by Epidermal Growth Factor and Activators of Protein Kinase C

Vol. 8. No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1988. p. 2247-2250 0270-7306/88/052247-04$02.00/0 Copyright © 1988, American Society for Microbiology

Independent Transcriptional Regulation of a Single VL30 Element by Epidermal Growth Factor and Activators of Protein Kinase C KARIN D. RODLAND, LESLIE L. MULDOON, THANH-HOAI DINH, AND BRUCE E. MAGUN* Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201 Received 18 September 1987/Accepted 3 February 1988 A single VL30 element present in the RVL-3 cell line was transcriptionaily induced by both epidermal growth factor (EGF) and the protein kinase C (pkC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and sn-1,2-dioctanoylglycerol within 5 min of stimulation. Following TPA-induced depletion of protein kinase C activity, EGF stimulation of VL30 transcription and accumulation was unaffected while TPA effects were inhibited, implying that EGF and TPA act by separable pathways.

VL30 is a proliferation-associated gene which was initially identified as a multigene family of approximately 200 homologous but not identical members in mouse (4, 15). Expression of the 30S VL30 RNA in mouse AKR-2B cells can be induced by either epidermal growth factor (EGF) or activators of protein kinase C (pkC) (11, 12, 23). However, the presence of multiple VL30 elements in AKR-2B cells raises the possibility that EGF and pkC act on distinct VL30 elements contained within the multigene family. Structurally, VL30 possesses many characteristics of a retroviral gene, including the presence of long terminal repeats and sequences homologous to known retroviral enhancers (12). VL30 elements can be packaged as pseudovirions by replication-deficient helper viruses, and this property was used to achieve the infection of Rat-I cells with single VL30 elements derived from AKR-2B cells (22, 25). A restriction endonuclease map illustrating the orientation of the integrated mouse VL30 element relative to flanking rat sequences in the cell line RVL-3 is presented in Fig. 1. In this study, we have used the cell line RVL-3, which contains a single mouse VL30 element with full responsiveness to both EGF and pkC activation (22), to study the ability of EGF and pkC activators to regulate the expression of a single VL30 element at the levels of RNA transcription and RNA accumulation. The ability of EGF to induce VL30 expression following the functional down regulation of pkC was also studied to determine whether induction by EGF required a competent pkC pathway. Temnporal characteristics of VL30 expression in RVL-3. The ability of the RVL-3 cell line to express VL30 RNA in response to EGF stimulation was monitored over a 3-h period and was compared with the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to elicit VL30 RNA accumulation over the same period. Northern analysis of cellular VL30 RNA accumulation was performed as previously described (22, 23). Significant accumulation was observed within 15 min of EGF treatment, and the high levels of VL30 RNA observed at 1 h were sustained throughout the remainder of the 3-h time course (Fig. 2). Treatment with TPA produced a similar elevation in cellular VL30 RNA levels (Fig. 2), although the response at 15 min was considerably less in TPA-stimulated RVL-3 cells than in EGF-stimulated RVL-3 cells. To determine whether the appearance of VL30 RNA induced by both EGF and pkC resulted from regulation at *

the transcriptional level, nuclear run-on analyses of VL30 transcription were conducted as described by Brown et al. (6), by using RVL-3 cells which had been stimulated with either EGF or the pkC activators TPA and sn-1,2-dioctanoylglycerol (DOG) (9, 26, 27) for varying times from 1 min to 1 h. All three agents were capable of inducing VL30 transcription very rapidly; transcripts were evident as early as 3 min after EGF stimulation and within 3 to 5 min of TPA or DOG stimulation (Fig. 3A and B). The short-lived transcriptional response to DOG may be attributed to oxidation and loss of active DOG during incubation (9, 10). Quantitation of 32P activity by liquid scintillation spectrometry demonstrated that the EGF-treated cells showed a peak in transcription at 10 min; these elevated levels of VL30 transcription were maintained for 60 min after stimulation (Fig. 4). Cytoplasmic accumulation of VL30 RNA lagged behind transcription by 15 to 30 min, but was maintained at peak levels for at least 4 h after EGF stimulation (Fig. 4). A similar lag period was observed between transcription and cytoplasmic accumulation following TPA stimulation (data not shown). Effect of pkC depletion on VL30 expression. Recent evidence suggests that the binding of EGF to cell surfaces can result in the activation of phospholipase C and in the generation of diacylglycerols which are believed to be the endogenous activators of pkC (9, 10, 21, 22). Therefore, the observed expression of VL30 RNA following EGF stimulation may reflect an EGF-mediated activation of pkC. Since prolonged exposure to TPA is known to result in the cytoplasmic translocation and subsequent degradation of pkC (13), the pkC activity of RVL-3 cells was depleted by exposing the cells to 100 ng of TPA per ml for 24 h. Both pkC activity and VL30 expression were then monitored separately. The ability of pkC to phosphorylate histone Ills in a Ca2+and phosphatidyl serine-dependent manner was monitored as previously described (20). While cytoplasmic extracts from naive, untreated RVL-3 cells displayed a substantial Ca2'- and phosphatidyl serine-dependent phosphorylation of histone Ills in vitro, Ca2+- dependent phosphorylation of histone Ills was not detectable in cytoplasmic extracts from RVL-3 cells which had been exposed to TPA for 24 h immediately prior to the preparation of the extracts (Table 1). The effect of the TPA-induced loss of pkC activity on VL30 expression in RVL-3 cells was monitored in cells which were exposed to TPA for 24 h prior to stimulation with

