©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Granulocyte Macrophage-Colony Stimulating Factor-dependent Replication of Polyoma Virus Replicon in Hematopoietic Cells
ANALYSES OF RECEPTOR SIGNALS FOR REPLICATION AND TRANSCRIPTION (*)

Sumiko Watanabe (1), Yoshiaki Ito (2), Atsushi Miyajima (3), Ken-ichi Arai (1)(§)

From the (1) Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan 108, the (2) Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan 606, and the (3) Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Granulocyte macrophage-colony stimulating factor (GM-CSF) stimulates proliferation of various hematopoietic cells. Using cytoplasmic deletion mutants of the human GM-CSF receptor (hGMR) subunit and tyrosine kinase inhibitors, we previously showed that distinct signaling pathways of hGMR are involved in the induction of c- fos/c- jun mRNAs and of c- myc mRNA/cell proliferation. We used polyoma virus (Py) replicon to analyze the initiation of DNA replication induced by hGM-CSF in mouse BA/F3 pro-B cells expressing hGMR. hGM-CSF efficiently stimulated Py replication in the presence of Py enhancer and Py large T antigen supplied in trans. Analyses of Py enhancer mutants revealed that hGM-CSF promoted Py replication and activated transcription of the Py early promoter through the PEA3/PEBP5 region of Py enhancer. The membrane proximal region of hGMR subunit is required for activation of PEA3/PEBP5-dependent replication which is also required for activation of DNA synthesis in the host cells. In contrast, a more distal region which is essential for activation of c- fos and c- jun genes is required for the PEA3/PEBP5-dependent transcription of Py early promoter. These results indicate that distinct signaling pathways of hGMR are required to activate PEA3/PEBP5-dependent replication and transcription although the same enhancer is required for both activities.


INTRODUCTION

Granulocyte macrophage-colony stimulating factor (GM-CSF)() induces early responsive genes and promotes proliferation in various hematopoietic cells (1, 2) . Interleukin-3 (IL-3) elicits similar, if not identical, activities. High affinity GM-CSF receptor (GMR) and IL-3 receptor consist of the subunit specific to each and of a common subunit. Although this high affinity GMR is mainly expressed in hematopoietic cells, reconstituted hGMR in fibroblasts transduces signals to activate early response genes and cell proliferation (3) . This means that hGMR can be linked to signal transduction pathways in fibroblasts and molecules specific to hematopoietic cells are not required to transduce these signals. Signal transducing molecules such as ras, raf, and microtubule-associated protein kinase required for the induction of c -fos mRNA linked with various growth factor receptors carrying tyrosine kinase activity have been extensively studied in fibroblast systems. These molecules are also involved in the GM-CSF/IL-3 signaling pathway for the induction of c- fos mRNA in hematopoietic cells (4, 5) .

Analyses of hGMR mutants carrying deletion from the cytoplasmic tail (C terminus) revealed that the region covering amino acid position up to 763 is required to activate c- fos and c- jun genes. In contrast, the region more proximal to membrane covering amino acid position up to 544 is required for activation of c- myc gene and proliferation (6, 7) . Furthermore, IL-3/GM-CSF induced c- fos and c- jun activation is insensitive to tyrosine kinase inhibitors such as herbimycin or genistein, whereas proliferation is completely suppressed by these reagents (6) . This indicates that the signaling pathway leading to DNA replication and activation of the c- myc gene is likely to be independent of the signaling pathway for the induction of c- fos/c- jun mRNAs. In contrast to the well characterized signaling pathway for activation of the c- fos gene, less is known of signaling mechanisms for cell proliferation and activation of the c- myc gene in mammalian cells. In yeast systems, characterization of cell cycle mutants led to identification of key molecules such as cdc2/28 or cyclin required for G/to S phase transition (8) . Subsequently, mammalian homologues of these kinases and cyclins were identified and their roles in regulation of cell cycle have been closely examined (9, 10) . While cell cycle regulators such as cyclin-dependent kinase/cyclins and anti-oncogene such as retinoblastoma or p53 are regulated by growth factors including IL-3/GM-CSF (11, 12) , the mechanisms by which these molecules regulate transition of Gto S phase remain unanswered. It should also be noted that the mechanism of initiation of DNA replication of mammalian chromosomes is largely unknown.

Analyses of the regulation of initiation of chromosomal DNA replication by growth factors have been hampered because an appropriate assay for DNA replication of host cells has not been available. Protocols for assaying cell proliferation generally make use of incorporation into DNA of nucleotide substrates or analogues, or binding of drugs to DNA, none of these approaches provide direct information regarding the initiation of DNA replication. For example, the incorporation of radioactive thymidine does not distinguish initiation and elongation steps of replicative DNA synthesis. In addition, despite extensive efforts to characterize the nature of chromosomal origins of DNA replication, the results are inconclusive and even the presence of a specific replication origin is controversial (13) .

Replication systems which make use of defined DNA templates such as small phages and plasmids provided excellent models to study DNA replication in bacteria (14) . Likewise, virus systems offer many advantages for studying mammalian DNA replication (13, 15) . The mechanisms of replication of SV40 and polyoma virus (Py), the closely related double stranded circular DNA virus, have been extensively studied in fibroblasts (14) . Several features of SV40 and Py make them attractive models to study chromosomal replication (15) . In both systems, except for the large T antigen (LTag), all of the factors required for execution of replication are present in the host cells (15) . Second, Py or SV40 replication is host cell cycle dependent (16) . The Py system in particular is very useful, where Py LTag does not cause tumorigenic transformation even though it induces immortality, and middle Tag is mainly involved in transformation of the host cell. (17) . Another interesting feature of Py replicon is enhancer dependence of Py replication (13) . There are several observations that implicate involvement of transcription factor/enhancer for eukaryote chromosomal DNA replication. Py may be a good model to elucidate the role of enhancer in DNA replication (18, 19) .

