isoproterenol treatment also causes extremely high induction of transcription and synthesis of the PRP [8-l 11. The proto-oncogene c-j&s, a cellular homolog of the FBJ murine osteosarcoma virus [12], is induced by a variety of stimuli including growth factors, agents that induce differentiation, and factors that interact with voltage-dependent calcium channels [ 13-171. In some cells CAMP was shown to be the second message for ’ To whom correspondence
should be addressed.
Copyright @ 1988 by Academic Press. Lnc. AU rights of reproduction in any form reserved 0014-4827/88 $03.00
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the induction of c-fos transcription [I& 191. These findings suggest the possibility that c-fos can be induced by membrane-originating stimuli such as receptor-ligand interactions. c-fos encodes a nuclear protein that exhibits DNA-binding activity in vitro and has been proposed to act as nuclear intermediary in the transduction and translation of cell-surface signals at the level of gene expression [2&22]. At present there is no information about oncogene expression in the highly regulated exocrine rat parotid acinar cell. Accordingly we have chosen to investigate the expression of the c-fos proto-oncogene in rat parotid cells in vitro after /Iadrenergic stimulation. In addition the expression of c-myc, ~53, c-abl, and c-sis proto-oncogenes, which have been shown to change during cell proliferation and differentiation [23-261, has also been studied in these cells.
MATERIALS
AND
METHODS
Animals and tissue preparurions. Male Wistar rats 3 months old were purchased from Harlan Sprague-Dawley. All animals were allowed water and NIH-Purina laboratory chow ad libitum. Rats were sacrificed, and parotid glands were removed and trimmed of fat and connective tissue. Enzymatically dispersed cell aggregates were prepared essentially as described earlier [2, 31. Following enzyme digestion, cell aggregates were washed three times with Ham’s F-12 medium containing 0.022% bovine serum albumin. Thereafter the cells were resuspended in fresh Ham’s F-12 medium and incubated in the presence or absence of the ,!I-adrenoreceptor agonist isoproterenol (10m5 M, Sigma) and/or [methyl-‘Hlthymidine (New England Nuclear) for various time intervals up to 4 h. For /I-adrenergic blocking experiments cell aggregates were preincubated (15 min) in the presence of 5~ 10e5 M propranolol (Sigma) prior to adding the agonist and/or [merhyL3H]thymidine and incubations were continued for another hour. When the effect of CAMP was to be studied, cells were exposed to 1 mM I-bromo-cyclic AMP (8-Br-CAMP, Sigma) for 60 min. At the appropriate time points, cells were separated from the media by centrifugation (15 s at 4Og), washed, frozen in liquid nitrogen, and stored at -70°C until use for RNA extraction. Incorporation of [methyl-‘Hlthymidine in cellular homogenates was detected by liquid-scintillation spectrophotometry and the DNA content was determined by the diphenylamine method [27]. In uivo treatmenr of ruts. The animals were anesthetized with 5 mg of sodium phenobarbital per kilogram body weight and a mini osmotic pump (Model 2002, Alza) was inserted subcutaneously in the back of the animal as per the manufacturer’s instructions. The minipumps delivered 1 mg/day of the /l-adrenergic agent isoproterenol over an I-day period. Analysis of steady-stare leuels of mRNA. Total cellular RNA was isolated from rat parotid acinar cells after incubation in either the presence or absence of isoproterenol by the guanidinium/hot phenol method [28] or by the method described in [29] with minor modifications. Equal amounts of RNA were used to select for poly(A)’ RNA using the method as described [30]. Aliquots of 8 ug of poly(A)+ RNA were denatured and separated by electrophoresis on 1.1% agarose12.2 M formaldehyde gels [28]. The mRNA was transferred and immobilized to GeneScreen (Dupont/New England Nuclear) membranes, as fust described [31], and hybridized with nick-translated probes [32]. Hybridization with the specific nick-translated probes was carried out at 42°C for 24-l8 h in 50% formamide, 0.02% polyvinylpyrolidone, 0.02 % bovine serum albumin, 0.02% Ficoll, 5x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1% SDS, and 100 &ml denatured salmon sperm DNA. Filters were washed and exposed for autoradiography. Sizes (kb) of mRNA were estimated by ,UHindIII DNA or by the 28 and 18 S ribosomal RNA markers. The DNA probes employed here were as follows: a fulllength rat-specific c-fos cDNA, [44] a full-length mouse ~53 cDNA (pSV53-c clone) [33], human c-myc genomic full-length clone, v-sis and v-abl were obtained from Oncor, Inc. (Gaithersburg, MD). Immunocytochemistry. Rat parotid acinar cells from 60- and 120-min incubations either with or without isoproterenol were fixed in 4% paraformaldehyde overnight. Next, the cells were immersed in 0.1 M sodium phosphate containing 4% sucrose for 2 days and cryoprotected with 30% sucrose. The cells were mounted, quick-frozen on dry ice, and sectioned on a cryostat by standard techniques. The buffer used for immunocytochemistry consisted of 75 mM sodium phosphate, pH 7.2, and 75 mM sodium chloride. The buffer used to dilute the antibodies and/or block the sections contained 0.2%
196 Kousvelari
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101112
131415
12
A
6
+ 2.3Kb -
Treatment ,so PROP. 8.BrcAMP Time imin)UPG
~
-
+
.-.