Corresponding author. 2247

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either EGF or TPA. In pkC-depleted RVL-3 cells, acute stimulation with fresh TPA at 100 nglml failed to produce a substantial elevation of cellular VL30 RNA levels whether monitored at 0.5, 1, or 3 h after stimulation (Fig. 5); naive RVL-3 cells displayed the normal response to TPA treatment. The EGF-mediated stimulation of cellular VL30 RNA accumulation was virtually unaffected by the TPA-induced depletion of pkC; both the time course and magnitude for the VL30 response were similar in naive and in pkC-depleted cells (Fig. 5). Comparison of VL30 transcription in pkC-depleted and in naive RVL-3 cells produced similar results. RVL-3 cells in which pkC had been depleted by a 24-h exposure to TPA showed a substantial induction of VL30 transcription in response to EGF stimulation, while VL30 transcripts were not observed following stimulation with fresh TPA for 30 min (Fig. 6). Naive RVL-3 cells which had not been previ-

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FIG. 1. Restriction endonuclease map of pRVL-3. pRVL-3 consists of a 6.25-kilobase-pair fragment produced by partial PstI digestion of the original genomic bacteriophage lambda clone containing the VL30 element from RVL-3. This fragment was inserted in the multiple cloning site of pGEM-4Z. The box represents the presumptive long terminal repeat (LTR), as determined by hybridization to long-terminal-repeat-specific fragment from pBVL-1 (11), while the hatched line represents intervening VL30 sequences, as determined by hybridization with intervening-sequence-specific probes (11). Solid lines represent Rat-1 specific sequences. Pst, PstI; H3, HindlIl; Hpa, HpaII; Xba, XbaI; Nde, NdeI; Bgl, BglI.

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FIG. 3. Transcriptional response of VL30 to EGF, TPA, and DOG. (A) Temporal response to EGF and TPA. Confluent 10-cmdiameter plates of RVL-3 cells (four plates per group) were serum deprived in DMEM for 24 h and then exposed to either EGF or TPA for 1, 3, 5, 10, 20, 30, or 60 min. Cells were harvested, and nuclear extracts were prepared as described by Brown et al. (6). The 0-min time point was obtained from cells which had been serum deprived but not exposed to either EGF or TPA. Initiated transcripts were elongated in the presence of [32P]CTP and hybridized against pRVL3 plasmid DNA which had been size fractionated by electrophoresis before capillary transfer and covalent immobilization on diazobenzyloxymethyl-paper by the method of Alwine et al. (1). Equal total activities (1 x 106 dpm) were used for each hybridization reaction. Hybridization was conducted for 4 days, and the autoradiogram was obtained after a 24-h exposure at -70°C with intensifying screens. (B) Temporal response to DOG and TPA. Confluent 10-cm-diameter plates of RVL-3 cells were serum deprived in DMEM for 24 h and then exposed to either DOG or TPA for 0, 1, 3, 5, 10, 30, or 60 min. Nuclear run-on analysis was performed as described for panel A. The autoradiogram represents a 30-h exposure at -70°C with intensifying screens.

ously exposed to TPA showed a normal induction of VL30 transcription in response to treatment with either EGF or TPA for 30 min (Fig. 6). These results indicate that pkC is not required for the EGF-mediated induction of either VL30 transcription or the cytoplasmic accumulation of VL30 RNA.

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FIG. 2. Time course of VL30 RNA accumulation in response to EGF and TPA. Confluent 10-cm-diameter plates of RVL-3 cells (five plates per group) were serum deprived in Dulbecco modified Eagle medium (DMEM) for 24 h and then stimulated with either EGF (10 ngtml) or TPA (100 ng/ml). Cells were harvested at 0.25, 0.5, 1, 2, and 3 h after stimulation, and the RNA was prepared for Northern analysis as described previously (22, 23). The 0-h time point was obtained from serum-deprived cells which had not received any additions. The vehicular control (VC) group was obtained from cells which were exposed to the TPA diluent dimethyl sulfoxide for 3 h. Total cytoplasmic RNA at 20 ,ug per lane was size fractionated by electrophoresis and transferred to uncharged nylon membranes (Micron Separations, Inc.). The hybridization probe used in this experiment was a 1.6-kilobase-pair HpaII fragment of the genomic VL30 clone obtained from RVL-3 (designated pRVL3; Fig. 1). The HpaII fragment was excised from a 1% low-melting-point agarose gel (Sea Plaque; FMC Corp.) and labeled to a specific activity of 9 x 108 dpm by random hexamer extension. Autoradiography was conducted for 6 h without intensifying screens. EGF was prepared as previously described (19, 24).