Origin of Py consists of two elements, the origin core and the enhancer. The core contains a palindrome that includes two repeats of the LTag recognition pentanucleotide sequence motif and A/T-rich sequence. The enhancer sequence in the Py genome is essential for Py replication (20) , and is replaceable with cellular, viral, and yeast enhancers (21, 22, 23) . Cell type specificity of Py replication is likely to be defined by the enhancer, because mutation or replacement of the enhancer region allow Py to replicate in types of cells different from the original (21, 24, 25, 26) . Py replicon has been studied almost exclusively in mouse fibroblasts either transformed or in the presence of fetal calf serum. However, the requirement of growth factors or cytokines for Py replication has remained to be determined. Based on our finding that hGMR is functional in both hematopoietic cells and fibroblasts, we asked whether Py could serve as a model replicon to examine the initiation of DNA replication induced by IL-3/GM-CSF in hematopoietic cells. We obtained evidence that hGM-CSF induces Py replication in BA/F3 cells expressing hGMR. The region of the subunit of hGMR required for Py replication is indistinguishable from that required for cell proliferation.


MATERIALS AND METHODS

Chemicals, Media, and Cytokines

Fetal calf serum was from Biocell laboratories Co. Ltd. Dulbecco's modified Eagle's medium and RPMI 1640 were from Nikken BioMedial Laboratories Co. Ltd. Recombinant hGM-CSF and recombinant mIL-4 produced in Escherichia coli were provided by Dr. R. Kastelein, DNAX Research Institute. Mouse IL-3 (mIL-3) produced by silkworm ( Bombyx mori) was purified as described elsewhere (27) . [-P]CTP, [-P]GTP, and [H]acetylcoenzyme A were from Amersham Japan Co. Ltd. Genistein was from Wako Pure Chemicals. Herbimycin and G418 were purchased from Life Technologies, Inc.

Cell Lines and Culture Methods

A mIL-3-dependent pro-B cell line, BA/F3 (28) was maintained in RPMI 1640 medium containing 10% fetal calf serum, 1 ng/ml mIL-3, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transfected BA/F3 cells expressing hGMR and subunits (BA/FGMR) were grown in the same type of media but supplemented with 500 µg/ml G418.

Plasmids and Genes

pPyOICAT contains the Py fragment (nt 5267-nt 152) which includes replication origin core sequence (nt 5267-nt 56) and early gene promoter but lacks enhancer region, and chloramphenicol acetyltransferase (CAT) coding sequence (29, 30) . Synthetic oligonucleotides or Py enhancer fragments were inserted at the BglII site of pPyOICAT for each plasmid. For pPyBPPCAT and pPyWACAT, the region from BclI site (nt 5021) to PvuII site (nt 5265) and A core (nt 5017-nt 5130) of Py enhancer was inserted, respectively, at the BglII site of pPyOICAT. The other inserted fragments are schematically shown in Fig. 3 A. pPyBPPCAT is an origin defective control plasmid with BPP fragment as an enhancer, lacking 4 base pairs (nt 5-nt 8) in the Py LTag binding site of the origin core. pRSVLTag is the expression plasmid, and contains the Py LTag coding region under the control of RSV-long terminal repeat.

Replication Assay

DNA replication of transfected plasmid was assayed by DpnI analysis (29) . Plasmids were introduced into semiconfluent BA/F3 cells (1.2 10cells/sample) by the DEAE-dextran method as described elsewhere (3) . Cells resuspended in factor-depleted media were incubated for 5 h and then stimulated with 5 ng/ml mIL-3, hGM-CSF, or mIL-4. After incubation for an additional 24 h, cells were harvested and washed twice with phosphate-buffered saline. Low molecular weight DNA was isolated by the Hirt extraction method (31) . Briefly, cells were resuspended in 1 ml of Hirt solution (10% SDS, 10 m M Tris, pH 7.8, 10 m M EDTA) and lysed by incubation for 5 min at room temperature. Then, 250 µl of 5 M NaCl was added and mixed by tumbling. After an overnight incubation at 4 °C, the extract was centrifuged for 60 min by a microcentrifuge. Supernatant was extracted twice with phenol/chloroform, and DNA was ethanol precipitated and resuspended in 20 µl of 10 m M Tris, pH 7.4, 1 m M EDTA. In separate experiments, radiolabeled plasmid DNA was used as an internal marker to determine the recovery of the low molecular weight DNA. Ten µl of DNA solution was digested with HindIII, that linearizes template plasmid, and DpnI. As DpnI digests only methylated or hemimethylated recognition sites of DNA, newly synthesized DNA is resistant to DpnI digestion. DNA were electrophoretically separated in a 0.8% agarose gel, and transferred to Hybond-N(Amersham) by alkaline blotting (32) . DNA blots were hybridized with denatured HindIII-digested pPyOICAT DNA labeled with P by the random priming kit (U. S. Biochemical Corp., Cleveland, OH) using QuikHyb rapid hybridization solution (Stratagene, La Jolla, CA) and according to the manufacturer's instructions. Blots were washed and exposed to an imaging plate for 15 min and intensity of bands was quantified using a FUJI Image Analyzer (Model BAS-2000).

CAT Assay

CAT activities were analyzed by diffusion analysis (33) . Briefly, BA/F3 cells were co-transfected with 10 µg of template DNA and 2 µg each of hGMR and subunits plasmids (3) by the DEAE-dextran method and cultured overnight with complete media. Cells were depleted of mIL-3 for 5 h, and restimulated with 5 ng/ml of either mIL-3 or hGM-CSF. After a 12-h incubation, cells were harvested and lysed in 50 µl of 0.25 M Tris, pH 7.4, by three cycles of freezing and thawing. Each sample containing approximately 50 µg of total protein was subjected to CAT activity.

