The cyclin-dependent kinases are a family of serine/threonine kinases that play a pivotal role in controlling progression through the cell cycle(1, 2, 3, 4) . The regulatory subunits of the cyclin-dependent kinases, known as cyclins, form complexes with their catalytic partners to function as kinases of specific proteins at different phases of the cell cycle(3, 4) . Cyclin D1 is induced in late G following the treatment of growth-arrested macrophage cell lines with colony-stimulating factor 1 (5) . Cyclin D1 is required for progression of the G phase (6) and is, therefore, a critical target for proliferative signals in G. Cyclin D1 is capable of shortening the G phase of the cell cycle, suggesting that cyclin D1 may be rate limiting in G progression(7, 8, 9) . The induction of cyclin D1 mRNA in response to cell cycle progression and the addition of growth factor is very rapid (6, 10, 11, 12) and is likely regulated at the level of transcription(5, 13, 14, 15) .

The ras gene, which is highly conserved in evolution, plays an essential role in cellular proliferation(16) , cellular development (17) , and cellular differentiation(18, 19, 20, 21) . The normal ras gene, activated by point mutations at amino acids 12, 13, or 61, is also capable of inducing cellular proliferation, development, and differentiation(18, 19, 20, 21) . Dominant negative mutants of p21block cellular proliferation of NIH3T3 cells (22, 23) and the induction of DNA synthesis and gene expression induced by serum(22) . p21 acts at several distinct phases of the cell cycle including early G, the G/S boundary(24) , and at G/M(25) . In evidence for a role of p21 in early G, ras activity is required soon after the release of cells from quiescence(24) . After the addition of serum, the proportion of Ras-GTP increases within 5 min, and the induction of immediate-early gene expression can be at least partially inhibited by previous injection of anti-Ras antibody(26) . In addition, anti-Ras antibody or the dominant Ras inhibitor proteins (27) efficiently inhibit DNA synthesis within the recipient cell as long as the injection occurs prior to the onset of the S phase. These findings suggest that p21 is also required late in the G phase of the cell cycle, presumably at the G/S boundary.

Recent studies have suggested functional interactions between p21 and cyclin D1. Cyclin D1 collaborates with p21 in primary rat kidney (28) or rat embryo fibroblasts transformation assays(29) . In cell lines overexpressing p21, cyclin D1 mRNA levels were induced(30) . In yeast, Ras activates transcription of the CLN genes(31) , which are analogous regulators of G phase progression(32) . p21 has the capacity to phosphorylate and/or activate target transcription factors including c-Jun(33, 34, 35) . The ability of p21 to augment the transactivation by c-Jun(36, 37) , likely involves a Jun kinase pathway distinct from p42()(36, 37, 38) . c-Jun, in conjunction with several related AP-1 proteins, promotes G phase progression (39, 40) and DNA synthesis(41) . For example, inhibition of Jun expression with antisense RNA (42) or microinjection of antibodies (40, 43) inhibits cell cycle progression induced by the addition of serum to G-arrested cells. Thus, several lines of evidence suggest that p21 activates c-Jun and that members of the c-Jun/AP-1 family are involved in promoting cellular proliferation(44) .

In addition to the Jun kinase pathway, a distinct MAPK pathway is involved in the intracellular transmission of growth factor signals (34) . Protein kinases including the MAPK or extracellular signal-regulated kinases (ERKs) (45) are induced in response to EGF. Induction of the MAPK pathway is associated with activation of several different transcription factors including the ETS family proteins(17, 46, 47, 48) . The synthesis of the relatively ubiquitous c-Ets-2 is induced upon growth factor stimulation, and its phosphorylation is increased in response to mitogenic stimulation(48) .

