PII: S0165-3806(98)00032-7
Developmental Brain Research 108 1998 7787.Research
reportRegulation of cyclin dependent kinase inhibitor proteins
during neonatal cerebella developmentGenichi Watanabe a, Pilar Pena
a, George E. ShambaughIII b, George K. HainesIII c,Richard G.
Pestell a,)aThe Albert Einstein Cancer Center, Department of
Medicine and Department of Deelopmental and Molecular Biology,
Albert Einstein College ofMedicine, Chanin 302, 1300 Morris Park
Ae., Bronx, NY 10461, USAbNorthwestern Uniersity Medical School,
Veterans Affairs Lakeside Medical Center, 303 E. Chicago Ae.,
Chicago, IL 60611, USAcDepartment of Pathology, Northwestern
Uniersity Medical School, 303 E. Chicago Ae., Chicago, IL 60611,
USAAccepted 3 February 1998
AbstractThe cyclin dependent kinase holoenzymes CDKs , composed
of catalytic cdk and regulatory cyclin subunits, promote cellular .
. . proliferation and are inhibited by cyclin dependent kinase
inhibitor proteins CDKIs . The CDKIs include the Ink4 family.
p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d. and the KIP family p21Cip1
and p27Kip1.. The sustained induction of p21 and p18 during
myogenesis implicates these CDKI in maintaining cellular
differentiation. Herein we examined the CDK cyclin D1, cdk5 and
CDKI expression . profiles during the first 24 days of postnatal
rat cerebella development. Cdk5 abundance increased and cyclin D1
decreased from day 9 through to adulthood. The CDKIs increased
transiently during differentiation. p27 increased 20-fold between
days 4 and 24, whereas p21 rose twofold between 6 to 11 days. p19,
p18 and p16 increased approximately two- to threefold, falling to
low levels in the adult. Immunostaining of cyclin D1 was localized
in the external granular cells, whereas p27, was found primarily in
the Purkinje cells. The period of maximal differentiation between
days 9 to 13 was associated with a change in p21 and p16 staining
from the external granular and Purkinje cells to a primarily
Purkinje cell distribution. Protein-calorie malnutrition, which was
previously shown to arrest rat cerebella development, reduced
cyclin D1 kinase activity and p27 levels. However, p16 and p21
levels were unchanged. We conclude that the CDKIs are induced with
distinct kinetics in specific cell types and respond differentially
to growth factors during cerebella development, suggesting discrete
roles for these proteins in normal cerebella development. q1998
Elsevier Science B.V. All rights reserved.
84G. Watanabe et al.rDeelopmental Brain Research 108 1998
7787()G. Watanabe et al.rDeelopmental Brain Research 108 1998
7787()85
Keywords: Cell-cycle; Cyclin dependent kinase inhibitor protein;
Cerebella
IntroductionThe family of cyclin dependent kinases CDKs and the
. associated cyclin dependent kinase inhibitor CDKI pro- . teins
play a critical role in cellular proliferation and differentiation
61 . The CDK holoenzymes are composed of aw x catalytic and
regulatory subunit which bind and phosphorylate target substrates
including the retinoblastoma protein pRB . The cyclin D1 gene
encodes a labile mitogen-in-. ducible regulatory subunit of the cdk
holoenzyme required for normal progression through the early G1
phase of the cell-cycle 44,46,61 . Cyclin D1 is expressed primarily
inw x
)
Correspondingauthor.Fax:q1-718-430-8674;E-mail:[email protected]$19.00
q 1998 Elsevier Science B.V. All rights reserved.the developing
brain, the sympathetic nervous system and in the external granular
layer of the developing cerebellum which gives rise to neurons
within the molecular layer w41,47,53 . Transgenic mice with
homozygous deletion ofx the cyclin D1 gene develop symptoms of
neurological impairment 47 and fail to develop normal retina 15,47
.w xw x The retina is reduced in thickness with hypoplasia of the
ganglion cells, nuclear and plexiform layers 15 , suggest-w x ing
cyclin D1 is required for normal cell number and neural development
47 . Cdk5 was previously shown tow x associate with cyclin D1 in WI
38 cells w63x and cdk5 prepared from brain exhibits pRB kinase
activity w55 .x Cdk5 abundance and activity are increased during
neuronal differentiation and in postmitotic neurons 25,26,56 .
