(Received for publication, October 11, 1995; and in revised form, February 15, 1996)
From the
Several hepatocyte mitogens have been identified, but the
signals triggering the G/G
transition and cell
cycle progression of hepatocytes remain unknown. Using hepatocyte
primary cultures, we investigated the role of epidermal growth
factor/pyruvate during the entry into and progression through the
G
phase and analyzed the expression of cell cycle markers.
We show that the G
/G
transition occurs during
hepatocyte isolation as evidenced by the expression of early genes such
as c-fos, c-jun, and c-myc. In culture,
hepatocytes progress through G
regardless of growth factor
stimulation until a restriction point (R point) in mid-late G
beyond which they cannot complete the cell cycle without
mitogenic stimulation. Changes in cell cycle gene expression were
associated with progression in G
; the cyclin E mRNA level
is low early in G
but increases at the G
/S
boundary, while the protein is constantly detected during cell cycle
but undergoes a change of electrophoretic mobility in mid-late G
after the R point. In addition, a drastic induction of cyclin D1
mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place
in mitogen-stimulated cells after the R point. In contrast, cyclin D3
mRNA appears early in G
, remains constant in stimulated
cells, but accumulates in unstimulated arrested cells, paralleling the
cyclin-dependent kinase 4 mRNA expression. These results characterize
the different steps of G
phase in hepatocytes.
In the normal liver, hepatocytes can remain for very long
periods in a quiescent G state. However, they have the
capacity to proliferate after chemical intoxication or partial surgical
resection of the liver (Higgins and Anderson, 1931). Following a
two-thirds hepatectomy (PHT), (
)hepatocytes rapidly enter
the cell cycle and begin their first round of DNA replication
18-20 h later (Fabrikant, 1968). An active field of research
during the last 15 years dealt with the identification of factors able
to promote hepatocyte DNA synthesis and to understand this compensatory
growth. It has been known for some years that hepatocyte growth factor
(HGF) and transforming growth factor-
(TGF-
) are primary
mitogens during liver regeneration after partial hepatectomy or
administration of CCl
(Mead and Fausto, 1989;
Michalopoulos, 1990). Mullhaupt et al.(1994) recently reported
a rapid increase of EGF levels in the immediate early phase of liver
regeneration. Furthermore, HGF, TGF-
, and EGF are well
characterized mitogens for hepatocytes in primary culture. However, how
the hepatocyte cell cycle is controlled by these external factors
remains to be clarified.
It is well established that, in
vivo, normal hepatocytes are largely unresponsive to growth
factors and become competent only after ``priming'' induced
by specific treatments such as partial hepatectomy, necrosis following
injury, metabolic stress, or any phenomenon leading to disruption of
cell-cell contacts (Etienne et al., 1988; Sawada, 1989; Ikeda et al., 1989) or digestion of the extracellular matrix (Liu et al., 1994). These metabolic events would trigger the
G/G
transition of hepatocytes in vivo and increase the expression of growth factors, which then induce
DNA synthesis. This hypothesis is based on the fact that induction of
immediate-early oncogenes such as c-fos or c-jun (Corral et al., 1985; Thompson et al., 1986;
Sobczack et al., 1989; Morello et al., 1990), takes
place 20-30 min after PHT, while HGF level rises around 2 h post
PHT (Lindroos et al., 1991). Previous in vitro studies have also shown that hepatocytes express immediate-early
oncogenes during cell isolation and in primary culture, in the absence
of mitogens. On the other hand, it was clearly established that these
``primed'' hepatocytes would undergo DNA synthesis only when
they are stimulated by growth factors (McGowan, 1986; Sawada 1989). We
therefore decided to determine whether unstimulated hepatocytes are
blocked at a given point in G
, and how stimulated
hepatocytes modulate their response to different mitogenic signals in
terms of DNA synthesis.