Preparation of Nuclear Extract

BA/F3 cells expressing various truncated subunit mutants and the wild type subunit (5 10) were depleted of mIL-3 for 5 h and stimulated with hGM-CSF or mIL-3. After incubation for 3 h, these cells were collected and nuclear proteins were extracted as described elsewhere (6, 34) .

Electrophoretic Mobility Shift Assay (EMSA)

EMSA was made with nuclear extracts prepared from BA/F3 cells, according to the method described above. PEA3/PEBP5 (5`-CGCAGGAAGTGACGG-5`) oligonucleotide was chemically synthesized and purified by Sephadex G-50 nick columns (Pharmacia, Upsala, Sweden) after being labeled with [-P]dGTP. The extracts containing 5 µg of protein were incubated with a labeled probe for 15 min at room temperature in a total volume of 15 µl containing 10 m M Tris, pH 7.5, 50 m M KCl, 1 m M dithiothreitol, 1 m M EDTA, pH 8.0, 12.5% glycerol, 0.1% Triton X-100, 5 µg of bovine serum albumin, and 0.5 µg of poly(dI-dC) as a nonspecific competitor. The DNA-protein complexes were electrophoresed on a 4% polyacrylamide gel in 6.7 m M Tris, pH 7.5, 3.3 m M sodium acetate, and 1 m M EDTA, pH 8.0. Then the gel was transferred to Whatman 3MM paper, dried, and analyzed by the FUJI Image Analyzer (model BAS-2000).


RESULTS

mIL-3 or hGM-CSF Induces Origin-dependent Py Replication in Mouse BA/F3 Cells Expressing a High Affinity hGMR

To determine whether or not IL-3 or GM-CSF induces the replication of Py, we analyzed the replication of transiently transfected plasmid pPyBPPCAT that contains the Py origin region, including the transcription initiation site with the entire Py enhancer BPP region (29) . The DNA template and pRSV-LTag, the expression plasmid supplying Py LTag in trans, were transfected using the DEAE-dextran method into BA/FGMR (6, 35) . This cell line extensively proliferates in response to mIL-3 or hGM-CSF and only transiently (up to 48 h) survives in the presence of mIL-4. Transfected cells were incubated without mIL-3 for 5 h and stimulated with mIL-3, hGM-CSF, or mIL-4. After a 24-h incubation, these cells were harvested and small molecular weight DNAs were extracted by the Hirt extraction method as described under ``Materials and Methods.'' DNAs were digested with DpnI which cleaves only unreplicated DNA. As shown in Fig. 1, mIL-3 and hGM-CSF but not mIL-4 induced replication of pPyBPPCAT. pPyBPPoriCAT, which lacks 4-base pair nucleotides (nt 5-nt 8) within the origin region and is unable to replicate in COP5 cells stably expressing Py LTag (29) , did not replicate with either stimulation. These results demonstrated that mIL-3 or hGM-CSF stimulated Py origin-dependent replication in BA/FGMR cells. The lack of Py replication by mIL-4 may be consistent with the observation that mIL-4 supports the survival but not the proliferation of BA/F3 cells.

Py Replication Induced by mIL-3 or hGM-CSF is Py LTag Dependent

Py LTag is a multifunctional protein essential for Py replication and regulation of Py early and late transcription (36, 37) . We next examined the dependence of mIL-3- or hGM-CSF-induced Py replication on Py LTag. BA/FGMR cells (5 10cells) were cotransfected with various amounts of pRSV-LTag DNA and 1 µg of pPyBPPoriCATand replication of the latter plasmid was analyzed as described above. As expected, Py replication was completely dependent on the presence of Py LTag (Fig. 2). pPyBPPoriCAT did not replicate in the absence of the LTag plasmid and the extent of replication was proportional to the amount of pRSV-LTag DNA cotransfected. In the following experiments, pRSV-LTag DNA, about 20-fold excess amount of template DNA containing Py replication origin, was used.

DNA Element in Py Early Enhancer Required for mIL-3 or hGM-CSF-dependent Replication

The Py enhancer BPP contains two core elements, A and B. The A element has been shown to respond to external growth factor stimuli (38) and has been extensively characterized. The A element contains binding sites for at least three transcription activators, PEA1/PEBP1 (AP-1 consensus), PEA2/PEBP2, and PEA3/PEBP5 (ets family consensus). Transcription factors shown to interact with these binding motifs are AP-1, PEBP2 (39) /CBF (core binding factor) (40) , and PEBP3 (41) /PEA3-91 (42) /PEBP5 (43) , respectively. To delineate DNA elements in Py enhancer responsible for mIL-3- or hGM-CSF-dependent replication, we employed various plasmids containing mutations in A element (30) . As shown in Fig. 3 A, A element, either the wild type or the mutant carrying various point mutations, was inserted at the BglII site of the pPyOICAT plasmid. These plasmids were cotransfected with pRSV-LTag into BA/FGMR cells and replication of plasmid induced by either mIL-3 or hGM-CSF was analyzed. Transfection efficiency of each sample as judged by luciferase activity of cotransfected SR-luciferase plasmid was nearly the same. pPyWACAT carrying the wild type A element replicate in response to mIL-3 (Fig. 3 B). pPyM3ACAT carrying two point mutations at the PEA3/PEBP5 site ( ets site) failed to replicate in response to mIL-3, indicating that this site is essential for mIL-3-dependent Py replication. In contrast, pPyM1ACAT carrying two point mutations at the PEA1/PEBP1 site (AP-1 site) showed only a slight decrease in replication (Fig. 3 B), and pPyM2ACAT carrying two point mutations at the PEA2/PEBP2 site (CBF site) elicited a significantly reduced replicative activity. The relative level of replicated pPyCAT DNA plasmids were presented in Fig. 3 A. These results suggest that the AP-1 site, only slightly, and PEBP2/CBF site, to much greater degree, contribute to mIL-3-dependent Py replication. Essentially the same results were obtained with hGM-CSF stimulation (data not shown).