The target DNA sequences of the ETS proteins includes a core motif with extensive variation at both the 5` and 3` sides of the invariant GGA core(48) . In a variety of promoters that lack TATA sequences, ETS binding sites have been localized close to the initiation site(48, 49) . Several mechanisms are likely to restrict or condition the activity of these sites as effective ETS-responsive elements. The binding of ETS family members to select promoter/enhancer sequences and their full transcriptional activity may require additional nuclear factors including Sp-1(50) . Sequences resembling the core motif (GGA) required for ETS protein binding (48) are located within the proximal cyclin D1 promoter.

We hypothesized that the recently identified pathways linking tyrosine kinase receptor growth factors, p21, and MAPK (18, 45) may be involved in transcriptional regulation of the cyclin D1 gene and thereby cell cycle progression. Cyclin D1 promoter fragments linked to the luciferase reporter gene were examined in the presence of activating or dominant negative, p21, MAPK, c-Jun, and c-Ets mutants, to determine their role in cyclin D1 transcription. Two distinct regions of the cyclin D1 promoter were identified as the site of activation by either p21/c-Jun, or EGF, p41/c-Ets-2. The identification of distinguishable mitogenic pathways regulating cyclin D1 transcription provides a mechanism for specificity in signal transduction cross-talk between proliferative pathways and a cell cycle regulatory pathway.

MATERIALS AND METHODS

Construction of Reporter Genes

Expression Vectors

Cell culture, DNA transfection, and luciferase assays were performed as described previously(58) . The trophoblast cell line JEG-3, the fibroblast cell line COS, and the mink lung epithelial cell line Mv1.Lu (CCl-64) were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 1% penicillin/streptomycin. Chinese hamster ovary (CHO) cells were maintained in -minimum essential medium with 10% fetal calf serum and 1% penicillin/streptomycin. Cells were transfected by calcium phosphate precipitation, the medium was changed after 6 h, and luciferase activity was determined after a further 24 h.

In cotransfection experiments, comparison was made between the effect of transfecting active expression vector with the effect of an equal amount of the parental empty expression vector. At least three different plasmid preparations of each construct were used. In cotransfection experiments, a dose response was determined in each experiment with 40, 60, 80, 100, and 200 ng of expression vector and the cyclin D1 promoter reporter plasmids (1.6 µg). The fold effect was determined for 100 ng of expression vector. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&, Berthold). Luciferase content was measured by calculating the light emitted during the initial 30 s of the reaction, and the values are expressed in arbitrary light units(58) . Background activity from cell extracts was typically < 150 arbitrary light units/30 s. Statistical analyses were performed using the Mann Whitney U-test. Significant differences were established as p < 0.05. EGF treatment was performed for 6-24 h at doses from 2 to 20 ng/ml to determine maximal responses. Subsequent experiments were conducted using EGF at 2.5 ng/ml for 24 h.

Oligodeoxyribonucleotides

EMSA

Reagents and Flow Cytometric Analyses

Flow cytometric analysis was carried out in a fluorescence-activated cell sorter (FACStar plus; Beckton Dickonson). DNA synthesis of the synchronized cells was determined by detection of 5-bromodeoxyuridine incorporation into DNA essentially as described(68) . The cells were grown in six-well culture dishes, and 5-bromodeoxyuridine was added with serum. All nuclei were counterstained with Hoechst 33258 (Sigma).

Western Blots

p42and p44Immune Complex Assays

RESULTS

p21Activates the Cyclin D1 Promoter

Figure 1: Activating p21 mutants stimulate cyclin D1 transcription. Panel A, the -1745CD1LUC reporter was transfected in conjunction with either activating (Ras Val-12 pMTEj, Ras Leu-61, Ras Val-12), dominant negative (Ras N17), or antisense (Rev3) p21 expression vectors into JEG-3 cells as described under ``Materials and Methods.'' The mutant Ras Leu-61,Ser-186, is incapable of inserting in the plasma membrane. The Ras Val-12 pMTEj or Rev3 vector was transfected into Mv.1Lu or CHO cells (panel B). Cells transfected with the p21expression vectors pMTEj or Rev3 (300 ng) and reporter plasmid (4.8 µg) were treated with Zn for 48 h. The mean data ± S.E. of either (panel A) n separate transfections indicated in figure or (panel B) n = three separate transfections are shown. Results are shown as the percent relative activity for the effect of the transfected expression vector on the reporter plasmid. Inset, Western blot analysis of whole cell extracts from JEG-3 cells either (left lane) mock transfected, or transfected with (right lane) the p21 antisense plasmid (Rev3). The blot was probed with the p21 antibody H-Ras (259) (Santa Cruz Biotechnology Inc.) as described under ``Materials and Methods.''