The G phase has been divided
into subphases (Pledger et al., 1977, 1978; Tushinski and
Stanley, 1985) during which external signals must impinge on the
machinery that regulates the G
to S phase transition. In
fibroblasts, Pardee(1992) has also described a point in G
at which the cells have acquired growth factor independence. He
called it the restriction point or R point. It was located near but not
concomitant with the initiation of S phase. However, comparison of the
data obtained from different in vitro models clearly
emphasizes the notion that there are several different regulatory
points in G
and that each cell type would be defined by a
specific behavior in G
characterized by its own check
point(s) (Pardee, 1992).
Many attempts have been made to identify
the proteins which control the progression of the cell cycle through
these G check points. Of the proteins characterized to
date, the cyclin-dependent kinases (Cdks) and their cyclin partners
play a crucial role in cell cycle regulation (Sherr, 1993). Cyclins
bound to Cdc2 or Cdk2 appear to be involved in regulating DNA
initiation and/or synthesis (Pagano et al., 1992; Zindy et
al., 1992) and G
/M transition (Pagano et al.,
1992; Sherr, 1993), respectively. The cyclin E-Cdk2 complex is
activated at the end of G
and is considered to be a
limiting step at the G
/S boundary (Koff et al.,
1991; Dulic et al., 1992; Sherr, 1993). The D type cyclins
also play a crucial role in G
via their association with
Cdk2, Cdk4, and Cdk6 (Xiong et al., 1992; Baldin et
al., 1993; Quelle et al., 1993; Meyerson and Harlow,
1994). The sequential activation of these complexes and their substrate
specificities could be the key to their regulatory function throughout
the G
phase (Ajchenbaum et al., 1993; Sherr,
1993).
Analysis of the expression and activation of Cdc2 and Cdk2 in
regenerating liver revealed that Cdc2 was expressed and active in S,
G, and M phases but not in G
, whereas Cdk2 was
constantly expressed during the cell cycle but inactive in G
(Loyer et al., 1994). The expression and role of the
different cyclins and their corresponding Cdk partners in the
hepatocyte cell cycle is poorly documented.
In the present report,
we used primary cultures of normal rat hepatocytes that were
unstimulated or stimulated by EGF to define the different subphases of
G and to examine the related expression of various cell
cycle markers including proto-oncogenes, cdks, and cyclins.
Figure 1:
Time course of
[H]methylthymidine incorporation into DNA in
unstimulated and EGF/pyr-stimulated primary hepatocyte cultures.
Hepatocytes were maintained under unstimulated conditions
(
-
) or in the presence of EGF/pyr
(
-
). Cultures were incubated with 2 µCi/ml
[
H]methylthymidine (2-h period) either at seeding
or every 8 h for 48 h; and thereafter every 2 h for 94 h. EGF/pyr was
added just after cell seeding and every day thereafter with renewal of
medium. Cultures were made in duplicate.
Figure 2: Levels of c-jun, c-fos, and c-myc mRNAs in hepatocytes during liver dissociation and in primary culture. Total RNAs (20 µg) extracted from normal liver (1), liver after Hepes perfusion (2), or after collagenase perfusion (3); hepatocytes after the first (4) and the second wash in Hepes buffer (5); freshly isolated hepatocytes (6) and hepatocytes 4 h after seeding (7) were analyzed by Northern blot hybridization with cDNA probes for c-jun, c-fos, c-myc, albumin (Alb), and 18 S as a control of the total amounts of RNAs in each lane.
Figure 3: Northern blot analysis of proto-oncogene activation in primary culture of rat hepatocytes continuously stimulated by EGF/pyr. Total RNAs (20 µg) extracted from normal liver (biop), freshly isolated hepatocytes (0), and primary hepatocyte cultures at 6, 24, 48, 72, and 96 h, were analyzed by Northern blot hybridization with cDNA probes for c-myc, c-jun, junB, junD, c-Ki-ras, and p53, and albumin (Alb) as control.
We next addressed the question whether the unstimulated as
well as the EGF-stimulated cells were able to progress through
G, by analyzing the mRNA levels of c-myc and p53
which were used as markers of early and mid G
respectively.