Figure 3: Enhancer requirement of Py replication by mIL-3 or hGM-CSF. A, schematic diagram showing various enhancers and the mutant inserted into the BglII site of pPyOICAT. Summary of activities of replication (%) and transcription (%) induced by mIL-3 in BA/FGMR cells are shown in the right column of each construct. Replicated band intensity or fold induction of transcriptional activation was normalized using the value of WA-CAT as 100%. B, replication of derivatives of pPyOICAT containing various enhancers by mIL-3 in BA/FGMR cells. Enhancer or its mutants inserted into pPyOICAT of the plasmids are given in the upper part of the figure. Locations of replicated ( R) or unreplicated ( U) plasmids are shown on the left.



To further confirm this finding, we next analyzed a series of plasmids that contain subfragments of either the wild type or the mutant A element in tandem and inserted at the BglII site of the pPyOICAT plasmid (Fig. 3 A). As expected, pPyALCAT containing six tandem repeats of the ets binding site replicated efficiently in response to mIL-3, and pPyAECAT containing six tandem repeats of the PEA2/PEBP2/CBF site replicated at a much lower level than pPyALCAT. pPyAMCAT, carrying six tandem repeats of the AP-1 site, showed almost no replicative activity in response to mIL-3 (Fig. 3 B). Essentially the same results were obtained with hGM-CSF stimulation. Taken together, these results show that the contribution to mIL-3 or hGM-CSF-induced Py replication is most prominent with the ets binding site, much less with the PEBP2/CBF site, and negligible with the AP-1 site.

Identification of the Region of hGMR Subunit Required for Activation of Py Replication

We previously showed that the cytoplasmic regions of hGMR subunit required for induction of c- fos/c- jun mRNAs and c- myc mRNA induction/cell proliferation differ (6, 7) . Mutant 544 carrying a deletion from the C terminus to amino acid position 544 is fully active, whereas mutant 517 is weakly active, and mutant 455 is completely inactive to induce c- myc mRNA induction/cell proliferation (6) . To determine whether or not the same region of hGMR subunit is required for hGM-CSF-induced Py replication, we used BA/F3 cells expressing hGMR composed of the wild type subunit and mutant subunit carrying a series of C terminus deletions (BA/F3GMR mutants) (35) . As shown in Fig. 4, mutant 544 is fully active as the original subunit, whereas mutant 517 is active but has a much reduced ability to support pPyWACAT replication. As expected, mutant 455 which lacks the potential to support proliferation of the host cells, failed to promote pPyWACAT replication. When pPyALCAT was used as the template, the requirement of hGMR subunit for replication was stricter and mutant 517 had lost the potential to support replication of this Py construct. These results are in good agreement with the findings of host chromosome replication monitored by thymidine incorporation, i.e. mutant 544 was fully active but mutants 517 and 455 were inactive in response to hGM-CSF (6) .

mIL-3 or hGM-CSF Activates Transcription through the PEA3/PEBP5 Site

Using CAT gene as the reporter, we then examined whether or not mIL-3 or hGM-CSF would stimulate transcription through PEA3/PEBP5, the ets binding site of Py enhancer in the absence of pRSV-LTag. BA/F3 cells transiently transfected with 10-µg derivatives of pPyOICAT containing various enhancer elements and 2 µg each of hGMR and subunits plasmids (3) were depleted of mIL-3 for 5 h and were re-stimulated with 5 ng of either mIL-3 or hGM-CSF. Cells were harvested after a 12-h incubation, as described under ``Materials and Methods.'' IL-3 induced fold induction of each construct in BA/F3 cells is shown in Fig. 5 A. Transcription (%) activities normalized using the value of pPyWACAT are summarized in the right column of each construct (Fig. 3 A). Essentially the same results were obtained by hGM-CSF stimulation. mIL-3- or hGM-CSF-induced CAT activity of pPyWACAT and this activity was lost by introduction of mutations in the PEA3/PEBP5 site (pPyM3ACAT). Mutations in either the PEA1/PEBP1 site (pPyM1ACAT) or PEA2/PEBP2 site (pPyM2ACAT) reduced CAT activities to some extent but not completely. CAT activity of pPyALCAT induced by mIL-3 or hGM-CSF was more than 10 times stronger than that of pPyWACAT. This may be caused by pPyALCAT containing 6 tandem copies of the PEA3/PEBP5 site. pPyALMCAT that contains point mutations in the AL region lost the potential to respond to mIL-3 or hGM-CSF. These results indicated that PEA3/PEBP5 is also functional as an enhancer responding to mIL-3 or hGM-CSF signals. Weak activity of pPyWACAT suggests that the PEA3/PEBP5 site in single copy is insufficient for activation or that other regions affect the PEA3/PEBP5 region in a negative manner.