cdk5w x activity is required for neurite outgrowth in cultured
cortical neurons 32 , and heterozygous deletion of the cdk5w x gene
w34x severely disrupted the cytoarchitecture of the cerebellum and
cerebrum. Together these findings indicate that cdk5 and cyclin D1
can play an important role in obtaining the differentiated neural
phenotype.The activity of the cyclin dependent kinases cdks. is
modulated in vitro by two families of inhibitor proteins. The Ink4
family, which contains a highly conserved motif of ankyrin rings,
includes p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d. The Ink4
proteins are specific inhibitors of Cdk4 and Cdk6 activity w20,40 .
The second family, the Cipx proteins, includes p27Kip1 and p21Cip1.
p27 and p21 are inhibitors of Cdk2 and Cdk4 in vitro and bind with
high affinity to cyclin D1 19,35,49,54 . The Cip proteins alsow x
function as assembly factors and regulate subcellular
compartmentalization of the cdks suggesting complex functions of
these proteins 24,39 . The abundance of both thew x Ink4 and Cip
proteins are induced during paradigms of cellular differentiation.
During oligodendrocyte differentiation p27 levels increase and
recent studies of mice homozygously deleted of the p27 gene have
suggested an important role for p27 as an inhibitor of neural
cellular proliferation 9 . p21 was induced during differentiation
ofw x myocytes w19,35,49 ,x hepatic and hematopoietic cells w23,51
and neural cell lines 38,64 . In myelomonocyticx w x cells,
differentiation induced by 1,25-dihydroxyvitamin D3 was associated
with an induction of p21 and overexpression of p21 induced a
component of the differentiated phenotype 28 . During myocyte
differentiation p18 levelsw x were increased and p19 levels were
decreased 16 . Cyclinw x D1 levels were decreased during myocyte
differentiation, and overexpression of cyclin D1 antagonized
components of the myocyte differentiation process 49 . These
studiesw x suggest that differentiation occurring in cultured cells
is the result of a dynamic interplay between the abundance of the
cdk holoenzymes and the CDKIs.The neonatal cerebellum represents an
ideal in vivo model to study the role of the CDKs and CDKIs in
normal neural development. Two main characteristics of this in vivo
system contribute to the utility of this model in understanding the
mechanisms of neural development. Firstly, the cerebellum undergoes
highly reproducible spatial and temporal profiles of neural
differentiation. Secondly, development of the cerebellum can be
arrested in a reproducible manner using maternal starvation; thus,
the mechanism of starvation induced developmental arrest can be
analyzed in detail. The developing cerebellum contains both
proliferating cells and differentiating cells which occupy specific
topographic positions 36,17 . During nor-w x mal postnatal
cerebellar development the cerebellar cortex is composed of the
external granular layer, molecular, Purkinje cell and internal
granular layers w36,17 . Thex external granular layer is composed
of proliferative and pre-migratory zones. Proliferation in the
external granular layer continues up to day 9 and then gradually
decreases up to day 21. Upon the completion of mitosis, the
external granular cell descends to the pre-migratory zone. The cell
soma then undergoes dramatic elongation perpendicular to the
previous cellular axis to pass through the molecular layer and
thereby reach the internal granular layer, whereupon it extends
axonal processes back into the molecular layer. Maternal starvation
is capable of delaying andror arresting cerebellar development
resulting in distinctive changes in the cerebellar cellular
topography w8,30,31,37,52 . Maternal protein restriction in the rat
durxing late gestation and the first 2 weeks of postnatal life
resulted in a 40% decrease in cerebellar DNA and protein levels and
a reduction in molecular layer thickness 42 .w xPrevious studies
have documented the induction of specific CDKIs during
differentiation in cultured cells. The aim of these studies was
firstly to systematically assess the expression profile of the
known members of the CDKI family in vivo. It was hypothesized that
the sustained induction of the CDKIs would be required for the
maintenance of the differentiated neural phenotype. These studies
also aimed to utilize the distinctive topographic changes that
occurred during postnatal cerebellar development to identify the
cell types expressing the CDKIs. It was hypothesized that the
induction of the CDKIs in the proliferating external granular
cells, expressing the CDKs, may be responsible for the cessation of
cellular proliferation and subsequent differentiation. Finally,
these studies aimed to determine whether starvation, that induced
developmental arrest, induced the CDKI, thereby directly blocking
proliferation in the affected cells.Materials and methods2.1.