The two cultures expressed c-myc and p53 mRNAs at similar
levels and at the same time. However, at day 3, both mRNAs gradually
disappeared in EGF-treated cells (Fig. 4A).
Figure 4:
A, comparison of c-myc, p53,
and cdc2 mRNA levels in unstimulated and stimulated
hepatocytes. Each well was loaded with 20 µg of total RNAs isolated
from unstimulated (1-4) or EGF/pyr-stimulated (5-8) hepatocytes at 24 (1, 5), 48 (2, 6), 72 (3, 7), and 96 h (4, 8) of culture. After electrophoresis and transfer
onto nylon sheet, RNAs were hybridized with c-myc, p53, and cdc2 cDNA probes and 18 S as a control of the total amount of
RNAs in each lane. EGF/pyr was added just after seeding and every day
thereafter with renewal of medium. B, cdc2 expression in
primary cultures of rat hepatocytes stimulated by EGF/pyr. cdc2 expression was investigated at the indicated times in hours, at
the mRNA level (cdc2 mRNA) by Northern blot, and protein level
(p34) by Western blot, and kinase activity was
determined using histone H1 as substrate.
We used
Cdc2 and the histone H1 kinase activity associated with p9-beads, as
markers to see whether unstimulated and EGF/pyr-stimulated hepatocytes
in culture underwent the G/S transition (Fig. 4, A and B). We also analyzed the cdc2 expression at the mRNA level. In the absence of EGF/pyr, cdc2 transcripts were detected as a faint band, if any (Fig. 4A). In contrast, in the continuous presence of
EGF/pyr, cdc2 mRNA was detected between 48 and 54 h post
plating and was highly expressed at 72 h, while the protein was first
detected at 60 h and maximally expressed at 84 h (Fig. 4B). Cdc2 binds to p9 regardless its
phosphorylation status or association with cyclins. Therefore, the Cdc2
Western blot after p9 purification represented the real Cdc2 expression
in total cell lysates. The kinase activity, measured by the ability of
the Cdc2-related proteins (Cdc2 and Cdk2) to phosphorylate histone H1,
was detected from 60 h and reached a maximum between 72 and 84 h,
correlating mainly with S and M phases as we previously observed in
regenerating liver (Loyer et al., 1994).
These results led
us to conclude that, under conditions of continuous EGF/pyr
stimulation, most hepatocytes reached the G/S boundary
approximately 58-60 h after seeding. In contrast, in the absence
of mitogenic factor, hepatocytes entered the G
phase,
progressed up to mid-G
but failed to complete the
G
/S transition.
Figure 5:
Effects on hepatocyte DNA synthesis of
24-hr EGF/pyr stimulations performed at different times of culture.
Hepatocyte primary cultures were exposed to EGF/pyr during 24 h at
different times: cell seeding (A) and 24 (B), 48 (C), and 72 h (D) of culture.
[H]Methylthymidine incorporation was performed
for 2-h periods in the unstimulated (
-
)
and EGF/pyr (
-
)-stimulated cultures and was
measured every 2 h during 120 h of culture. Values are expressed as
counts/min/µg of protein and are means of triplicate
cultures.
These data clearly show that hepatocyte entry in
S phase could be delayed by late mitogenic stimulation. The results
also suggest that the cells were blocked and arrested at an R point
presumably located between 24 and 48 h. In addition, they indicate that
hepatocytes remained responsive to EGF/pyr treatment for at least three
days of culture. To further define this R point, we performed the
following experiment. Hepatocytes were exposed to EGF/pyr for a 12-h
period and at different times between 24 and 54 h of culture.
[H]Methylthymidine incorporation was measured
every 6 h from 24 to 84 h of culture (Fig. 6). It appeared that,
irrespective of the time of EGF/pyr stimulation prior to 42 h, DNA
synthesis always started at 58-60 h of culture. In contrast,
later addition of the mitogen, for example at 48 and 54 h, delayed cell
entrance in S phase, indicating that the R point was located between 42
and 48 h, in mid-late G
.