Requirement of the Cytoplasmic Region of hGMR Subunit for mIL-3- or hGM-CSF-dependent Transcription through the PEA3/PEBP5 Site

We then analyzed the ability of hGMR mutants to induce CAT activity of pPyALCAT in response to hGM-CSF by cotransfection of various GMR mutants plasmids (35) and reporter plasmid pPyALCAT (Fig. 5 B). hGM-CSF stimulated CAT activity through mutant 826 (data not shown) and mutant 763 in response to hGM-CSF at levels similar to the wild type construct. In contrast, mutants 626 and 544 failed to transduce signals to induce CAT activity of pPyALCAT even though these mutant receptors can induce replication of the same Py construct in response to hGM-CSF. As expected, mutants 517 and 455 did not activate CAT activity or the replication of pPyALCAT. Essentially the same results were obtained with BA/F3GMR mutants cells (data not shown). Thus, the region between positions 455 and 517 of the hGMR subunit is required for the replication induced by hGM-CSF, and that between positions 763 and 626 is required for the transcriptional activation of pPyALCAT. It should be noted that the latter region corresponds to that required for c- fos/c- jun mRNA induction. These results suggested that signals generated by the hGMR subunit for transcription and for the replication of pPyALCAT differ.


Figure 5: Transcriptional activity of Py-CAT by mIL-3 and/or hGM-CSF. A, derivatives of pPyOICAT containing various enhancers and hGMR and subunits plasmids were introduced into BA/F3 cells and the CAT activity induced by 5 ng/ml mIL-3 were analyzed. B, transcriptional activity of pPyALCAT by hGM-CSF through hGMR subunit mutants. pPyALCAT and hGMR and mutants was transfected to BA/F3 and cells were depleted of mIL-3 for 5 h. Cells were re-stimulated by either mIL-3 (5 ng/ml, open bar) or hGM-CSF (5 ng/ml, hatched bar) and harvested after 12 h incubation. CAT activity was analyzed by diffusion assay as described under ``Materials and Methods.'' A and B, all the values represent the relative amount of H activity to that of unstimulated cells and are the average of three samples with standard deviations. Numbers below the figure indicate position of the C-terminal amino acid residue of each truncated mutant subunits.



Effects of Tyrosine Kinase Inhibitors on PEA3/PEBP5-dependent Transcription and Py Replication

Involvement of distinct pathways for the induction of c- fos/c- jun mRNAs and for the activation of c- myc gene/cell proliferation was also supported by the finding that two pathways showed different sensitivities to tyrosine kinase inhibitors (6) . Although mIL-3- or hGM-CSF-dependent induction of c- fos/c- jun mRNAs is not apparently affected by herbimycin or genistein, activation of the c- myc gene and cell proliferation are almost completely inhibited by these drugs. Therefore, we examined the sensitivity of mIL-3- or hGM-CSF-dependent transcription and replication of pPyALCAT to tyrosine kinase inhibitors. Neither herbimycin nor genistein had any effect on mIL-3- or hGM-CSF-induced CAT activity of pPyALCAT (Fig. 6 A). In contrast, both drugs almost completely suppressed the mIL-3- or hGM-CSF-induced replication of pPyALCAT (Fig. 6 B). The effects of tyrosine kinase inhibitors on CAT activity of the RSV-CAT plasmid was examined. Neither herbimycin nor genistein suppressed CAT activity (data not shown). These results exclude the possibility that these inhibitors affect replication by suppressing the expression of LTag. Taken together, these results suggest that signals required for activation of c- fos/c- jun mRNAs and PEA3/PEBP5-dependent transcription are similar. Likewise, the signals required for PEA3/PEBP5-dependent Py replication and cell proliferation/c- myc mRNA induction are related.


Figure 6: Effects of tyrosine kinase inhibitors on transcription ( A) and replication ( B) of pPyALCAT induced by mIL-3 or hGM-CSF. A, pPyALCAT was transfected to BA/FGMR cells and CAT activity induced by 5 ng/ml of either mIL-3 ( open bar) or hGM-CSF ( hatched bar) in the presence of tyrosine kinase inhibitors was analyzed by diffusion analysis as described in the legend to Fig. 5. Closed bar represents value of nonstimulated cells. All the values are the average of three samples with standard deviations. B, pPyALCAT or pPyWACAT (1 µg) and pRSV-LTag (10 µg) were transfected to BA/F3GMR cells and stimulated by 5 ng/ml of either mIL-3 or hGM-CSF in the presence of tyrosine kinase inhibitors. Replication activity was analyzed as described in legend to Fig. 1. Herbimycin (1 µg/ml) was added 24 h prior to stimulation and genistein (10 µg/ml) was added 15 min prior to stimulation.



EMSA of PEA3/PEBP5 Oligonucleotides

The results described above indicate that distinct regions of hGMR subunit are required for transcription and replication, even though both processes depend on the same PEA3/PEBP5 site. They also suggested that the roles of PEA3/PEBP5, probably through interaction with a set of binding proteins, in replication and transcription differ. To examine the properties of proteins interacting with the PEA3/PEBP5 site, we carried out EMSA using oligonucleotide probes corresponding to PEA3/PEBP5. BA/F3GMR cells depleted of mIL-3 for 5 h were re-stimulated with 5 ng/ml mIL-3 or hGM-CSF. After a 3-h incubation, cells were harvested and nuclear extracts were prepared and used for EMSA as described under ``Materials and Methods.'' The results showed that nuclear extracts of unstimulated cells interacted with the PEA3/PEBP5 oligonucleotide and generated a specific band (Fig. 7). The mobility as well as the intensity of the complexes formed with nuclear extracts prepared from cells stimulated by either mIL-3 or hGM-CSF were much the same. Likewise, no significant difference was observed with extracts of BA/F3 cells expressing various deletion mutants of hGMR subunit regardless of the stimulation (data not shown).