+
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+
+ +
~
-
0
15 15 30 30
60
60
60
90 120240
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90 120 240
mal
Fig. I. Effect of isoproterenol on c-fos expression. (A) Parotid acinar cells were incubated (in vitro) in the presence or absence of lo-’ M isoproterenol (ISO) for times indicated in the figure. Eight micrograms of poly(A)+ RNA was applied per lane and analyzed by Northern blot hybridization. Lane I, poly(A)’ RNA from an untreated parotid gland (UPC?). Lane 9, cells were first preincubated (15 min) with propranolol(5~ IO-’ M) (PROP) and then incubated for 60 min in the presence of 10e5M isoproterenol. The autoradiograph was exposed overnight for lanes 1,2, IO-15 and 6 h for lanes 3-9. (II) Poly(A)+ RNA was extracted from parotid acinar cells after 60 min incubations without (lane I) and with (lane 2) 1 rnkf Sbromo-cyclic AMP.
Nonidet-P40 and 20% normal goat serum. ARinity-purified primary antibody, specific for a synthetic peptide corresponding to amino acids 127 to 152 of the c-fos sequence [20], was used at a 1: 20 dilution for 1 h. For controls, sections were incubated with normal rabbit serum. Detection of antigen-antibody complexes was accomplished with the Zymed streptavidin-biotin system (Zymed Laboratories). This system utilizes a horseradish peroxidase-streptavidin conjugate as biotinylated secondary antibody and a substrate
RESULTS Expression of c-fos mRNA at Various Time intervals after /3-Adrenergic Receptor Stimulation
The steady-state level of c-fos mRNA was examined in rat parotid acinar cells in vitro after incubation for various time periods in either the absence or presence of 10m5 M isoproterenol, and also in cells from parotid glands before and after Equal amounts (8 kg) of chronic (8 days) in vivo treatment with isoproterenol. poly(A)+ RNA from each experimental point (0 to 240 min) were analyzed by Northern blot hybridization to 32P-labeled rat-specific c-fos full-length cDNA. A representative autoradiograph of the Northern blotting analysis is shown in Fig. 1 A. In freshly excised parotid glands, c-fos transcripts were barely detectable (Fig. IA, lane I). However, at 0 min, which is after mincing and treatment of parotid cells with collagenase and hyaluronidase, the c-fos mRNA was higher in comparison with the levels seen in freshly excised parotid glands (Fig. 1A, lane 2). This result is not surprising since it has been shown, in amnion cells [32], that
/?-Adrenoreceptors 12
12
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B
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3
4
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+
C
- 24Kb + 2.2Kb
23Kb-+
Treatment IS0
-
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-
+
Timeknin)
60
60
60
60
60
90
120
240
+
+
60
90
+
+
120 240 &
+ 120
240
120 240
Fig. 2. Effect of isoproterenol on ~53, c-myc, c-&t, and c&s mRNA levels. Parotid acinar cells were incubated in vitro in the presence or absence of 1O-5 M isoproterenol for the times indicated in the figure. Eight micrograms of poly(A)+ RNA was applied to each lane and analyzed by Northern blot hybridization with different probes: (A) ~53, (B) c-myc, (C) c-abl, and (0) c-k. For (C) lane 9 poly(A)+ RNA was from rats treated chronically (8 days) with isoproterenol.
