(Received for publication, April 14, 1995)
From the
Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V.(1993) Mol. Cell. Biol. 13, 7358-7363). In the present reseach, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.
Mitogenic stimulation leads to increased rates of protein
synthesis, which is required for entry of resting cells into the cell
cycle(2, 3, 4, 5) . The increase in
net protein synthesis after mitogenic stimulation of resting cells is
connected with mitogen-induced expression of genes coding for
translation initiation
factors(6, 7, 8, 9, 10) .
In addition to the total increase in protein synthesis, it is
reasonable to expect that there should be selective increases in the
synthesis of growth-promoting proteins. One of the translation
initiation factors whose levels are increased after mitogenic
stimulation of resting cells is the rate-limiting mRNA cap-binding
protein eukaryotic translation initiation factor 4E (eIF-4E), ()which may be involved in unwinding of mRNA 5` secondary
structures, mRNA splicing, mRNA 3` processing, and mRNA
nucleocytoplasmic transport (11, 12, 13) . An
important role for eIF-4E in cell growth has been demonstrated in
experiments in which microinjection of this translation initiation
factor into quiescent NIH 3T3 cells induced them to enter the S
phase(14) . Furthermore, overexpression of eIF-4E transforms
both established and primary
cells(15, 16, 17) . It has also been
demonstrated that the c-myc oncoprotein increases the expression of
eIF-4E by a transcriptional mechanism(6) , while ras and src
oncoproteins increase the function of eIF-4E by increasing its
phosphorylation(34, 35) .
We have examined
previously the role of eIF-4E in the expression of cyclin
D1/PRAD-1/bcl-1(1) . One of the functions of cyclin D1 is to
bind the tumor suppressor protein, pRB, which allows
hyperphosphorylation and inactivation of pRB by a cyclin D1-dependent
kinase that is necessary for G/S
transition(18, 19) . Cyclin D1 is expressed late in
G
and its induction by growth factors requires new protein
synthesis(20, 21) . In addition, cyclin D1 is
identical to the PRAD1 oncogene that is overexpressed in parathyroid
and breast tumors and is located near the bcl-1 breakpoint in some
lymphomas(21, 22, 23) . We have found that
the levels of cyclin D1 protein, but not pRB, are specifically and very
strongly increased in continuously growing NIH 3T3 cells overexpressing
eIF-4E(1) . We did not observe a proportional increase in
cyclin D1 mRNA levels, a finding which suggests that the effect of
eIF-4E on cyclin D1 expression involves a post-transcriptional
mechanism. In the present report we describe our progress on
identifying the nature of regulation of cyclin D1 gene expression by
translation initiation factor 4E.
Figure 4: The effect of eIF-4E on cyclin D1 gene transcription. Nuclei from equal numbers of cells continuously growing in 10% FCS (10) or cells cultured in 0.5% FCS for 3 days (0.5) were isolated, and a run-on assay was performed as described under ``Materials and Methods.''
Figure 1: Expression of cyclin D1 protein in serum-deprived eIF-4E-overexpressing cells. Cells were plated in 15-cm culture dishes in 10% FCS and grown to three-quarters confluence, cultured for 3 days in 0.5% serum, and treated with fresh 10% FCS added directly to the culture media for the indicated periods before protein extraction. For Western blot analysis 40 µg of protein/lane were analyzed for each sample.
Figure 2: Amount of cyclin D1 mRNA associated with polysomes is increased in wild-type eIF-4E-overexpressing cells. A, cells were plated in 15-cm culture dishes in 10% FCS, grown to three-quarters confluence, and cultured for 3 days in 0.5% serum; and 4 h before RNA extraction, they were treated with 10% FCS added directly to culture media (A) or not treated (R). Polysomal fractionation and Northern blot analysis of polysomal mRNA were performed as described under ``Materials and Methods.'' Membranes were sequentially analyzed with indicated probes. B, after sucrose gradient centrifugation total cytoplasmic subpolysomal and polysomal fraction were separately pooled and equal amounts of RNA from subpolysomal (SP) and polysomal fractions (P) from resting (R) and serum-activated (A) cells were analyzed by Northern blot.