The effect of several transforming p21 mutants on cyclin D1 promoter activity was examined in JEG-3 cells. The magnitude of cyclin D1 reporter induction by the activating p21 mutant expression vector was dependent upon the amount of transfected expression vector and increased from 24 to 48 h. The activating p21 mutants (Ras Val-12 and Ras Leu-61) and pMTEj stimulated the cyclin D1 promoter in several different cell lines. The induction by the pMTEj Ras vector was dependent upon the addition of Zn (not shown). The constitutively active p21 mutants induced cyclin D1 reporter activity 4-5-fold in JEG-3 cells (Fig. 1A). The expression vector encoding the double mutant Ras Leu-61,Ser-186, which is incapable of insertion in the plasma membrane, did not affect cyclin D1 promoter activity (Fig. 1A). The expression vector cassettes driving Ras Val-12 and Ras Leu-61 did not affect cyclin D1 promoter activity (not shown).

The constitutively active p21 mutant induced cyclin D1 reporter activity 22-fold in Mv1.Lu cells and 5-fold in CHO cells (Fig. 1B). The induction by the pMTEj vector was dependent upon the addition of Zn (not shown). Rev3 reduced basal -1745CD1 reporter activity in CHO cells but not in Mv1.Lu cells (Fig. 1B).

To determine the region of the cyclin D1 promoter required for regulation by p21, the cyclin D1 5` promoter deletions (Fig. 2A) were transfected in the presence of either the sense or antisense p21 expression vectors (Fig. 2B). Mutation of sequences resembling an AP-1 site at -954 abolished transcriptional activation by p21 (pMTEj) (Fig. 2B) and the -964mtDCLUC reporter was repressed to less than 40% by the activating p21 mutant.

Figure 2: p21 activation of the cyclin D1 promoter requires the -954 region in JEG-3 cells. Panel A, schematic representation of the series of cyclin D1 5` promoter constructs in the vector pALUC. The area homologous to the AP-1 (striped oval), and E2F elements (black oval), and the INR (gray square) are represented schematically. Panel B, p21 expression vector (pMTEj 300 ng), was transfected with 4.8 µg of -1745CD1LUC or an equal molar amount of the other 5` promoter constructs including the wild type -964CD1 promoter or the site-directed mutant of the AP-1 site (-964 mtCD1LUC), into JEG-3 cells. The data are shown as the mean ± S.E. for n separate transfections as indicated in parentheses adjacent to the designated construct. Dose-response curves using 100-300 ng demonstrated similar trends (not shown). * represents a significant difference from the adjacent 5` deletion construct for p < 0.05.

c-Jun Activates the Cyclin D1 Promoter

Figure 3: c-Jun activation of the cyclin D1 promoter requires the -964 region in JEG-3 cells. Expression vectors encoding wild type or c-Jun mutant DNA (300 ng) were transfected with 4.8 µg of -1745CD1LUC into JEG-3 or Mv.1Lu cells. For comparison with the effect of c-Jun on -1745CDLUC, the canonical AP-1 reporter pTP-LUX was transfected into JEG-3 cells with c-Jun. Panel B, cotransfection experiments were conducted using the -1745CD1LUC reporter construct (or an equal molar amount of the other 5` promoter constructs including the wild type -964CD1 promoter or the site-directed mutant of the AP-1 site (AP-1mtCD1LUC)) with the RSV c-Jun expression vector in JEG-3 cells. The mean data ± S.E. of 7-14 separate transfections, are shown. * represents a significant difference from the adjacent 5` deletion construct for p < 0.05. Panel C, expression vectors encoding wild type or mutant c-Jun proteins (300 ng) were transfected with 4.8 µg of -1745CD1LUC into JEG-3 cells. The data are shown as the mean ± S.E. for six separate transfections. Dose-response curves using 100-300 ng demonstrated similar trends (not shown).