DISCUSSION

Py Replicon Replicates in Response to GM-CSF Signals

In the present work, we showed that the Py origin was activated in mouse hematopoietic cells expressing high affinity hGMR in response to mIL-3 or hGM-CSF stimulation. To our knowledge, this is the first demonstration that Py replication is triggered by defined growth factors. Previously, most if not all work on Py replication was done using proliferating mouse fibroblasts. In such systems, it is difficult to assess the requirement for growth signals because fetal calf serum rather than the combination of defined growth factors was used to maintain cell proliferation. In addition, the extent of cell proliferation in the absence of growth factors is relatively high. In contrast, in hematopoietic cells, there was only a low level of Py replication in the absence of added cytokines. BA/F3 cells died quickly in the absence of mIL-3 but mIL-4 transiently maintained viability of the cells. After depletion of mIL-3 followed by readdition of mIL-3 or hGM-CSF, Py replicated efficiently in the BA/FGMR cells. Replication was absolutely dependent on Py LTag and Py origin. All molecules except for Py LTag required for Py replication are supplied by host cells. Thus, Py replicon is a useful tool to characterize the GM-CSF-dependent signaling pathway for the initiation of DNA replication.

Molecules Involved in GMR-dependent Signaling for Py Replication

How does mIL-3 or hGM-CSF activate Py replication? To initiate Py replication, both Py LTag as well as the replication machinery of host cells need to be activated. The function of SV40 LTag has been known to be regulated by the state of phosphorylation of LTag (43, 44) . SV40 LTag is phosphorylated at threonine 124 by cdc2 kinase and this phosphorylation is essential for its function (45, 46) . Cyclin-dependent kinase and cyclins have been shown to participate in IL-3-dependent signaling pathway in hematopoietic cells (11) . In view of the high degree of structural conservation between LTags of SV40 and Py, it is tempting to speculate that, in BA/FGMR cells, Py LTag is also activated by phosphorylation through cyclin-dependent kinase and cyclins by mIL-3 or hGM-CSF signals.

Interestingly, the regions of hGMR subunit required for LTag-dependent replication of Py origin and for proliferation of host cells are indistinguishable. In addition, sensitivity of Py replication and cell proliferation to tyrosine kinase inhibitors is similar. These results suggest that hGM-CSF promotes Py replication through a signaling pathway leading to the activation of cellular machinery for chromosomal replication. It appears that Py replicon serves as a model system to dissect GMR signals for initiation of chromosomal replication. It should be noted that the induction of c- myc mRNA is also sensitive to tyrosine kinase inhibitors and depends on the same region of hGMR subunit required for proliferation (6) . In addition, this region was also found to be associated with activation of Janus protein tyrosine kinase (47) and induction of cyclin mRNAs() (Fig. 7). The role of c- myc in cell proliferation has been implicated, but its exact role is largely unknown. As tyrosine kinase inhibitors block Py replication as well as host chromosomal replication, tyrosine kinase(s) appears to be involved in the signal transduction pathway for replication although the nature of tyrosine kinase(s) remains to be clarified. More detailed analysis of the role of these molecules in initiating Py replication is ongoing in our laboratory.


Figure 7: Binding activity of nuclear proteins to PEA3/PEBP5 of BA/FGMR. Nuclear proteins extracted from BA/FGMR stimulated by 5 ng/ml of either mIL-3 or hGM-CSF were analyzed for binding activity by gel retardation assay using PEA3/PEBP5 oligonucleotides as described under ``Materials and Methods.''



Enhancer Specificity for Py Replication Induced by mIL-3 or hGM-CSF

SV40 and Py replication origins have similar structures containing LTag binding site, stretch of AT base pairs, and palindrome (13) . However, the requirement of enhancer region for Py replication in vivo is stricter than that for SV40 replication (20, 21) . The Py enhancer region can be replaced by various cellular enhancers and can result in an altered potential for replication in different species of cells (21, 26) . These observations suggest that cell type specificity of Py replicon depends on the enhancer sequence which contributes to activate replication. Our results also indicate that Py replicates in hematopoietic cells in a manner dependent on the Py enhancer.

Mutation analyses of Py early enhancer revealed PEA3/PEBP5 to be a main region responding to mIL-3 or hGM-CSF signals although the nature of PEA3/PEBP5 binding protein(s) in BA/FGMR cells remains to be elucidated. The protein binding to PEA3 was molecularly cloned from the mouse mammary carcinoma cell line FM3A (41) and was shown to be a member of an ets family. Although ets family proteins are expressed mainly in hematopoietic cells, expression of PEA3 appears to be restricted in fibroblasts and epithelial cells (41) . Studies on sequence specificity of Ets-1 and elf-1 (48) suggest that PEA3/PEBP5 has features which can be recognized by both proteins. It is tempting to speculate that PEA3/PEBP5 interacts with the ets family protein(s) other than PEA3 in hematopoietic cells. Mutations within the PEBP2 site of pPyWACAT considerably decreased the level of Py replication. However, tandem repeats of the PEBP2 site showed only weak replicative activity in response to mIL-3 or hGM-CSF signals. Interestingly, PEBP2 has been shown to be identical to CBF and cooperative binding of Ets-1 and CBF was reported (49) . It is possible that, in BA/FGMR cells, mIL-3 or hGM-CSF induces cooperative binding of Ets-1 at the PEA3/PEBP5 site and CBF at the PEBP2 site. PEBP1 which contains a AP-1-like motif failed to respond to mIL-3 or hGM-CSF signals, although c- fos and c- jun are induced by these signals (3, 6) .