Figure 6:
Effects on hepatocyte DNA synthesis of
12-h EGF/pyr stimulations initiated serially in mid and late
G. EGF/pyr was added to primary cultures at different
times: 24 (
-
), 30
(
-
), 36 (
-
), 39
(
-
), 42 (
- - - - -
), 45
(
- - - - -
), 48 (
- - - - -
), and 54 (
-
- - - -
) h after seeding. For each condition,
[
H]methylthymidine incorporation was performed
for 6-h periods and followed for 30 h. Cultures were made in duplicate. Inset, comparison of DNA synthesis in hepatocyte primary
cultures unstimulated or stimulated by EGF/pyr, TGF-
/pyr, or
FCS/pyr. Hepatocytes were maintained in basal conditions for 48 h, then
stimulated during 24 h by EGF/pyr (
-
),
TGF-
/pyr (
-
), or FCS/pyr
(
-
). For each condition, 2 µCi/ml
[
H]methylthymidine were incubated for 8-h
periods, and incorporation was measured every 8 h during 104 h;
(
-
): unstimulated cultures as control.
Cultures were made in duplicate.
In order to address the
question whether different growth factors, known to be overproduced
during liver regeneration process, were like EGF, capable of overriding
the hepatocyte block in mid-late G, we compared the DNA
synthesis of primary cultures stimulated by EGF/pyr, TGF-
/pyr, and
FCS/pyr. Four h after seeding, cells were maintained in a mitogenic
factor-free medium for two days and then, stimulated by EGF/pyr,
TGF-
/pyr, or FCS/pyr for a 24 h period. An active DNA replication
simultaneously started in the EGF- and TGF-
-stimulated cultures
approximately 64 h after seeding and displayed similar kinetics, with
maximum DNA synthesis occurring 80-88 h post seeding. In
contrast, FCS did not significantly induce DNA synthesis (Fig. 6, inset).
Figure 7:
Hepatocyte DNA replication in response to
EGF/pyr pulse stimulations of increasing lengths. Hepatocyte primary
cultures were maintained during 48 h in basal conditions and then,
stimulated by EGF/pyr for 1, 6, 12, 18, 24, 30, 36, or 42 h (abscissa). [H]Methylthymidine
incorporation was measured every 6 h up to 90 h. Each point corresponds
to the cumulative values of incorporation in counts/min/µg of
protein, obtained for each stimulation condition, during the culture
period between 48 and 90 h; C, control of basal incorporation,
defined as the cumulative values of incorporation, in counts/min/µg
of protein, obtained in unstimulated cells during the same culture
period (48-90 h). Cultures were made in
triplicate.
Differences in the amounts of
[H]methylthymidine incorporated into the cells
were also correlated with the time of culture at which the mitogenic
factor was added (see Fig. 5and Fig. 6), suggesting that
the hepatocyte response to EGF/pyr could vary during the G
phase. Indeed, when EGF/pyr was added for a 24-h period at
different times after seeding, the DNA synthesis was low in cultures
stimulated just after plating and increased for later stimulations, and
the maximal incorporation was observed when stimulation took place at
36 or 42 h, near the R point (data not shown).
These data indicate
that the ability of hepatocytes to enter S phase, in response to
mitogen, varied according to the length of stimulation and the location
of the cells in G. This led us to consider the possibility
that these variations could be associated with changes in the major
cell cycle control proteins which regulate the G
progression.
Figure 8: A, Northern blot analysis of the different Cdk and cyclin mRNA levels in unstimulated and EGF/pyr-stimulated hepatocytes during 72 h of culture. Total RNAs (20 µg) were extracted from freshly isolated hepatocytes (0), unstimulated primary cultures (-EGF/pyr) at 18, 24, 36, 48, 60, 66, and 72 h, and stimulated primary cultures (+EGF/pyr) at 27, 30, 36, 42, 48, 60, 66, and 72 h. EGF/pyr was added at 24 h and maintained during culture. RNAs were analyzed by Northern blot hybridization with cdc2, cdk2, cdk4, cyclin A, cyclin E, cyclin D1, D2, and D3 cDNA probes and 18 S as a control of the total amounts of RNAs in each lane. B, Western blot analysis of cyclins D1 and E in primary hepatocyte cultures either unstimulated or stimulated by EGF/pyr at 24 h. a, total proteins at the indicated times were subjected to Western blotting with anti-cyclin D1 antibodies. b, proteins were purified with p9CKShs1-Sepharose beads and subjected to Western blotting with anti-cyclin E antibodies. C+, cyclin E control from p13suc1 Xenopus extracts; C-, bovine serum albumin bound to p13suc1-Sepharose beads; 24*, regenerative rat liver extract at 24 h.