To determine the minimal region of the cyclin D1 promoter activated by c-Jun, the series of 5` promoter deletion constructs were transfected into JEG-3 cells with either wild type or mutant expression vectors. The region of the cyclin D1 promoter activated by c-Jun was localized to the region between -964 and -485. Site-directed mutagenesis of the AP-1 site at -954 abolished activation by c-Jun in JEG-3 cells (Fig. 3B).

The domains of c-Jun required for activation of cyclin D1 transcription were determined using a series of expression vectors encoding wild type and mutant c-Jun proteins(58, 66, 74) . Previous studies demonstrated that the DNA binding activity of these mutants, other than DNA, were similar to wild type(66) . Western blot analyses (74) demonstrated that all the mutant proteins used in these studies were expressed similarly in transfected cells. Deletion of the amino-terminal 91 amino acids, deletion of the A2 activation domain, or mutation of the leucine zipper reduced activation of the cyclin D1 promoter by c-Jun to less than 20% of wild type (Fig. 3C).

AP-1 Proteins Bind the Cyclin D1 -954 Region in Primary and Transformed Cells

Figure 4: Binding of AP-1 to the cyclin D1 -954 region characterized using EMSA. EMSA were performed with nuclear extracts from either Mv1.Lu cells (panels A and B), JEG-3 cells (panel C), or rat adrenal medulla cells (panels D and E). Panels A and B, EMSA were performed with in vitro translated c-Fos with bacterially expressed c-Jun (Promega) (lane 1) and comparison made with the mobility of the complexes binding the cyclin D1 AP-1 site or wild type (collagenase) AP-1 site in Mv1.Lu cells. The specific band binding the cyclin D1 AP-1 site or the collagenase AP-1 site probe is marked with an arrow. Panel C, the -P-labeled cyclin D1 -954 region (CD1 AP-1) probe was incubated with (lane 1) cellular nuclear extracts alone or (lane 2) with the addition of 100-fold excess of cold self competitor, (lane 3) 100-fold excess of mutant cyclin D1 AP-1 cold competitor. Comparison was made with the binding of cellular nuclear extracts with the -P-labeled wild type collagenase AP-1 site probe. The complexes binding the cyclin D1 AP-1 site and wild type AP-1 site is labeled 1. Panels D and E, nuclear extracts prepared from the adrenal medulla of rats were incubated with competitor or antibodies as indicated in the figure. The specific complex binding the -P-labeled cyclin D1 -954 region is indicated by 1. The complex shifted by the c-Fos or c-Jun antibodies is indicated by *. Details of the antibodies are outlined under ``Materials and Methods.''

To determine whether the cyclin D1 AP-1 site was capable of binding AP-1 proteins in nontransformed cells, nuclear extracts were prepared from rat tissues and the abundance of AP-1 proteins binding these sequences examined. The c-Jun antibody (Jun Ab2) inhibited binding of most of the complex binding the cyclin D1 AP-1 site, and the JunB and JunD antibodies (40, 67) induced a minor supershift of the complex binding either the cyclin D1 AP-1 site (Fig. 4D) or the wild type AP-1 site (Fig. 4E). The c-Fos antibody shifted most of the complex binding either AP-1 site (indicated by an asterisk in Fig. 4, D and E). These studies demonstrate that the cyclin D1 AP-1 site is capable of binding AP-1 proteins from cultured cells and primary tissue.