Roles of Enhancer in Py Replication and Transcription

Our results indicate that the roles of Py enhancer in replication and transcription differ. There are several instances where the functions of enhancer for transcription and for replication can be uncoupled. This has been demonstrated either by analyzing the cis-acting enhancer or trans-acting factor interacting with the enhancer. Analysis of cell specificity of individual enhancer revealed uncoupling of transcriptional activation from replication in several cell types (50) or enhancer stimulates replication in a position-dependent manner (30, 51) . Mutation analysis of P53 and Rel proteins revealed that these proteins elicit differential effects on stimulation of Py replication and transcription (52, 53) . These results suggest that the enhancer has different functions in activating transcription and replication. Our results which are in line with these observations are unique in that differential requirements of enhancer for replication and transcription are demonstrated in terms of signal transduction through hGMR. These events beg the question as to why an enhancer such as PEA3/PEBP5 is required for Py replication even though this sequence does not stimulate Py replication through transcriptional activation? It should be noted that the binding activity was observed in cell extracts not exposed to mIL-3 or hGM-CSF. Activation of transcription requires additional GMR signals for the modification of the PEA3/PEBP5 binding protein or the replacement of PEA3/PEBP5 binding protein with other enhancer binding proteins. In contrast, it is possible that the PEA3/PEBP5 binding protein promotes Py replication by facilitating recruitment of Py LTag or other essential molecules such as replication protein A to replication origin. Replication protein A is a single stranded DNA binding protein physically interacting with SV40 LTag (54) and is required for unwinding of the origin by SV40 LTag (55) . Recently, enhancer binding proteins such as VP16 of HSV or P53 has been shown to play a role in recruitment of replication protein A through protein-protein interaction (19, 56, 57) . Whether or not PEA3/PEBP5 binding protein stimulates Py replication by attracting replication protein A remains to be determined. The Py replication system responding to the hGMR signal described in this paper will be useful to characterize various cellular components required for DNA replication and transcription.


FOOTNOTES

*
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and Cancer Research from the Ministry of Education, Science and Culture of Japan. DNAX Research Institute for Molecular and Cellular Biology is supported by the Schering-Plough Corp. 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.

§
To whom correspondence should be addressed. Tel.: 81-3-5449-5660; Fax: 81-3-5449-5424.

The abbreviations used are: GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; mIL, mouse interleukin; hGMR, human GM-CSF receptor; nt, nucleotide(s); CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; Py, polyoma virus; LTag, large T antigen; CBF, core binding factor.

S. Watanabe and K. Arai, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. F. Hanaoka, Y.-Y. Iwai, and Y. Murakami for helpful discussions and technical advice, and M. Ohara for comments on the manuscript.