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TIME (min) FIG. 4. Comparison of the time course of VL30 transcription and VL30 RNA accumulation observed following EGF treatment. The appropriate bands representing VL30 RNA expression in response to EGF treatment were excised from the hybridization membranes illustrated in Fig. 2 and 3A. The bands were placed in 20-ml vials with 10 ml of EcoScint aqueous scintillation cocktail and quantitated by liquid scintillation spectrometry. Counting efficiency was 96 to 98%. 32P disintegrations per minute were plotted versus time for transcription (-) and accumulation (0).

VOL. 8, 1988

NOTES

TABLE 1. Cytoplasmic pkC activity in naive and TPA-down-regulated RVL-3 cells'

d

Mean 32P incorporation (dpm) ± SD

Cells and treatment

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Naive RVL-3 cellsb Minus Ca2+ and phosphatidyl serine ..................... 434 ± 56 Plus Ca2' and phosphatidyl serine ....................... 2,418 ± 400

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TPA-down-regulated RVL-3 cells' Minus Ca2+ and phosphatidyl serine ..................... 440 ± 41 Plus Ca2' and phosphatidyl serine ....................... 363 ± 52 a RVL-3 cells were serum-deprived in DMEM and then exposed to either 100 ng of TPA per ml or 0.01% dimethyl sulfoxide for 16 h. Cells were then harvested, and a cytoplasmic extract was prepared for analysis of pkC activity in vitro as described by Muldoon et al. (20). Control values for histone Ills incubated with [y-32P]ATP in the absence of cell extracts was 160 dpm, while cell extracts incubated with [y-32PJATP in the absence of exogenous histone Ills contained 294 dpm. After the in vitro phosphorylation reaction, the extracts were run on sodium dodecyl sulfate-polyacrylamide gels and autoradiographed, and the bands corresponding to histone Ills were excised and quantitated by liquid scintillation counting. bn =4. n = 3.

In mice, VL30 is a gene whose expression is induced very rapidly in response to stimulation with either EGF or activators of pkC. The rapidity of the VL30 transcriptional response demonstrated in these experiments is comparable with that observed for c-fos expression following mitogen stimulation (18), and the transcription of VL30 was sustained for comparable periods. Previous work in this laboratory has demonstrated that protein synthesis is not required for the accumulation of VL30 RNA in response to either pkC activators or EGF (23), indicating that the induction of VL30 is a primary response to both pkC activation and EGF

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FIG. 6. Transcription of VL30 RNA in pkC-depleted cells treated with either EGF or TPA. pkC activity was depleted in RVL-3 cells by exposing confluent 10-cm cultures of RVL-3 to serum-free DMEM containing TPA for 24 h prior to the addition of either EGF or TPA; naive cells were incubated for 24 h in serumfree DMEM containing 0.1% dimethyl sulfoxide. At 5 min after the addition of either EGF or fresh TPA, the cells were harvested and nuclear extracts were prepared as described in the legend to Fig. 3. Control extracts (cont.) were obtained from cell cultures after the 24-h incubation in serum-free DMEM either with or without TPA (pkC-depleted and naive cells, respectively) but before the addition of EGF or fresh TPA. Nuclear run-on analysis was performed as described earlier (6) with 3 x 106 dpm per hybridization reaction. A 30-h exposure at -70°C with intensifying screens was used to produce the autoradiogram.

