(Received for publication, February 2, 1995; and in revised form, June 5, 1995)
From the
Changes in the levels of cyclins A, D, and E, p21, and cyclin-dependent kinase 2 (CDK2) were examined in rat pheochromocytoma PC12 cells during neuronal differentiation induced by nerve growth factor (NGF). Expression of cyclin A decreased to an undetectable level after 5 days of exposure to NGF, while expression of CDK2 decreased gradually after day 3. In contrast, the levels of cyclins D1 and E increased gradually through day 10, yet the amount of cyclin E associated with CDK2 decreased concomitant with a decrease in the CDK2 protein level. p21 expression increased gradually after day 7, while the level of CDK2-associated p21 remained unchanged. When human cDNAs encoding cyclins and CDK2 were introduced into PC12 cells, only CDK2 overexpression inhibited NGF-induced differentiation. The cell lines overexpressing CDK2 showed stable and high levels of CDK2 kinase activity during differentiation, whereas parental and vector-transfected cell lines displayed a marked decline in CDK2 kinase activity 1 day after NGF treatment. In cell lines overexpressing cyclins A, D, and E, this reduction of the kinase activity was not apparent until day 3. These results suggest that down-regulation of CDK2 activity is a crucial event for the neuronal differentiation of PC12 cells.
The commitment to cellular differentiation is a highly
controlled stochastic process consisting of successive steps that
require specific signals for survival and simultaneous loss of
proliferative potential. The determination as to whether cells continue
to proliferate or differentiate appears to be executed during the
G phase of the cell cycle when several types of G
cyclins and cyclin-dependent kinases (CDKs) (
)interact
in various combinations (1, 2, 3, 4, 5, 6, 7) .
The D-type cyclins assemble primarily with CDK4, whose activity is
detectable at mid-G
and which increases as cells approach
the G
-S boundary(8, 9, 10) .
Cyclin E associates with CDK2 and induces maximal levels of kinase
activity at the G
-S
transition(11, 12, 13) . In addition, cyclin
A expression peaks at the G
-S boundary and accumulates in
early S phase, activating both CDK2 and
Cdc2(14, 15, 16) .
Cyclin-CDK complexes
are believed to play an essential role in the G-S
transition. Constitutive ectopic expression of cyclin D or E in normal
fibroblasts has been reported to shorten G
and reduce the
dependence of cells on growth factors (17, 18, 19, 20) . In addition,
microinjection of antibodies against cyclin D1 during G
phase prevents cells from entering S phase(17) .
Inhibition of the function of CDK2 has also been reported to prevent
the entry of cells into the S
phase(21, 22, 23) . The fact that cyclin-CDKs
play a crucial role in the G
phase implies that the
regulation of their functions is also critical for the commitment to
cell differentiation. Indeed, ectopic expression of D-type cyclins and
CDK4 was reported to inhibit granulocyte colony-stimulating
factor-induced differentiation of murine myeloid cells and
erythroleukemia cells, respectively(24, 25) .
In the present study, we investigated the roles of cyclins and CDK2 in the process of the neuronal differentiation of PC12 cells. In addition, we examined the expression of p21, a negative regulator of several cyclin-CDK complexes, including cyclin D-CDK4, cyclin E-CDK2, and cyclin A-CDK2(26, 27, 28) . Rat pheochromocytoma PC12 cells have proven to be a good model for neuronal differentiation due to their characteristic responsiveness to NGF. This response consists of partial growth arrest and acquisition of phenotypic properties typical of sympathetic ganglion, such as prominent neurite outgrowth(29) . Herein, we demonstrated that suppression of CDK2 activity is a critical step in the NGF-induced differentiation of PC12 cells.
Figure 1: Protein levels of cyclins A, D1, and E, CDK2, and p21 during NGF-induced neuronal differentiation of PC12 cells. a, lysates were prepared from cells that had been cultured in medium containing 20 ng/ml of NGF and harvested at the indicated times and were subjected to immunoblotting analysis to detect cyclins A, D1, and E, CDK2, and p21. b, histograms were generated by quantitating the intensity of the bands on nitrocellulose filters in panel a with a dual wavelength flying spot scanner. All values are indicated by ratios relative to those obtained on day 0. In the case of cyclin E and CDK2, the most slowly migrating bands were subjected to quantification.
Figure 2: Forced expression of ectopic cyclins A, D1, and E, CDK2, and p21 in PC12 cells. Representative clones and control clones (parental and vector-introduced cell lines) were lysed as described under ``Experimental Procedures.'' Lysates (50 µg of protein) were subjected to immunoblotting analysis to detect cyclins A, D1, and E, CDK2, and p21. The positions of three different forms of endogenous rat cyclin E are indicated by arrowheads. Ectopically expressed human cyclins, CDK2, and p21 are indicated by arrows.