To study the correlation
between mRNAs and corresponding proteins we further analyzed the
expression of cyclins D1 and E by Western blotting (Fig. 8B). These two cyclins were chosen since their
mRNA levels were increased at two different times of G phase, respectively the R point (mid-late G
) and the
G
/S boundary. Cultures were performed following the same
protocol as in Fig. 8A. Cyclin D1 was very low in
unstimulated cultures. In stimulated cells, it strongly increased at
around 60 h and until 96 h, appearing shortly after the induction of
the mRNAs. Cyclin E was expressed in both unstimulated and stimulated
cultures with very low quantitative variation but displayed a shift in
electrophoretic mobility in mitogen-stimulated cells only.
To
determine whether induction of cyclin D1 was associated with the
ability of the cells to progress in late G after the R
point, hepatocytes were exposed to EGF/pyr for a 24-h period as early
as 6 h or 24 h after plating, or as late as 48 or 60 h (Fig. 9).
These late stimulations resulted in a delayed transition to S phase, as
expected by data shown in Fig. 5. The amounts of cyclin D1 mRNAs
drastically increased in all conditions, and their location in G
and the levels of the peaks of expression varied according to the
stimulation: when cells were stimulated at 6 h, the peak was clearly
seen at 24-30 h, but at a low rate because of the poor synchrony
and the lower number of cells, which progressed up to S phase in these
conditions. The maximal expression was observed for exposition to the
mitogen at 48 h, a condition corresponding to maximal DNA synthesis.
The occurrence of induction was always located after mitogenic
stimulation and, therefore, was delayed in cultures lately exposed (at
24, 48, and 60 h) to the mitogen. Interestingly, the time between
stimulation and increased cyclin D1 mRNA level was dramatically
reduced, for instance from a 24-h to a 6-h period, as this stimulation
occurred later in G
(Fig. 9, A and D). All of these data clearly showed the association of cyclin
D1 induction with progression in late G
after the R point.
In addition, no induction of cyclin D1 could be observed either after
collagenase treatment, cell or tissue washing in Hepes buffer, or cell
seeding in culture medium (data not shown).
Figure 9:
Modulation of cyclin D1 induction
according to the R point overcrossing. Hepatocytes were exposed to
EGF/pyr for a 24-h period either early, at 6 (A) and 24 (B) h, or late, at 48 (C) and 60 h (D) of
culture. [H]Methylthymidine incorporation was
measured every 6 h up to 84 h. Total RNAs (10 µg) were extracted
from culture samples corresponding, for each condition, to mid and late
G
and early S phase, and analyzed by Northern blot
hybridization with cyclin D1 cDNA probe.
In order to understand the molecular and cellular mechanisms
involved in liver regeneration and its controls by growth factors, we
isolated normal rat hepatocytes and analyzed their cell cycle
progression under mitogenic stimulation in an in vitro system.
Using this experimental approach, we confirmed that collagenase
perfusion of the liver triggers the G/G
transition of quiescent normal rat hepatocytes. In addition, we
show that cultured hepatocytes were able to progress from early G
to a restriction point located in mid-late G
,
regardless of growth factor addition. We demonstrate that this
progression in G
is essential to make hepatocytes entirely
competent to EGF and TGF-
signals. We also demonstrate that
mitogens do play a crucial role in allowing the cells to override this
restriction point and enter the S phase. Furthermore, there was
characteristic kinetics of expression of the G
cell cycle
proteins that were associated with cell cycle progression.