Animals and tissue preparationAll dietary manipulations and
procedures described were approved by the Animal Care Committee of
the Veterans Administration Lakeside Medical Center. The details of
nutritional manipulation of the Female SpragueDawley rats weighing
150250 g Holtzman, Madison, WI. used in these studies were
previously described 41 . Virus-freew x female SpragueDawley rats
weighing 150250 g Holtz- man, Madison, WI were housed at 21. "18C
with alternating lightdark cycles of 12 h each. Animals were
received on gestational day 17 delivery day 21 r22.. The standard
diet PROLAB-3000 Agway Country Foods, Syracuse, NY , contained 22%
protein. The control diet,.a960260, ICN Biochemicals, Cleveland, OH
, was a 20% protein. pellet diet and the test experimental diet,
a960254., contained 4% protein supplemented with additional
carbohydrates to make equal weights of the pellets isocaloric with
the test control.Pregnant rats were randomly assigned on
gestational day 21 to either the experimental group, assigned a 4%
protein diet, or the controls which were given a 20% protein diet.
For refeeding, 8-day-old animals from malnourished mothers were
foster fed to a control mother.Non-pregnant females at 60 days of
age and nursing mothers were anesthetized with carbon dioxide prior
to sacrifice. Pups were weighed, then sacrificed on postnatal days
4 through 24 days as indicated in the figures. Animals were
anesthetized with carbon dioxide prior to sacrifice and following
decapitation, the brain including the cerebellum was removed and
tissues placed on a block of dry ice. Following decapitation, the
brain including the cerebellum was removed and tissues placed on a
block of dry ice. Frozen tissues were stored at y708C until ready
for sectioning. Sectioning of the lateral cerebellar hemispheres
was performed in the coronal plane beginning at the caudal surface
of the cerebellum at the vermis. Contiguous sections were stained
using specific antibodies as described below. Tissues were
sectioned in 8 mm thickness at y178C, and mounted on silan coated
slides. Slides were stored at y808C until ready for
immunohistochemical staining.2.2. Tissue cultureC2C12 myoblasts CRL
1772; American Type Culture Collection, Rockville, MD. were
maintained in growth media Dulbeccos modified Eagles medium with
4500 mgrl glucose wDMEM-H , 15% fetal bovine serum andx
antibiotics. Cells were grown on 150-mm tissue culture dishes at
low density and induced to differentiate by switching medium to
DMEM-H with 2% horse serum and 10 mgrml bovine insulin Life
Technologies, Gaithersburg, MD ..2.3. Western blots and
immune-complex assaysWestern blotting was performed as previously
described w59x using antibodies to p21 C-19 , p21 187 , p27 M-197,
. . C-19 , p16 C-20 -G, p16 M-156 and cyclin D1 HD-11 ,. . . .
Santa Cruz, CA , HD-1 a gift from Dr. J. Koh and Dr. E.. Harlow.
and horse radish peroxidase conjugated second antibody. In
addition, the p35 antibody 5H8, a gift from Li-Huei Tsai and Dr. M.
Nicolic , the p21 antibodies. Cp36 and CP68. a gift from Dr. B.