stimulation. Whether VL30 expression plays a role in modulating the expression of other gene products involved in the mitogenic response to EGF or phorbol esters is unknown. The observation that EGF could still induce VL30 transcription in RVL-3 cells which had experienced a loss of over 90% of their pkC activity after prolonged exposure to TPA indicates that the initial steps in the EGF-mediated induction of VL30 do not require the participation of pkC. Similarly distinct pkC-dependent and -independent pathways have been observed in several other systems, including EGF Treaitment the stimulation of prostaglandin production in human osteod(oN n-reagullIter( diw. n-re.u laIterl naive a~ i V sarcoma cells (17), the TPA-induced phosphorylation of threonine 654 on the EGF receptor in human fetal lung 1 3 1Vl z} s).> I) n.> l S l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ fibroblasts (8), and the TPA-induced phosphorylation of ribosomal protein S6 and the induction of c-fos and c-mnyc mRNA in human astrocytoma cells (5). In each of these systems, a separate pkC-independent pathway persists even 0 in pkC-down-regulated cells. Alternatively, the effects observed following chronic TPA exposure may still be attributed to pkC, perhaps via activation of a TPA-insensitive isoform. At least three distinct isoforms of pkC have been identified by both chromatographic and DNA sequencing methods (7, 14, 16), and it is possible that these isoforms may differ in their sensitivities to either activation or inactiFIG. 5. Accumulation of VL30 RNA in naive and pkC-downvation by phorbol esters. regulated RVL-3 cells in response to either EGF or TPA. pkC While EGF and pkC appear to function through pathways activity was depleted by incubating confluent 10-cm-diameter plates which are at least initially separate, the point at which these of RVL-3 cells in serum-free DMEM containing TPA for 24 h. Naive pathways converge to modulate expression of VL30 and cells were exposed to serum-free DMEM containing 0.1% dimethyl other genes is not known. While the VL30 element present in sulfoxide (the TPA diluent) for 24 h. Each experimental group was then treated with either EGF or TPA freshly added to the same RVL-3 appears to contain a heptameric sequence homolomedium used for conditioning. Cells were harvested at 0.5. 1. and 3 gous to the TPA-responsive element of Angel et al. (2, 3; K. h after stimulation; the 0-h time point was obtained from cells after Rodland, unpublished observations), the current experithe 24-h incubation in serum-free DMEM but before stimulation ments cannot determine whether the induction of VL30 by with either EGF or fresh TPA. RNA extraction and Northern both EGF and pkC activation is targeted on a single regulaanalysis were performed as previously described, by using the tory sequence within the VL30 element or whether the 1.6-kilobase-pair HpaIl fragment of pRLV3 labeled with 32P to a inducing agents act on multiple sequences within VL30. specific activity of 7 x 108 dpm/,ug as the hybridization probe. The These questions are being addressed by experiments curautoradiogram was produced from a 16-h exposure at -70°C with rently underway. intensifying screens. VC, Vehicular control. )

0.5

1

3

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This work was supported by Public Health Service grant CA39360 from the National Cancer Institute to B.E.M. and by a grant from the Medical Research Foundation of Oregon to K.D.R. We thank Shall Jue, Roy Garvin, and Jean Aschenbach for excellent technical assistance. LITERATURE CITED 1. Alwine, J. C., D. J. Kemp, B. A. Parker, J. Reiser, J. Renart, G. R. Stark, and G. M. Wahl. 1979. Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol. 68: 220-242. 2. Angel, P., I. Baumann, B. Stein, H. Delius, H. J. Ramsdorf, and P. Herrlich. 1987. 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol. Cell. Biol. 7:2256-2266. 3. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J. Ramsdorf, C. Jonat, P. Herrlich, and M. Karin. 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729-739. 4. Besmer, P., U. Olshevsky, D. Baltimore, D. Dolberg, and H. Fan. 1979. Virus-like 30S RNA in mouse cells. J. Virol. 29:11681176. 5. Blackshear, P. J., D. J. Stumpo, J.-K. Huang, R. A. Nemenoff, and D. H. Spach. 1987. Protein kinase C-dependent and -independent pathways of proto-oncogene induction in human astrocytoma cells. J. Biol. Chem. 262:7774-7781. 6. Brown, A. M. C., J.-M. Jeltsch, M. Roberts, and P. Chambon. 1984. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proc. Natl. Acad. Sci. USA 81:6344-6348. 7. Coussens, L., P. J. Parker, L. Rhee, T. L. Yang-Feng, E. Chen, M. D. Waterfield, U. Francke, and A. Ullrich. 1986. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signalling pathways. Science 233:859-866. 8. Davis, R. J., and M. P. Czech. 1987. Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts. J. Biol. Chem. 262:6832-6841. 9. Davis, R. J., B. R. Ganong, R. M. Bell, and M. P. Czech. 1985. sn-1,2-Dioctanoylglycerol: A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis. J. Biol. Chem. 260:1562-1566. 10. Ebeling, J. G., G. R. Vandenbark, L. J. Kuhn, B. R. Ganong, R. M. Bell, and J. E. Neidel. 1985. Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation. Proc. Natl. Acad. Sci. USA 82:815-819. 11. Foster, D. N., L. J. Schmidt, C. P. Hodgson, H. L. Moses, and M. J. Getz. 1982. Polyadenylated RNA complementary to a mouse retrovirus-like multigene family is rapidly and specifically induced by epidermal growth factor stimulation of quiescent cells. Proc. Natl. Acad. Sci. USA 79:7317-7321. 12. Hodgson, C. P., P. K. Elder, T. Ono, D. N. Foster, and M. J. Getz. 1983. Structure and expression of mouse VL30 genes.

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