It is
generally assumed that division of mammalian cells is mainly controlled
during the G phase by signals from the external environment
(Pardee, 1992) varying from one cell type to another, defining for each
cell type, different characteristic check points. In normal liver, in vivo, hepatocytes are arrested in G
. One
hallmark of the G
/G
transition is the
sequential overexpression of immediate early and early proto-oncogenes
such as c-fos, c-jun, c-myc, and p53 (Corral et al., 1985; Thompson et al., 1986; Sobczack et
al., 1989; Morello et al., 1990). However, the factors
which control this transition remain poorly understood. One hypothesis
is that hepatocyte re-entry into G
after PHT is a
consequence of metabolic changes regardless of growth factors (Corcos et al., 1987; Fausto, 1992). These metabolic changes could be
associated with alterations in cell-cell interactions (Etienne et
al., 1988). Also the possibility that early activation of a growth
factor occurs via a cascade of events related to alteration of the
extracellular matrix, cannot be ruled out. Indeed, Liu et
al.(1994) have shown that partial degradation of the extracellular
matrix of the liver in vivo could also trigger the
G
/G
transition.
It may be argued that normal
rat hepatocytes, when seeded in culture, have already entered the early
G. In this study, we show that they also progress up to
mid-late G
, in the absence of growth factor and serum in
the medium. This is consistent with the sequential overexpression of
the early G
oncogenes c-fos and c-jun during collagenase perfusion, followed by c-myc and junB expression, 4-6 h after seeding, and finally, of
c-Ki-ras and p53 expression, after 24 h of culture. In
addition, in the absence of mitogen signal, these cells were found to
be arrested in mid G
and further progression to the
G
/S boundary was strikingly dependent on growth factor
addition. This was supported by the following observations: 1) in the
absence of mitogen, cyclins D1 and D2 were low, cyclin A and Cdc2 were
not expressed, and DNA synthesis was not observed; and 2) the onset of
DNA synthesis was delayed by late addition of EGF on days 2 and 3, and
a sharp peak of labeling was observed in these conditions, reflecting
the high synchrony of the hepatocyte population arrested at the R
point.
From our data, we estimate that the mitogen-dependent R point
in rat hepatocytes occurs at the end of the first two-thirds of the
G phase, approximately 42-48 h after seeding under
our conditions. Considering effects of early and late exposure of
hepatocytes to EGF/pyr, the lag time between the R point and the onset
of DNA synthesis appears to be approximately 18-20 h. This R
point has some similarities with the start point of the yeast
G
/S transition; in both systems, the R point occurs late in
G
, close to but distinct from the G
/S boundary,
and both are dependent on external signals (Reed, 1992).
In
fibroblasts, G progression requires growth factors (Campisi et al., 1982; Croy and Pardee, 1983), but after the R point,
they become growth factor-independent (Yen and Pardee, 1978; Zettenberg
and Larsson, 1985). In addition, fibroblasts require at least two
factors, platelet-derived growth factor and EGF or insulin-like growth
factor, to stimulate the transition of quiescent cells from G
to G
and from G
to S phases,
respectively. In contrast, in murine macrophages, one growth factor,
the colony-stimulating factor 1, is sufficient to induce transition
from quiescence to S phase (Tushinski and Stanley, 1985). Moreover, in
this model, colony-stimulating factor 1 must be present throughout
G
in order to maintain cell cycle progression until S
phase. Here we demonstrate that, for cell cycle progression of
hepatocytes, growth factor is required in G
for awhile, to
override the R point. The fact that G
/G
transition of hepatocytes occurs spontaneously during collagenase
perfusion and that G
/S transition takes place only after
stimulation of the cells by growth factors strongly argues for a dual
factor requirement to trigger these two transitions. A first
unidentified factor, synthetized and/or secreted or released from the
extracellular matrix (Liu et al., 1994) during the tissue
disruption, would induce G
/G
transition but not
G
/S transition. This last step would require a growth
factor acting later in mid-late G
. Taken together, these
results define a specific behavior of hepatocytes regarding the cell
cycle progression in G
in relation with growth factor
stimulation.