Dynlacht and tubulin. monoclonal antibody 5H1 . w60x were used in
Western blot analysis or immunoprecipitation kinase assays as
previously described. The p18 antibody 11256 and p19 12077 . .
antibodies 16 were a generous gift from Dr. Y. Xiong.w x Reactive
proteins were visualized by the enhanced chemiluminescence system
Amersham, Arlington Heights, IL. and quantitation was performed by
densitometry using a Bio-Rad Molecular Analysis.Immunoprecipitation
kinase assays were performed essentially as previously described
w29,58,59x using either Histone H1 Sigma, St. Louis, MO. or pRB
substrate prepared from the vector pGEX-Rb 14w x a gift from Dr. E.
Harlow . Cells were suspended at 1. =106 to 5=106rml in
immunoprecipitation RIPA buffer 150 mM NaCl, 1% . NP-40, 0.5%
Deoxycholate, 0.1% SDS, 50 mM Tris pH7.5. with 0.1 mM
phenylmethylsulfonylfluoride, 1 mg of leupeptin per ml, and 1 mM
sodium orthovanadate Sigma , . at 48C. Lysates were centrifuged at
10,000=g for 5 min and the supernatants were precipitated for 4 h
at 48C with protein A- sepharose beads precoated with saturating
amounts of the cyclin D1 antibody, DCS-11 NeoMarker, Fremont, CA .
Phosphorylated proteins were visualized. after exposure to KODAK
XAR film and quantitation performed by densitometry Image Quant
version 1.11, Molecular Dynamics Computing Densitometer, Sunnyvale,
CA ..2.4. Immunohistochemical stainingImmunohistochemical analysis
was performed with the cyclin D1 and the CDKI p27, p21 and p16.
antibodies essentially as previously described w41x. The cyclin D1
antibody clone DSC-6 was purchased from Vector laboratories
Novacastra Laboratories, Newcastle upon Tyne, UK and this antibody
was used at a 1:10 dilution. The p16. M-156. and the p21 C-19.
antibodies were purchased from Santa Cruz Biotechnology Santa Cruz,
CA . The. CDKI antibodies were used at a 1:50 dilution.
Immunohistochemical analysis was carried out using a biotinylated
secondary antibody, and an avidinrbiotin-linked horseradish
peroxidase Vectastain ABC system, Vector laboratories, Burlingame,
CA . The complex was stained. with diaminobenzamidine
tetrahydrochloride DAB. substrate obtained from Kirkegaard and
Perry laboratories Gaithersburg, MD . The sections were wiped dry
then. fixed in either acetone for 20 min at 48C, followed by
rehydration in phosphate buffered saline PBS , or fixation . with
formalinracetonermethanol 2r19r19 vrv. for 5 min, at 48C, followed
by a PBS rinse, then in methanol for 10 min followed by rehydration
in PBS 41 . Rehydratedw x samples were blocked with serum directed
against the species producing the secondary antibody. Control sera
included either normal rabbit serum or isotype matched monoclonal
antibodies . Following a 15 min incubation at. 378C, the primary
antibody was added and sections incubated for 24 h at 48C.
Following incubation with the primary antibodies, slides were
rinsed in PBS then treated with a biotinylated secondary antibody
and held at 378C for 15 min, washed again in PBS, and incubated
with horseradish peroxidase in an avidinbiotin matrix. Following a
15-min incubation with a biotinylated secondary antibody and horse
radish peroxidase, the slides were immersed in DAB 100 mg in 200 ml
0.1 M phosphate buffer pH 7.3 to which was added 5 ml 1% cobalt
chloride then 4.0 ml of 1% nickel ammonium sulfate 1 . Optimalw x
incubation time was found to be 15 min. Then 0.66 ml 3% hydrogen
peroxidase was added and slides were incubated at room temperature
for 5 min. The slides were then placed in water for 2 min and
stained for 3 min with neutral red or 1 min with light green.
Slides were then placed in alcohol and dehydrated with
isopropanol.Results3.1. Cdk5 and cyclin D1 leels are inersely
related during cerebellar deelopmentPrevious studies had examined
the expression of cdk5 mRNA in the developing embryonic brain and
in the adult cerebellum w22,55 . Western blotting was performed tox
examine the cdk5 protein abundance in the developing cerebellum.