Thus, cyclin D1
mRNA was found predominantly associated with polysomes in resting cells
and no change in the distribution of cyclin D1 mRNA was seen following
mitogenic activation. Another important finding (Fig. 2A) is that polysomal distribution of
translationally regulated mRNA for elongation factor 1 (EF-1
)
is not affected by eIF-4E at all. As demonstrated
previously(25, 26) , we have found that EF-1
mRNA
is localized in subpolysomal fractions of resting cells, but is shifted
to heavy polysomes upon mitogenic stimulation. However, overexpression
of functional eIF-4E has no effect on polysomal distribution of
EF-1
mRNA in both resting or serum-activated cells (Fig. 2A). These findings suggest that translational
regulation of EF-1
is independent of eIF-4E.
Figure 3: Increased expression of cyclin D1 mRNA in serum-deprived eIF-4E transformed NIH 3T3 cells. Cells were plated in 15-cm culture dishes in 10% FCS and grown to three-quarters confluence, cultured for 3 days in 0.5% serum, and were treated with fresh 10% FCS added directly to the culture media for the indicated time periods before RNA extraction. Northern blot analysis was performed as described under ``Materials and Methods.''
Figure 5: Increased levels of cyclin D1 mRNA in serum-deprived cells are not sufficient for elevation of cyclin D1 protein. NIH 3T3 cells transfected with MMTV-driven cyclin D1 expression vector (CD-10, (1) ) and control parental cells (NIH) were serum deprived as described above. For the last 24 h of serum deprivation, cells were treated (+) or not treated(-) with dexamethasone to induce MMTV promoter. Left, Northern blot analysis of total cyclin D1 mRNA level. Right, Western blot analysis of cyclin D1 protein level.
Figure 6: The overexpressed cyclin D1 mRNA is accumulated in the nuclei of CD-10 cells. Cells were made quiescent by serum deprivation as described above, and total (T), nuclear (N), and cytoplasmic (C) RNA were extracted and analyzed by Northern blot.
Figure 7:
Cell cycle analysis of
eIF-4E-overexpressing cells during serum deprivation. Cells were plated
at 200 10
/well in 6-well culture plates in 10% FCS.
The following day they were washed once with serum-free
Dulbecco's modified Eagle's medium and further cultured in
Dulbecco's modified Eagle's medium containing 0.5% FCS.
Cells were harvested by trypsinization at indicated days, stained with
propidium iodide, and cell cycle distribution was determined by flow
cytometry.
We have found previously that increased expression of cyclin D1 protein in continuously growing eIF-4E-overexpressing cells cannot be explained simply by elevated cyclin D1 mRNA levels, suggesting the involvement of post-transcriptional regulation(1) . In this paper we provide data suggesting the complex involvement of eIF-4E in the regulation of cyclin D1 gene expression. First, the levels of eIF-4E protein become critical in determining the amount of total cyclin D1 mRNA when cells are deprived of growth factors (Fig. 3). Further, the levels of total cyclin D1 mRNA (Fig. 3) and the amount of cyclin D1 mRNA in polysomes of serum-deprived and stimulated eIF-4E-overexpressing cells are greatly increased (Fig. 2). These increases correlate with the elevated levels of cyclin D1 protein in these cells (Fig. 1). Furthermore, the transcription rate of cyclin D1 gene remains high when 4E(P2) cells are deprived of growth factors, but it is readily down-regulated in control Ala cells (Fig. 4). It has been demonstrated recently that eIF-4E increases the levels of transcription factors responsible for interleukin-2 gene expression in T-lymphocytes (40) . It is likely that eIF-4E facilitates translation of some transcription factor which is rate limiting for cyclin D1 mRNA synthesis under conditions of serum deprivation.
However, the failure of increased cyclin D1 mRNA levels to provide for elevated cyclin D1 protein levels (when mRNA is overexpressed by exogenous cyclin D1 expression vector in serum-deprived or continuously growing cells, Fig. 5and (1) ) suggests that the post-transcriptional step is also involved in the control of cyclin D1 expression. The role of post-transcriptional events is also suggested by findings that strong overexpression of cyclin D1 mRNA in transgenic mice from the same construct as in our NIH 3T3 cells provides for much less pronounced (although significant) increase in cyclin D1 protein in the mammary glands of those mice(38) . Also, dramatic increase in cyclin D1 protein levels ((1) ) and the lack of changes in both the transcription rate of cyclin D1 gene (Fig. 4) and its mRNA levels ((1) ) in 4E(P2) cells continuously growing in 10% FCS, suggest the important role for post-transcriptional control. The molecular interactions of eIF-4E protein and cyclin D1 mRNA in post-transcriptional regulation are not understood yet. However, high GC content in the 5`-untranslated region and possible secondary structures in the 5`-untranslated region probably do not play a crucial role in this regulation because most of the 5`-untranslated region is deleted in the MMTV cyclin D1 construct providing high levels of cyclin D1 mRNA (cD-10 cells, Figs 5 and 6) without corresponding increase in cyclin D1 protein levels.