c-fos proto-oncogene expression is under the regulation of many external factors. The expression of c-fos decreased to the basal levels after 15 min incubation. A dramatic increase (20- to 30-fold) was observed at 60 min following incubation of parotid acinar cells with isoproterenol (Fig. 1 A, lane 8). Following this induction, steady-state levels of mRNA gradually declined and showed a second induction at 240 min (Fig. 1A, lane 15). This induction was 5- to lo-fold higher than the control 240-min sample (Fig. 1 A, lane IS). Pretreatment of rat parotid acinar cells with the fi-adrenergic antagonist propranolol (5x 10m5 M) blocked the effect of isoproterenol on c-fos induction (Fig. 1A, lane 9). c-fos mRNA was undetectable in parotid cells from chronically treated animals. Since the effect of isoproterenol on c-fos induction was inhibited by proprano101, we hypothesized that cyclic AMP might be the second message involved in cfos gene induction, The results are shown in Fig. 1B. Addition of 1 miI4 %bromoCAMP for 60 min increased c-fos mRNA levels above basal levels. However, cfos expression was not elevated to a level equivalent to that of isoproterenol. This result suggests a weaker potency of this CAMP analog in c-fos induction. by Isoproterenol on Effect of /3-Adrenergic Receptor Stimulation c-myc, ~53, c-abl, and c-sis Gene Expression To determine whether there was any degree of specificity in the induction of cfos expression after /?-adrenergic receptor stimulation in rat parotid acinar cells, we examined ~53, c-myc, c-abl, and c-sis mRNA levels following treatment of cells with isoproterenol from 0 to 240 min. ~53, which has also been implied to play a role in normal cellular processes [24, 321, showed increased levels (5- to lofold) of mRNA only after 60 min of incubation of rat parotid acinar cells with isoproterenol (Fig. 2A, lane 2). ~53 transcripts were not detected at any other time points tested. The untreated (control) 60-min cells reproducibly exhibited a
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smear in the high-molecular-weight region in the autoradiograph of the Northern blots (Fig. 2A, lane 1). The c-fos and c-myc genes have been shown to be a set of coordinately regulated genes which cooperatively contribute to the mitogenic response in lymphocytes [ 131. Moreover c-fos expression has been shown to precede that of c-myc in these cells. In our studies c-myc gene expression was induced at the same time (60 min) as that of c-fos (Fig. 2B, lanes 1 and 2). The pattern of induction of both c-abl (Fig. 2 0 and c-sis (Fig. 20) oncogenes was different from that of c-fos at all time points studied (0, 15, 30, 60, 90, 120, 240 min and 8 days). c-abl and c-sis transcripts were undetectable at all time points tested (Fig. 2C, lanes l-4 and Fig. 20, lanes I, 2) in cells from control incubations. The c-sis transcripts were present at later (120 and 240 min) but not at earlier time points (0, 15, 30, 60, 90 min) following isoproterenol treatment (Fig. 20, lanes 3,4). Low c-abl mRNA levels were detectable at 60, 90, 120, and 240 min (Fig. 2 C, lanes 5, 6, 7, 8). Surprisingly, high levels of c-abl were present in cells from parotid glands of animals chronically (8 days) treated with isoproterenol (Fig. 2 C, lane 9). The transcripts were detected as two bands of 1.5 and 1.3 kb. The 1.3-kb transcript was not present in the in vitro incubated cells. In our study we have observed the transcripts of c-abl and c-sis to be of smaller sizes. However, low molecular sizes (2.6 and 1.3) for c-sis have been reported [26] in HUT 102 cells and were attributed to differences in methods adopted for extraction. However, it is not clear at this point if the low molecular sizes observed are tissue specific. The effect of /?-adrenergic receptor stimulation on the above oncogenes (~53, c-myc, c-abl, and c-sis) was blocked by the /I-adrenergic antagonist, propranolol (data not shown). Immunological Studies of the c-fos Protein in Rat Parotid Acinar Cells To determine if the induction of c-fos mRNA is accompanied by an increase in the expression of its gene product we used rat parotid acinar cells following 60and 120-min incubations with and without isoproterenol. A comparison of immunoperoxidase staining for c-fos protein in control and isoproterenol-treated cells is shown in Fig. 4. In both control samples (60 and 120 min; Fig. 4A and B) most of the staining reaction was observed in the nucleus of cells of the striated and intercalated ducts. Weakly stained nuclei were occasionally observed in some acinar cells. The cytoplasm of these cells was not stained at all, as in the case of the 60-min sample, or exhibited a faint, diffuse staining which is likely to be associated with endogenous peroxidase activity known to occur in the parotid gland and is also present in the specimens treated with normal rabbit serum (Fig. 3A). A dramatic induction of c-fos protein is observed in isoproterenol-treated cells after 60 and 120 min (Fig. 4C and D). Strong and homogenous immunoperoxidase staining was associated with the nuclei of the acinar cells at 120 min, while at 60 min some acinar cell nuclei had a pale appearance. The cytoplasm, in cells from both time points, displayed peroxidase staining mainly localized in the basal portion of the acinar cells. The intensity of the cytoplasmic staining was slightly reduced at 120 min as compared to that of cells treated for 60 min with