REFERENCES
  1. Miyajima, A., Kitamura, T., Harada, N., Yokota, T., and Arai, K. (1992) Annu. Rev. Immunol. 10, 295-331 [CrossRef][Medline] [Order article via Infotrieve]
  2. Watanabe, S., Nakayama, N., Yokota, T., Miyajima, A., and Arai, K. (1991) Curr. Opin. Biotechnol. 2, 227-237 [Medline] [Order article via Infotrieve]
  3. Watanabe, S., Muto, A., Mui, A.-F., Miyajima, A., and Arai, K. (1993) Mol. Cell. Biol. 13, 1440-1448 [Abstract]
  4. Carroll, M. P., Clark-Lewis, I., Rapp, U. R., and May, W. S. (1990) J. Biol. Chem. 265, 19812-19817 [Abstract/Free Full Text]
  5. Satoh, T., Uehara, Y., and Kaziro, Y. (1992) J. Biol. Chem. 267, 2537-2541 [Abstract/Free Full Text]
  6. Watanabe, S., Muto, A., Yokota, T., Miyajima, A., and Arai, K. (1993) Mol. Biol. Cell 4, 983-992 [Abstract]
  7. Sato, N., Sakamaki, K., Terada, N., Arai, K., and Miyajima, A. (1993) EMBO J. 12, 4181-4189 [Abstract]
  8. Forsburg, S. L., and Nurse, P. (1991) Annu. Rev. Cell Biol. 7, 227-256 [CrossRef]
  9. Matsushime, H., Roussel, M. F., Ashmun, R. A., and Sherr, C. J. (1991) Cell 65, 701-713 [Medline] [Order article via Infotrieve]
  10. Koff, A., Giordano, A., Desai, D., Yamashita, K., Harper, J. W., Elledge, S., Nishimoto, T., Morgan, D. O., Franza, B. R., and Roberts, J. M. (1992) Science 257, 1689-1694 [Medline] [Order article via Infotrieve]
  11. Ando, D., Ajchenbaum-Cymbalista, F., and Griffin, J. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9571-9575 [Abstract]
  12. Borellini, F., and Glazer, R. I. (1993) J. Biol. Chem. 268, 7923-7928 [Abstract/Free Full Text]
  13. DePamphilis, M. L. (1993) Annu. Rev. Biochem. 62, 29-63 [CrossRef][Medline] [Order article via Infotrieve]
  14. Kornberg, A., and Baker, T. A. (1992) DNA Replication, W. H. Freeman and Co., New York
  15. Challberg, M. D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717 [CrossRef][Medline] [Order article via Infotrieve]
  16. D'Urso, G., Marraccino, R. L., Marshak, D. R., and Roberts, J. M. (1990) Science 250, 786-791 [Medline] [Order article via Infotrieve]
  17. Rassoulzadegan, M., Naghashfar, Z., Cowie, A., Carr, A., Grisoni, M., Kamen, R., and Cuzin, F. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4354-4358 [Abstract]
  18. Fangman, W. L., and Brewer, B. J. (1992) Cell 71, 363-366 [Medline] [Order article via Infotrieve]
  19. Dutta, A. (1993) Curr. Biol. 3, 709-712 [Medline] [Order article via Infotrieve]
  20. Tyndall, C., La Mantia, G., Thacker, C. M., Favaloro, J., and Kamen, R. (1981) Nucleic Acids Res. 9, 6231-6250 [Abstract]
  21. De Villiers, J., Schaffner, W., Tyndall, C., Lupton, S., and Kamen, R. (1984) Nature 312, 242-246 [Medline] [Order article via Infotrieve]
  22. Baru, M., Shlissel, M., and Manor, H. (1991) J. Virol. 65, 3496-3503 [Medline] [Order article via Infotrieve]
  23. Bennett-Cook, E. R., and Hassell, J. A. (1991) EMBO J. 10, 959-969 [Abstract]
  24. Herbomel, P., Bourachot, B., and Yaniv, M. (1984) Cell 39, 653-662 [Medline] [Order article via Infotrieve]
  25. Fujimura, F. K., Deininger, P. L., Friedmann, T., and Linney, E. (1981) Cell 23, 809-814 [Medline] [Order article via Infotrieve]
  26. Campbell, B. A., and Villarreal, L. P. (1986) Mol. Cell. Biol. 6, 2068-2079 [Medline] [Order article via Infotrieve]
  27. Miyajima, A., Schreurs, J., Otsu, K., Kondo, A., Arai, K., and Maeda, S. (1987) Gene ( Amst.) 58, 273-281 [Medline] [Order article via Infotrieve]
  28. Palacios, R., and Steinmetz, M. (1985) Cell 41, 727-734 [Medline] [Order article via Infotrieve]
  29. Murakami, Y., Asano, M., Satake, M., and Ito, Y. (1990) Oncogene 5, 5-13 [Medline] [Order article via Infotrieve]
  30. Murakami, Y., Satake, M., Yamaguchi-Iwai, Y., Sakai, M., Muramatsu, M., and Ito, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3947-3951 [Abstract]
  31. Hirt, B. (1967) J. Mol. Biol. 26, 365-369 [Medline] [Order article via Infotrieve]
  32. Reed, K. C., and Mann, D. A. (1985) Nucleic Acids Res. 13, 7207-7221 [Abstract]
  33. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Watanabe, S., Yssel, H., Harada, Y., and Arai, K. (1994) Int. Immunol. 6, 523-532 [Abstract]
  35. Sakamaki, K., Miyajima, I., Kitamura, T., and Miyajima, A. (1992) EMBO J. 11, 3541-3550 [Abstract]
  36. Cogen, G. (1978) Virology 85, 222-230
  37. Farmerie, W. G., and Folk, W. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 68, 6919-6923
  38. Yamaguchi, Y., Satake, M., and Ito, Y. (1989) J. Virol. 63, 1040-1048 [Medline] [Order article via Infotrieve]
  39. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863 [Abstract]
  40. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, N. A. (1993) Mol. Cell. Biol. 13, 3324-3339
  41. Xin, J., Cowie, A., Lachance, P., and Hassell, J. A. (1992) Genes & Dev. 6, 481-496
  42. Martin, M. E., Yang, X. Y., and Folk, W. R. (1992) Mol. Cell. Biol. 12, 2213-2221 [Abstract]
  43. Cegielskaa, A., and Virshup, D. M. (1993) Mol. Cell. Biol. 13, 1202-1211 [Abstract]
  44. Wang, E. H., Bhattacharyya, S., and Prives, C. (1993) J. Virol. 67, 6788-6796 [Abstract]
  45. McVey, D., Brizuela, L., Mohr, I., Marshak, D. R., Gluzman, Y., and Beach, D. (1989) Nature 341, 503-507 [CrossRef][Medline] [Order article via Infotrieve]
  46. Prives, C. (1990) Cell 61, 735-738 [CrossRef][Medline] [Order article via Infotrieve]
  47. Quelle, F. W., Sato, N., Witthuhn, B. A., Inhorn, R. C., Eder, M., Miyajima, A., Griffin, J., and Ihle, J. N. (1994) Mol. Cell. Biol. 14, 4335-4341 [Abstract]
  48. Wang, C., Petryniak, B., Ho, I., Thompson, C. B., and Leiden, J. M. (1992) J. Exp. Med. 175, 1391-1399 [Abstract]
  49. Wotton, D., Ghysdael, J., Wang, S., Speck, N. A., and Owen, M. J. (1994) Mol. Cell. Biol. 14, 840-850 [Abstract]
  50. Campbell, B. A., and Villarreal, L. P. (1988) Mol. Cell. Biol. 8, 1993-2004 [Medline] [Order article via Infotrieve]
  51. Hassell, J. A., Muller, W. J., and Mueller, C. R. (1986) Cancer Cells 4, 561-569
  52. Ishikawa, H., Asano, M., Kanda, T., Kumar, S., Gelinas, C., and Ito, Y. (1993) Oncogene 8, 2889-2896 [Medline] [Order article via Infotrieve]
  53. Kanda, T., Segawa, K., Ohuchi, N., Mori, S., and Ito, Y. (1994) Mol. Cell. Biol. 14, 2651-2663 [Abstract]
  54. Dornreiter, I., Erdile, L. F., Gilbert, I. U., von Winkler, D., Kelly, T. J., and Fanning, E. (1992) EMBO J. 11, 769-776 [Abstract]
  55. Borowiec, J. A., Dean, F. B., Bullock, P. A., and Hurwitz, J. (1990) Cell 60, 181-184 [Medline] [Order article via Infotrieve]
  56. Li, R., and Botchan, M. R. (1993) Cell 73, 1207-1221 [Medline] [Order article via Infotrieve]
  57. Zhigang, H., Brinton, B. T., Greenblatt, J., Hassell, J. A., and Ingles, J. (1993) Cell 73, 1223-1232 [Medline] [Order article via Infotrieve]

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