Cdk5 protein was readily detectable at day 4 and cdk5 abundance
increased between postnatal days 9 and 13 Fig. 1a,b . We examined
the abundance of cyclin . D1 using a specific antibody HD-1 .
Cyclin D1 protein. abundance by Western blotting increased
transiently between days 4 and 9 postnatally, then decreased
between days 9 and 11 and subsequently into adulthood Fig. 1a,c , .
consistent with previous studies in which cyclin D1 mRNA levels
began to decrease between days 9 and 11 41 .w x3.2. p27 and p21 are
induced transiently during cerebellar deelopmentTo examine the
expression profile of p27 and p21 in the developing cerebellum,
Western blotting was performed. p27 levels increased 10-fold from
day 4 to day 9 Fig. 2a . . The induction of p27 was sustained
through day 24. In the
Fig. 1. Expression of cdk5 and cyclin D1 proteins during
postnatal cerebellar development. The mean values for Western blot
analysis of either b . cdk5 and .c cyclin D1 from cerebella or from
differentiated C2C12 cells a . The adjacent figures show the raw
data for the animals . at each time point. Fold induction is shown
normalized to the abundance of cyclin D1 in the adult as 1.
Fig. 2. Expression of p27 and p21 during postnatal cerebellar
development. Western blot analysis of a p27 and b p21 in the
developing rat . . cerebellum. The data are also shown as
normalized mean data for the separate animals at each time
point.adult, p27 levels were reduced to levels approaching that
seen at day 4 Fig. 2a . p21 abundance increased from. days 6
through 9 and then decreased to very low levels in the adult Fig.
2b . .3.3. p18 leels are relatiely constant during cerebellar
deelopmentp19 and p18 were readily detected in the day 4
cerebellum. Between days 4 and 9, the abundance of p19 and p18
changed relatively little compared with the changes in p27 Fig. 2a
observed during this time Fig. 3a,b . p19 levels. . increased
twofold between days 4 and 9 and remained elevated until day 24,
decreasing by 50% in the adult Fig. 3a . Experiments were also
conducted in C2C12 myocytes. induced to differentiate by switching
to differentiation medium Fig. 3a, inset . The abundance of p19 in
cerebel- . lar samples was significantly less than that observed in
equal amounts of C2C12 cell extracts Fig. 3a inset . p19 . levels
decreased with the addition of differentiation medium in C2C12
cells Fig. 3a inset, and data not shown. con-Fig. 3. Expression of
Ink4 proteins during postnatal cerebellar development. Western blot
analysis of either a p19 b p18 from cerebella or c p16 levels . . .
from rat cerebella. The adjacent figures show data for the mean of
two to three separate animals. The inset of a shows a comparison of
the relative . abundance of p19 from differentiated C2C12 cells and
cerebella in which equal amounts of total protein were loaded. The
C2C12 cells were induced to
differentiate as described in Section 2.firming recently
published observations w16 . p18x levels were relatively constant
from days 4 through 24 then decreased by 50% from days 24 to the
adult levels Fig. 3b . These results contrast with the dramatic
increases in. p18 levels that occurred during C2C12 myocyte
differentiation 16 . p16 levels increased twofold between days 6w x
and 9 and remained elevated until day 16, after which time levels
decreased to barely detectable in the adult Fig. 3c . . p15 was
detected at low levels and increased between days 13 and 16 data
not shown . The peak induction of p15 at . days 13 to 16 was much
later than found with the other Ink4 family members. Together these
results indicate that the Ink4 proteins are induced at different
times in the developing cerebellum.Fig. 4. Immunohistochemical
localization of p27 in the developing cerebellum. Cyclin D1 a and
p27 b immunostaining is shown in the rat cerebellum at . . 11 days.