Our findings that exogenously
overexpressed cyclin D1 mRNA accumulates in the nucleus (Fig. 6)
and the demonstration that the cytoplasmic/nuclear ratio for cyclin D1
mRNA is increased in eIF-4E overexpressing cells ()suggest
that expression of cyclin D1 is regulated in part by eIF-4E-facilitated
transport of cyclin D1 mRNA from the nucleus to cytoplasm. This is
consistent with the localization of eIF-4E in both the nucleus and
cytoplasm(33) . In addition, our findings that the total level
of cyclin D1 mRNA is greatly increased in serum-deprived NIH 3T3 cells
when they overexpress wild-type eIF-4E and the lack of down-regulation
of cyclin D1 gene transcription in serum-deprived 4E(P2) cells suggest
that eIF-4E is involved in the regulation of cyclin D1 gene by
increasing its transcription (Fig. 3, 4). Mitogen-inducible
regulatory elements in the cyclin D1 promoter have been identified (27) and transcription factors that bind to those elements
could be possible candidates for regulation by eIF-4E. In summary,
there seem to be at least two distinct steps where eIF-4E regulates the
expression of cyclin D1 gene: first is the level of cyclin D1 mRNA and
second is recruitment of this mRNA from the nucleus into polysomes.
The overexpression of cyclin D1 has been shown to accelerate the
transition of fibroblasts through the G period(28) . In addition, microinjection of cyclin D1
antibodies or treatment with cyclin D1 antisense oligonucleotide
prevented mitogen-activated NIH 3T3, Rat 2, or human lung fibroblasts
from entering the S phase of the cell cycle(28, 29) .
However, introduction of cyclin D1-overexpressing Rat 6 cells into nude
mice did not readily produce tumors because an increase in the number
of injected cells from 2
10
to 1
10
was required for raising the occurrence of tumors from 25 to 100%
of cases(30) . Cyclin D1 by itself was not able to transform
rat embryo fibroblasts and required cooperation with Ha-ras for
transformation(31) . Recent findings demonstrate that
overexpression of cyclin D1 in mammary glands of transgenic mice leads
to tumor formation in mice with a latency of 550 days(38) .
These data taken together suggest that increased cyclin D1 levels per se may be necessary but not sufficient for cell cycle
progression and tumorigenicity, and additional events have to occur to
bring about cell proliferation and transformation. Our analysis of the
cell cycle in serum-deprived cells demonstrated that marked increase of
cyclin D1 protein in cells overexpressing eIF-4E does not prevent them
from efficient cessation of their proliferation during first 6 days of
serum deprivation (Fig. 7). These data are in agreement with the
demonstration that cyclin D1-overexpressing Rat 6 fibroblasts withdraw
from the cell cycle almost as efficiently as control cells (30) and with the findings that increased expression of cyclin
D1 in response to the treatment of human diploid fibroblast with
defined mitogens is not sufficient for their entry into S
phase(32) . Remarkably, wild-type eIF-4E-overexpressing cells
seem to re-enter the cell cycle after 11 days of culture in 0.5% serum.
At this time control Ala and parental NIH 3T3 cells died out (data not
shown). One explanation is that eIF-4E-transformed cells secrete growth
factor(s) into the media(39) , which provide for their survival
and re-entry into the cell cycle when a threshold mitogenic
concentration is reached. In conclusion, our findings demonstrate that
eIF-4E can increase the expression of the important cell cycle
regulating protein, cyclin D1, by acting through both transcriptional
and post-transcriptional regulatory pathways. Finally, it is most
likely that cyclin D1 is only one of many growth-promoting proteins
whose expression is directly and/or indirectly regulated by translation
initiation factor 4E.