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Fig. 3. Light micrographs of rat parotid acinar cells treated with normal rabbit serum. Cells after treatment with or without isoproterenol reacted with normal rabbit serum. Faint but not specific immunoperoxidase staining is present in the cytoplasm of the cells. The nuclei of ductal (arrow) or acinar cells (arrowhead) are not stained. Magnifications: (A) x350; (B) x350.
isoproterenol. It is likely that some of the cytoplasmic staining observed is due to endogenous peroxidase activity since it is also present in the tissue specimens treated only with normal rabbit serum (Fig. 329. Also, the antiserum to c-fos synthetic peptide may recognize products of antigenically related cellular genes. Isoproterenol caused very little change in the immunoperoxidase staining of parotid striated and intercalated ductal cells. DISCUSSION The results presented in this study strongly indicate that stimulation of rat parotid acinar cells at the level of /I-adrenergic receptors by isoproterenol has a profound effect on c-fos mRNA transcription and c-fos protein levels in these cells. Elevation in the steady-state levels of c-fos mRNA was dramatic at 60 min followed by a decrease in expression at 2 h with a second peak at 4 h. Induction of c-fos appears to be specific since increased levels of ~53 and c-myc were detected only at one time point (60 min), while c-abl and c-sis were induced by isoproterenol treatment in a pattern quite different from that seen with c-fos. c-abl was the only proto-oncogene in rat parotid gland which Furthermore, showed increased expression after animals were treated chronically with isoproterenol. Similar results on c-fos expression have been reported by others in NIH 3T3 cells following serum stimulation [13, 361. These workers observed a strong correlation of c-fos expression with mitogenic responses of cells. In rat parotid cells we observed no correlation between DNA synthesis and c-fos induction. Incorporation of [methyL3H]thymidine/ug DNA was studied in two separate experiments for O-240 min. The results were variable but detectable differences were not observed between treated and untreated cells (data not shown). Although the induction of c-fos is coincident with the onset of mitogenesis in many
200 Kousvelari
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cell types, it has been reported that the expression of c-fos can be induced even in the absence of DNA synthesis and cell proliferation [18, 37, 38, 391. Moreover, the hypertrophy and hyperplasia of the parotid gland produced by chronic administration of isoproterenol were not correlated with an elevation of c-fos mRNA. Our results are consistent with those reported previously [38] for submandibular gland of 5- and 14-day-old and adult mice. Incorporation of [methyl3Hlthymidine was not significant but variable and was not correlated with c-fos induction. The low levels of constitutively expressed c-fos detected in parotid cells from control incubations, and in glands from untreated rats, may indicate a physiologic regulation of this gene. Interestingly, we observed slightly elevated levels of c-fos mRNA at 0 time and this increase could be attributed to a number of external stimuli [35]. For example, expression of c-fos was increased in amnion cells at early times after plating, but when cells were transferred into normal cell culture medium, c-fos expression was barely detectable. In rat parotid glands, P-adrenoreceptor stimulation elicits effects on a spectrum of processes including protein biosynthesis, phosphorylation, glycosylation, and exocytosis, all of which are mediated by cyclic AMP [l-3, 5, 6, 8, 9, 391. Our present results suggest that induction of c-fos gene expression in these cells is also mediated by cyclic AMP. It has been reported by Bravo et al. [18] that cyclic AMP induces c-fos gene transcription in macrophages, and that the cyclic AMP induction pathway may be cell type specific. The gene product, c-fos protein, has been shown by immunocytochemical techniques to occur