The external granular cells EG , the molecular layer M , and the
Purkinje cells P are shown. The cyclin D1 immunopositive cells,
indicated by. . . the arrow, are shown primarily in the external
granular cell layer and the p27 immunopositive cells, which are
yellowrbrown, are shown predominantly in
the Purkinje cells. The magnification is 400=.3.4.
Immunolocalization of p27, p21 and p16 in the deeloping rat
cerebellumImmunohistochemical studies were performed to identify
the cell type in which the CDKIs were expressed. The layers of the
cerebellum, external granular EG , molecular . .M , and the
Purkinje P are shown Fig. 4 . In cerebella . . at 11 days, cyclin
D1 staining was seen only in the external granular layer Fig. 4a .
p27 staining at this time . of development was seen predominantly
in the Purkinje cells with no staining apparent in the molecular or
external granular layers Fig. 4b . The predominant distribution of
. cyclin D1 and p27 are therefore in different cell types at the
same time during cerebellar development. p21 and p16 staining were
next examined in the developing cerebellum Fig. 5 . Comparison was
made between days 9 and 13, as. considerable differentiation during
this time results in increasing thickness of the molecular layer
Fig. 5a vs. b . . p21 staining at 9 days was seen in scattered
cells within the external granular layer, in occasional cells
migrating across the molecular layer and in Purkinje cells Fig. 5a
. . By 13 days, the predominant staining for p21 was local-
Fig. 5. Immunohistochemical analysis of p21 and p16 in the
developing cerebellum. The cell types expressing p21 a, b and p16
c, d in the developing . . rat cerebellum are shown. Immunostaining
is shown for days 9 a, c and 13 b, d . Positively staining cells
are brown cells indicated by the arrow. p21 . . staining at 9 days
a is found predominantly in the Purkinje cells P with some
immunopositive cells in the external granular layer EG . At 13
days, p21 . . . staining is found in the Purkinje cells b . The p16
positive cells at day 9 are located in several different cell
types, including the external granular cells . EG , the molecular
layer M , the Purkinje cells P and the internal granular cells IG .
At day 13, p16 staining is observed primarily in the Purkinje
cells. . . . with infrequent staining in the internal granular
layer. The magnification is 400=.
ized to the Purkinje cell layer Fig. 5b . The external. granular
layer was essentially free of p21 staining, with occasional cells
immunopositive for p21 found migrating across the molecular layer
Fig. 5b . In the sections of. 9-day cerebella Fig. 5c , p16
immunostaining was found . in both the external granular and the
Purkinje cell layer Fig. 5c . At 13 days, p16 staining was found
only in the. Purkinje cells, with no remaining staining in the
granular layer Fig. 5d . Together these studies indicate that in
the . developing cerebellum, cyclin D1 levels decrease as
expression of the CDKI p16 and p21 is induced and subsequently
maintained in the differentiated cells.3.5. Reduced cyclin D1
kinase actiity in protein-calorie malnutrition associated with
deelopment delayMalnutrition during critical periods of brain
development has adverse effects on the developing brain and can
Fig. 6. The effect of protein calorie malnutrition on cyclin D1
kinase activity and cdki abundance in the developing cerebellum. a
Cyclin D1 . immune kinase assays were performed on cerebella from
fed 20% and . protein calorie malnourished 4% animals. The cyclin
D1 immunoprecip- . itation was performed using the DCS-11 antibody
and GST-pRB substrate as described in Section 2. The phosphorylated
pRB band is shown for animals either fed 20% or starved 4% animals.