Figure 3: Growth rates of cell lines. Growth rates of parental PC12 cells and derivative clones expressing cyclins A, D1, and E, CDK2, and p21 are shown in the absence (a) or presence (b) of NGF (20 ng/ml). For CDK2-expressing cell lines, only the growth curve of the representative clone K7 is shown because all 3 CDK2-overexpressing cells revealed quite similar growth rates. Cell numbers represent the mean values of triplicate experiments.
Figure 4: Morphological changes in cell lines. Morphological changes in PC12 cells and derivative clones in the absence (a) or presence (b, c, d) of NGF (20 ng/ml) are shown. Since the changes in morphology of the cell lines overexpressing cyclins A, D1, and E and p21 were quite similar, only representative photomicrographs of cyclin A-expressing cells are shown. For CDK2-expressing cell lines, only the photographs of clone K7 are shown because all 3 CDK2-overexpressing clones exhibited similar morphological features. e, f, 500 cells from the parental, vector-transfected, and cyclin A-overexpressing cell cultures were counted at the indicated times, and the number of cells having neurites with length more than twice (e), or 3 times (f) the diameter of the cell body were calculated as a percentage of cells counted. Since the data obtained with cell lines overexpressing cyclins A, D1, and E and p21 were quite similar, only the data obtained with cyclin A-expressing cells are shown.
Figure 5: Kinase activities associated with CDK2. a, CDK2-associated kinase activity in parental PC12 cells and derived cell lines expressing cyclins A, D1, and E, CDK2, and p21 during NGF treatment (20 ng/ml) are shown. Lysates prepared at the indicated times were immunoprecipitated with anti-CDK2 antibody. The immunocomplex was assayed for kinase activity using GST-RB fusion protein as a substrate. Since the changes in kinase activity of the cell lines overexpressing cyclins A, D1, and E and p21 were quite similar, only the results of a representative cyclin A-expressing cell line are shown. b, The histograms were generated by quantitating the intensity of radioactive signals on the x-ray films in panel a with a dual wavelength flying spot scanner. All values are indicated by ratios relative to those obtained on day 0.
Figure 6:
Levels of
CDK-associated cyclin A, cyclin E, and p21 during differentiation. a, lysates prepared from PC12 cells treated with NGF for the
indicated time periods were precipitated with p13 (for cyclin A and cyclin E) or immunoprecipitated with
anti-CDK2 antibody (for p21). The immunocomplex was subjected to
immunoblotting analysis with antibodies against cyclin A, cyclin E, and
p21. b, the histograms were generated by quantitating the
intensity of the bands on nitrocellulose filters in panela with a dual wavelength flying spot scanner. All values
are indicated by ratios relative to those obtained on day 0. In the
case of cyclin E, the most slowly migrating bands were subjected to
quantification.
In the present study, we examined alterations in the expression levels of cyclins, CDK2, and p21 during the NGF-induced neuronal differentiation of PC12 cells. Furthermore, we examined the effects of overexpression of these cell cycle regulators on the differentiation of PC12 cells. The results obtained from these experiments suggest that CDK2 is a key regulator of neuronal differentiation. The activity of CDK2 dramatically decreased following the addition of NGF, and constitutive overexpression of CDK2, but not of any cyclins tested, significantly blocked differentiation of PC12 cells. Ectopically expressed CDK2 probably exerted its effect by forming complexes with endogenous cyclins E and A and possibly with cyclins D2 and D3, i.e. those cyclins whose expression may be high enough to interact with CDK2(2, 8, 32, 35) . On the other hand, overexpression of cyclins A, D1, and E had a weaker effect. These findings are different from those previously reported using myeloid and erythroleukemia cell lines. Ectopic overexpression of cyclins D2 and D3 was reported to have inhibited granulocyte colony-stimulating factor-induced differentiation of murine myeloid cells. The mechanism of these results was attributed to the interaction of ectopically expressed cyclin D2 or D3 with CDK2, which was persistently expressed in that cell line(24) . Kiyokawa et al.(25) determined that the suppression of CDK4 expression is a critical event in the pathway of terminal differentiation of the erythroleukemia cell line MEL. In addition, Jahn et al.(36) reported that CDK2 activity does not change during differentiation of the mouse skeletal myogenic cell line C2C12. These differences may be due to cell type-specific molecular mechanisms of cell cycle modulation and differentiation.
Although the levels of CDK2 and cyclin A were found to be suppressed during NGF-induced differentiation, neurite outgrowth apparently preceded this decrease. Hence, suppression of the expression levels of CDK2 or cyclin A may not be of primary importance in the induction of differentiation.
In
contrast to CDK2 and cyclin A, the levels of cyclins D1 and E were
found to increase gradually during differentiation of PC12 cells.
