From the
Granulocyte macrophage-colony stimulating factor (GM-CSF)
stimulates proliferation of various hematopoietic cells. Using
cytoplasmic deletion mutants of the human GM-CSF receptor (hGMR)
Granulocyte macrophage-colony stimulating factor
(GM-CSF)
Analyses of hGMR
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
Interestingly, the regions of hGMR
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) .
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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) .
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 G
to S phase remain
unanswered. It should also be noted that the mechanism of initiation of
DNA replication of mammalian chromosomes is largely unknown.
subunit of hGMR required for Py replication is
indistinguishable from that required for cell proliferation.
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 10
cells/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).
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
10
cells) 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
We previously showed
that the cytoplasmic regions of hGMR Subunit
Required for Activation of Py Replication
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
We then analyzed the ability of hGMR
Subunit for mIL-3- or hGM-CSF-dependent Transcription through the
PEA3/PEBP5 Site
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).
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.