c-Fos Represses the Cyclin D1 Promoter

Figure 5: AP-1 proteins have distinct transcriptional effects on the cyclin D1 promoter. Panels A and B, cotransfection experiments were conducted using the -1745CD1LUC reporter construct with the designated AP-1 expression vectors in JEG-3 and COS cells. Data are shown as the mean ± S.E. for n separate transfections as indicated in parentheses. Dose-response curves using 100-300 ng demonstrated similar trends (not shown). * represents a significant difference from the adjacent 5` deletion construct for p < 0.05.

p41Activates Cyclin D1 Transcription through the Proximal Promoter Region

Figure 6: MAPK stimulates cyclin D1 reporter activity through the proximal promoter. Panel A, expression plasmids encoding either p41 or the dominant negative mutant p41 (400 ng) were transfected with reporter plasmid (4.8 µg) into JEG-3 (panel A) or Mv1.Lu cells (panel B). The reporter plasmids were (panel A) the -1745CD1LUC reporter, or (panel B) the cyclin D1 5` promoter deletion constructs (-1745CD1LUC, -141CD1LUC, -22CD1LUC, or -22RevCD1LUC reporter). The basal level activity of -22 CD1LUC was >1,000 relative light units/s with background 3-6 relative light units/s). The mean data ± S.E. of (panel A) JEG-3, n = 6, Mv.1Lu, n = 3 (panel B). n = 5 separate transfections are shown. Panel C, MAPK activity was measured in JEG-3 cells treated with EGF (10 ng/ml) for the time points as indicated.

Several growth factors, including EGF, stimulate MAPK activity in a cell type-specific manner. Previous studies have demonstrated that EGF induced cyclin D1 mRNA levels in fibroblast cell lines(10) . Trophoblast cell lines, such as JEG-3 cells, express EGF receptors, and EGF promotes trophoblast outgrowth and blastocoel expansion(57) . The effect of EGF on MAPK activity was therefore determined in JEG-3 cells. EGF (10 ng/ml) was used to treat JEG-3 cells, and cells were harvested at time points from 30 min to 24 h. MAPK activity was induced 6-fold within 30 min (Fig. 6C). Using fluorescence-activated cell sorter analysis EGF stimulated a 12-15% increase in the proportion of JEG-3 cells moving from G/G to S phase (not shown).

EGF Activates Transcription of the Cyclin D1 Promoter

Figure 7: EGF activates the cyclin D1 promoter. Panel A, the -1745CD1LUC reporter was transfected into JEG-3 cells, and the cells were treated with EGF (0.125-20 ng/ml) for 12 h. The data of a representative experiment determined by comparison with untreated cells are shown as fold induction. Panel B, JEG-3 cells were transfected with a variety of native or synthetic promoters in the pALUC plasmid, and EGF treatment (2.5 ng/ml) was conducted for 24 h. The data are shown for the mean ± S.E. of at least four separate transfections. Panel C, JEG-3 cells transfected with the series of cyclin D1 5` promoter constructs were treated with EGF (2.5 ng/ml). The mean data ± S.E. of six separate transfections are shown. * represent significant differences from the adjacent 5` promoter construct (p < 0.05).

The DNA sequence requirements for EGF-induced transcription of the cyclin D1 promoter were determined using a series of 5` promoter fragments. Cells were treated for 24 h with EGF (2.5 ng/ml), and the effect was compared with untreated cells. The induction by EGF (3-4-fold) was conveyed by the minimal -22-bp promoter fragment. The -22 bp in the inverted orientation was not activated by EGF (Fig. 7C).

c-Ets-2 Activation of Cyclin D1 Requires the Proximal Promoter Region

Figure 8: c-Ets-2 specifically activates the minimal cyclin D1 promoter. The -1745 CD1LUC reporter and a variety of native or synthetic promoters in the pALUC plasmid were transfected into JEG-3 cells with either c-Ets-2 or ets-LacZ expression plasmids. The data are shown for the mean ± S.E. of at least four separate transfections. Panel B, the 5` promoter deletions of the cyclin D1 promoter were transfected with the c-Ets-2 expression vector into JEG-3 cells. Expression vector (300 ng) was transfected with 4.8 µg of -1745CD1LUC (or an equal molar amount of the other 5` promoter constructs). The data are shown as the mean ± S.E. for five separate transfections. The effect of c-Ets-2 on the -1745CD1LUC reporter was also determined in (panel C) Mv.1Lu and (panel D) COS cells. The mean data ± S.E. of n = four separate transfections are shown.