It is generally assumed that G cyclins and
their corresponding Cdk(s) are integrators of growth factor-mediated
signals that drive the cell cycle. However, with the exception of three
studies (Lu et al., 1992; Zindy et al., 1992;
Albrecht et al., 1995), there is no report describing the
kinetic expression of different G
proteins in hepatocytes
and their association with different steps in G
has never
been defined in these cells. We confirm the expression of cyclin A and
Cdc2 at the S phase entry and showed that cdk2 mRNA is
expressed throughout G
with an important increase late
after growth factor stimulation, whereas the H1 kinase activity was
mainly detected in S and M phases. The cyclin E mRNAs were found as a
weak band in unstimulated culture and their level greatly increased in
late G
only after mitogenic stimulation. It may reflect an
activation of this cyclin only in cells which have overcrossed the R
point. In addition, cyclin E Western blot was performed on
p9-Cdk-cyclin affinity purified complexes. Since this protein does not
bind p9 directly but via its binding to a Cdk, likely Cdk2 as reported
by Koff et al.(1991) and Dulic et al.(1992), we may
assume that the kinetic corresponded to the levels of cyclin E
complexed with Cdk2 along the cell cycle. We did not find significant
variation in the level of cyclin E protein, but interestingly, a change
in the electrophoretic mobility, probably related to a modification of
its phosphorylation status, was evidenced in mid-late G
after mitogenic stimulation. This likely reflects an activation
of the complex in cells which have overcrossed the R point.
In
contrast, we show that cyclin D1 mRNA levels and protein expression are
both greatly increased after mitogenic stimulation. Accumulation of
mRNAs correlated with the R point onset, whereas the cyclin D1 protein
was detected 10-15 h later. The induction of cyclin D1 mRNAs
correlated well with that located in mid-late G in
regenerating liver (data not shown). Albrecht et al.(1995)
have also reported that cyclin D1 protein was abundantly induced in
vitro in HGF stimulated hepatocytes. A similar induction of cyclin
D1 has also been reported in macrophages (Matsushime et al.,
1991); the peptide could not be immunoprecipitated from
colony-stimulating factor-1-starved macrophages blocked in G
while it accumulated early after the mitogen addition. However,
we do not know whether between cyclin D1 mRNA and protein appearance
cells progress in cell cycle or if they are arrested until the protein
is expressed.
Interestingly, we observed that this drastic cyclin D1
mRNA activation is dependent on the growth factor stimulation and
coincided with hepatocyte progression beyond the R point in response to
this stimulation. Thus, if progression beyond the R point was delayed
by late mitogen stimulation, cyclin D1 induction was delayed in
parallel. This result is in agreement with the hypothesis that cyclin
D1 plays a critical role in cell progression in late G after the R point, and contributes to the so-called cell cycle
clock (Sherr, 1993) by ensuring the commitment of cells to enter S
phase.
In contrast, cyclin D3 appears to accumulate in the absence of mitogen signal, whereas cyclins D1 and D2 are weakly detected, indicating that the three D type cyclins are differentially activated throughout the cell cycle. These D type cyclins have been previously reported to be differentially expressed in various cell lineages, and it has also been suggested that their roles are distinct (Ajchenbaum et al., 1993; Kato and Sherr, 1993; Sherr, 1994). Our results are in agreement with these hypotheses. It is intriguing to note the correlation between the accumulation of cyclin D3, cdk4, and to a lesser extent cdk2 mRNAs, in the absence of mitogen stimulation. The role of these cyclins accumulated in unstimulated cells must be further analyzed.
Altogether, these observations are consistent with the idea that the D-type cyclins may be multifunctional regulators that could target different Cdk partners (Sherr, 1993, 1994). Experiments are now in progress to determine which of these Cdks directly associate with the cyclins to form complexes that serve to link growth factor stimulation with cell cycle progression of hepatocytes and which activators or inhibitors may control this process.