Although overexpression of G cyclins has been reported to
accelerate G
phase in several cultured cell lines (17, 18, 19, 20) , the increase in
cyclin D1 and E expression observed at the later stages of
differentiation of PC12 cells may not be involved in the acceleration
of the G
/S transition; indeed, our experiments using
p13
precipitates showed a gradual decrease in the amount
of cyclin E associated with CDK. Similar up-regulation of cyclins has
been reported to occur during the differentiation of various cell
lineages. For example, the expression of cyclin D1 has been reported to
increase in neurons at the onset of rat brain maturation as well as
during differentiation of PC12 h cells (37, 38) .
During 12-O-tetradecanoylphorbol-13-acetate-induced
differentiation of human promyelocytic leukemia HL60 cells into
macrophage-like cells, cyclin D1 expression is also up-regulated,
although its expression is down-regulated in Me
SO-induced
granulocytic cells(39) . The expression of cyclin D3 is also
known to increase during differentiation of mouse erythroleukemia MEL
cells (25) and rat myoblast L6 cells(40) . In the
latter case, the kinase activity of the cyclin D3 complex, which
presumably includes CDK2 and CDK4, has been shown to be markedly
suppressed in the differentiated myotubes. A similar phenomenon was
also observed in senescent human diploid fibroblasts(41) .
Cyclin E-associated CDK2 activity is very low in senescent cells,
although the amounts of cyclins D1 and E are 10-15-fold higher
than observed in quiescent early passage cells. Taken together, these
findings raise the possibility that G
cyclins may play some
roles other than G
/S acceleration during cellular
differentiation. For example, it has been observed that the G
cyclins can form complexes with pRB or p107, although the
significance of these complexes is still
unclear(2, 42, 43, 44) .
Since
the changes in the expression levels of CDK2 and cyclin E do not seem
to be of primary importance in the down-regulation of CDK2 activity
during differentiation of PC12 cells, an alternative mechanism may
involve CDK inhibitor proteins(6, 7) . However,
expression of p21 was not significantly changed during differentiation
of PC12 cells. Furthermore, overexpression of p21 neither induced
differentiation nor growth retardation. Consistent with these results,
no significant suppression of CDK2 kinase activity was observed in
p21-overexpressing cells. One possible reason may be that the levels of
ectopically expressed p21 in these established cell lines was not high
enough to block the function of CDK2, although there was an
approximately 20-fold increase in p21 level as compared with the
parental cells. Presumably, cell lines overexpressing p21 at higher
levels were not established due to its cell proliferation-inhibitory
activity. In addition to p21, a growing number of CDK inhibitors, such
as p16, p15
, p27
and
p57
, have recently been
reported(6, 7, 45, 46) .
Interestingly, the TGF-
-mediated cell cycle block has been shown
to involve p15
and
p27
(6, 7, 47) . Additionally,
it has been reported that a protein, SNT, that appears as a doublet of
78-90 kDa in SDS-polyacrylamide gel electrophoresis gels and
which coprecipitates with p13
-agarose is rapidly
phosphorylated on tyrosine in neurons and PC12 cells treated with
differentiation factors but not in those treated with
mitogens(48) . Since p13
associates with
cyclin-CDK, this finding raises the possibility that SNT may link the
differentiation signal mediated by receptor tyrosine kinases to the
cell cycle regulator CDKs; i.e. SNT may act as a negative
regulator of CDKs. Thus, the contribution of CDK inhibitors as well as
of SNT to the NGF-induced inhibition of CDK2 activity remains to be
elucidated in future studies.
Cyclin-CDK is believed to
phosphorylate cellular proteins important for cell cycle
regulation(6, 7) . One of the main targets of
cyclin-CDK is pRB, which negatively regulates cell cycle progression
through the G phase(31, 43) . pRB also
plays a crucial role in early neuronal and hematopoietic development as
demonstrated by the analysis of mice carrying a targeted mutation in
the RB gene(49, 50, 51) . The amount of
underphosphorylated pRB, which is believed to be the active form of
pRB, is increased by extracellular signals, which induce cell cycle
arrest and differentiation(52, 53) . The results
obtained in this study suggest that the NGF-induced reduction in CDK2
activity may be responsible for the accumulation of the
underphosphorylated form of pRB during differentiation of PC12 cells.
Accordingly, constitutive overexpression of CDK2 may block
differentiation by driving the phosphorylation of pRB. Consistent with
this notion, ectopic overexpression of adenovirus E1A, which associates
with and inactivates pRB as well as p107, has been demonstrated to
inhibit NGF-induced neuronal differentiation of PC12
cells(54, 55, 56) . Further detailed analysis
of the function of pRB, as well as the identification of other CDK2
substrates, may prove important for our understanding of the precise
mechanism by which PC12 cells differentiate.