The series of cyclin D1 5` promoter deletions was transfected into JEG-3 cells with c-Ets-2 to determine the minimal ETS-responsive region of the cyclin D1 promoter. The 5` promoter deletion constructs were activated 7-10-fold by c-Ets-2. The minimal c-Ets-2-responsive region was located within 22 bp of the transcriptional start site in JEG-3 cells (Fig. 7B). To determine whether regulation of the cyclin D1 promoter by c-Ets-2 was observed in other cell types, transient expression studies were performed in Mv1.Lu and COS cells. c-Ets-2 activated -1745CD1LUC 18-fold in Mv1.Lu cells and 4-fold in COS cells (Fig. 8, C and D).

As the regions activated by EGF, MAPK, and c-Ets-2 colocalized within the proximal promoter region, studies were performed with activating or dominant negative expression vectors to determine whether sequential or parallel pathways linked EGF, p41, and c-Ets-2 in the context of the cyclin D1 promoter. Overexpression of p41 induced the -141 cyclin D1 promoter fragment 3-6-fold (Fig. 9A). EGF activated -141CD1LUC 3-5-fold. MAPKi or ets-LacZ reduced the induction of the cyclin D1 promoter by EGF 60-80%, suggesting MAPK and ETS functions downstream of EGF in the context of the cyclin D1 promoter (Fig. 9, A and B). The induction of -141CDLUC by p41 was reduced or abolished by ets-LacZ in JEG-3 (Fig. 9C). Activation by c-Ets-2 was increased only 25% by the overexpression of MAPK, suggesting sequential rather than parallel pathways link MAPK and ETS signaling in JEG-3 cells (Fig. 9C).

Figure 9: EGF activation of the proximal cyclin D1 promoter is antagonized by dominant negative MAPK and ETS expression vectors. Panels A-D, the -141 cyclin D1 reporter was transfected into JEG-3 cells with expression vectors encoding proteins for MAPK (p42 or p42), p21(activating (pMTEj) or p21 antisense (Rev3)), or ETS (the activating (c-Ets-2) or dominant negative (ets-LacZ)). Expression vector (300 ng) was transfected with 4.8 µg of -141CD1LUC. In panel A the effect of MAPKi and ets-LacZ on EGF-induced -141CDLUC activity is shown as a percent of wild type. The data from a representative experiment from at least three separate transfections are shown. In panel C the induction of the -141CDLUC reporter by MAPK overexpression (3-4-fold) was normalized to 1 for the purpose of showing in the same figure the additional effect of c-Ets-2. In panel D the induction by EGF was normalized to 1 for comparison with the additional effects of c-Ets-2.

DISCUSSION

In these experiments, activating p21 mutants stimulated cyclin D1 promoter activity in a DNA sequence-dependent manner. The activation of cyclin D1 transcription by p21 is consistent with recent observations in which the abundance of cyclin D1 mRNA was increased in v-Ha-Ras-transformed pre-B cell lines(30) . p21 promotes cellular proliferation and early G phase progression(16, 23, 24, 77) . As cyclin D1 may be rate-limiting in promoting G phase progression, the induction of cyclin D1 transcription by p21 may provide, in part, a mechanism by which p21 promotes G phase progression. The observation that distinguishable regions of the cyclin D1 promoter were targeted by either p21 and c-Jun through one region and by MAPK and ETS through another are consistent with recent observations suggesting that a specific Jun kinase, distinguishable from MAPK, modulates activity of c-Jun(37) .