throughout the nucleus in a variety of cells [ 13,2 1,22, 381. In our studies with control preparations, the c-fos protein was primarily localized in the nuclei of duct cells. After incubations (60 and 120 min) with isoproterenol, the levels and distribution of this protein increased dramatically. As shown in Figs. 4 C and D there was an increase in the number and intensity of stained acinar cell nuclei between 60 and 120 min. c-fos protein has also been shown by Barka et al. to be localized in the nuclei of duct cells in mouse submandibular gland and in the nuclei of acinar cells after 30 and 60 min of in vivo administration of isoproterenol 1371. Degradation of c-fos mRNA occurs very rapidly [ 13, 211. The half-life of the induced c-fos protein in NIH 3T3 cells has been shown [ 131 to be about 2 h. In rat parotid acinar cells the levels of c-fos protein appeared stable at 120 min poststimulation. The c-fos, ~53, and c-myc genes are members of the “nuclear” class of oncogenes. They all encode proteins which are located in the nucleus, phosphorylated, and metabolically unstable. All three genes are found to be induced at the same time (60 min) after /3-adrenergic stimulation. In our studies the induction of all of these genes occurs in the absence of DNA synthesis and cell proliferation. In general, our results support the notion [17,40] that these genes are involved in the intranuclear transduction of extracellular signals by participating in the regulation of other genes [40-42]. We have also shown that c-abf is the only oncogene whose expression seems to correlate with the hypertrophy and hyperplasia of the salivary gland after chronic isoproterenol treatment of the rat. Northern blot analysis showed that the size of the transcripts observed under these experimen-
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Fig. 4. Immunocytochemical localization of c-fos protein in rat parotid acinar cells. A comparison of immunoperoxidase staining of c-fos in control [A (60 min) and B (120 min)] and isoproterenoltreated cells [C (60 min) and D (120 min)] in vitro. Immunoreactivity in controls at 60 min (A) and 120 min (B) is present in the nuclei of the duct cells (arrowhead) while the nuclei of the acinar cells exhibited no (A, arrow) or sporadic (B) staining. The amount of immunoreactivity is greatly increased in the nuclei (arrow) and to some extent in the cytoplasm of the acinar cells after 60-min (C) or 120min (D) incubations with isoproterenol. Magnifications: (A) x440; (B) x300; (C and D) x350.
tal condition was of a lower molecular size than those observed with cells from in vitro experiments. This could suggest a differential splicing or translocation of the c-abl gene after in uiuo treatment of rats with isoproterenol. This gene (c-ab0 has a tyrosine kinase activity and on the basis of its remarkable conservation throughout evolution has been suggested to play an essential role in cellular metabolism 1431. The c-h oncogene, which encodes for a PDGF-like molecule, in our studies was induced at 2 and 4 h after /3-adrenergic receptor stimulation. This finding may
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indicate a coordination between c-sis induction and the second peak of induction observed for c-fos. Thus, the second peak of c-fos induction at 240 min may be in part attributed to the presence of PDGF-like factors (such as c-k) after isoproterenol treatment, The role of c-fos protein in rat parotid acinar cells is not clear. As noted c-fos protein is a nuclear protein that at least in one study has been shown to complex with trans-acting factors which act as negative regulators of a P2 gene activity in preadipocytes [39]. It is possible that c-fos protein acts with putative regulatory complexes of other parotid proteins. Such interactions by c-fos with other nuclear proteins may be important in the differentiation of the parotid gland. In this context a particularly important consideration follows the suggestion 191 that cyclic AMP may be the key second messenger modulating PRP gene expression in rat parotid acinar cells. The product of the c-fos gene, the c-fos protein. has been proposed to act as a nuclear intermediary in the transduction and translation of cell-surface signals at the level of gene expression [2&22]. Our results permit the speculation that the c-fos protein may play the key linking role as suggested [9], mediating fi-adrenoreceptor-cyclic AMP coupled events with gene expression in rat parotid acinar cells. We are grateful to Dr. Bruce J. Baum for his encouragement and helpful discussions. We also thank Dr. Connie Oliver for helping us with the immunocytochemistry and Dr. Peter T. Mora for discussions.
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