Several lines of evidence indicate a role for c-Jun and the AP-1 proteins in promoting G phase progression(40, 67, 78) . In these studies, c-Jun activated the cyclin D1 promoter in all cell lines examined. Mutation of the cyclin D1 AP-1 site abolished the induction by c-Jun in JEG-3 cells, consistent with a role for this site in mediating transactivation by c-Jun. The DNA binding domain of c-Jun was required for induction of the cyclin D1 promoter. In vitro translated c-Fos with c-Jun bound the cyclin D1 AP-1 site in EMSA. The cyclin D1 AP-1 site bound nuclear complexes from JEG-3 and Mv1.Lu cells, and the mobility of these complexes was similar to the complexes binding the wild type AP-1 site in EMSA. Together these findings suggest that the cyclin D1 -954 region is capable of binding AP-1 proteins and conveys c-Jun-induced transcription in the cell line examined. The signal transduction pathway modulating c-Jun-induced transcription of cyclin D1 is currently unknown; however, the recently identified Jun kinase(38) , which phosphorylates the amino terminus of c-Jun, is a likely candidate. Recent studies suggested a role for the -58 sequences (``cAMP response element'' TGAGGTAA) in c-Jun-mediated transactivation of cyclin D1 in fibroblast cells using the pOLUC vector (79) . A residual 5-fold induction by c-Jun was observed by Herber et al.(79) . after mutation of the cAMP response element site, and no deletion or mutation abolished transactivation by c-Jun. Cell type-specific differences may be responsible for the different localization of the site of c-Jun action.

AP-1/ATF Proteins Differentially Regulate the Cyclin D1 Promoter

MAPK and c-Ets-2 Activate the Cyclin D1 Promoter through the Minimal Promoter

It remains to be determined whether the effect of c-Ets-2 to activate the cyclin D1 promoter is mediated directly through binding target sequences within the minimal promoter or is mediated indirectly through interaction with the basal transcription apparatus. The ETS protein, PU.1, binds the basal level transcription factor TFIID(86) , which in turn binds either TATAA or Inr sequences(87) . Recent studies demonstrated that the cyclin D1 Inr is sufficient for negative regulation by c-Myc(88) , and that the Inr may also function to convey regulation by several other factors including ETS.

EGF Activation of the Cyclin D1 Promoter Required the Proximal 22 bp

()

These studies demonstrate that EGF, activating p21 mutants, p42, c-Jun, and c-Ets-2 augment cyclin D1 promoter activity. These findings suggest that the cyclin D1 promoter may be an important target for several distinct signal transduction pathways involved in conveying proliferative signals during G. c-Jun and c-Ets-2 target distinct regions of the promoter providing a mechanism for collaborative interactions between these two proto-oncogenes in stimulating abundance of this cell cycle regulatory kinase subunit. These studies demonstrate an avenue for signal transduction cross-talk between the MAPK and cell cycle regulatory pathways and a mechanism by which different mitogenic and transforming factors may interact to promote cellular proliferation.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant CA55909, by an American Cancer Society faculty research award (to A. A.), and National Cancer Institute Clinical Investigator Award KO8 CA 620008 01 and Grant 94-27 from the American Cancer Society (Illinois Division, Inc.) (to R. G. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of the Neil Hamilton Fairley postdoctoral fellowship from the Australian Medical Research Council and the Royal Australian College of Physicians Winthrop travelling fellowship. To whom correspondence should be addressed: Division of Endocrinology, Metabolism, and Molecular Medicine, Tarry 15, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-0644; Fax: 312-908-9032; pestell{at}merle.acns.nwu.edu.

()

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; bp, base pair(s); wt, wild type; RSV, Rous sarcoma virus; CMV, cytomegalovirus; CHO, Chinese hamster ovary; LUC, luciferase; EMSA, electrophoretic mobility gel shift assays; Inr, initiator element.

()

C. Albanese and R. G. Pestell, unpublished data.

ACKNOWLEDGEMENTS

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