In b d Western . . . . blotting was performed on cell extracts
derived from the cerebella of fed or starved animals. The abundance
of p27, p21, and p16 is shown graphically as mean data.result in
lasting deficits 12 . In the neonatal rat, cerebellarw x growth
increases more rapidly during the first two postnatal weeks than
the remainder of the brain. Changes in cerebellar weight during
malnutrition are relatively greater than in extracerebella regions
of the brain w36,42x and malnutrition results in defective
maturation of Purkinje cell circuitry 43 . In previous studies,
nutritional deprivationw x markedly reduced cerebella mass, reduced
cellular proliferation and resulted in a delay in neural maturation
7,41,48 .w x The effects of protein-calorie deprivation were
manifested by a marked decrease in the width of the molecular
layer, and thinning of the external granular layer 41 . To deter-w
x mine whether nutritional deprivation-induced growth arrest was
associated with changes in cyclin D1 kinase activity,
immune-precipitation kinase assays were performed on the cerebella
of either wild-type or nutritionally-deprived animals. Cyclin
D1-immune kinase activity CD1K was de- . termined in the developing
rat cerebellum from fed controls 20% . A direct comparison between
the malnour.ished and control animals at day 11 demonstrated a 50%
decrease in CD1K in the malnourished animals Fig. 6a . .CD1K
activity is determined in part by the relative abundance of cyclin
D1 and the CDKIs. Because CD1K activity was reduced with
malnutrition we examined the abundance of the CDKIs to determine
whether an induction of the CDKIs contributed to the reduction in
CD1K activity during starvation. p27 levels were reduced to 30%,
and p16 and p21 levels were unchanged Fig. 6bd . As . previous
studies had shown a reduction in cyclin D1 abundance in starved
animals at 11 days w41x together these studies suggest the
reduction in CD1K activity during starvation may be due to the
reduction in cyclin D1 abundance.DiscussionThe current studies were
performed in order to characterize the temporal and spatial profile
of the CDKIs in vivo during cerebellar development. It was
hypothesized that the sustained induction of the CDKIs may be
required for the maintenance of the differentiated phenotype. These
studies demonstrated that the induction of differentiation but not
the maintenance of the differentiated phenotype was associated with
the expression of the CDKIs. Secondly, we had hypothesized that the
induction of the CDKIs within the proliferating external granular
cells may attenuate their proliferation resulting in cell-cycle
arrest and subsequent differentiation. Although the p27 levels
increased by Western blotting, the results of the immunostaining
suggested that the induction of the CDKI, in particular p27, did
not occur in the external granular cells, either within the
proliferating external layer or the inner postmitotic layer. These
results suggest that p27, although previously described as capable
of inducing cellular arrest in other cell types, is not directly
involved in this process of cellular proliferative arrest in the
external granular cells of the cerebellum. The induction of p16 and
p21 may contribute to the cellular arrest and the subsequent
induction of differentiation. Finally, we hypothesized that
maternal starvation may induce the CDKIs, and thereby contribute to
the growth arrest observed in vivo. Although the phosphorylation of
pRB, assessed through immune-precipitation kinase assays, decreased
with starvation, the CDKIs were not induced. In conjunction with
previous studies demonstrating a reduction in cyclin D1 protein
levels upon maternal starvation 41 , these results suggest the
growthw x arrest may involve a reduction in proliferation secondary
to a fall in the abundance of cyclin D1.The relative abundance of
cyclin D1 compared with the CDKIs, decreased during
differentiation, is consistent with several other models of
differentiation including cultured myocytes. Cyclin D1 protein
abundance gradually decreased in the developing cerebellum
consistent with previous studies showing a reduction in cyclin D1
mRNA levels during postnatal cerebellar development. In previous
studies cyclin D1 was expressed in the proliferating external
granular cells, but not in the molecular layer derived from the
external granular cells 41 . Recent studies performedw x with
cultured rat neural cells demonstrated a decrease in cyclin D1
levels as the oligodendrocyte precursor cells differentiated into
non-dividing oligodendrocytes w13 .x Similar types of observation
have been made during myocyte differentiation, wherein cyclin D1
mRNA levels decreased as myotubules form 19,35,49 . cdk5 levels
in-w x creased gradually in the developing cerebellum, with
increased levels maintained in the adult. cdk5 associates with
cyclin D1 and p35 63 . When bound to cyclin D, cdk5w x associates
with p27, p21 and p57 63 .w xIn our studies, p27 levels increased
in postmitotic neurons, commensurate with increased cdk5 activity
and reduced cyclin D1 levels. p27 was found in the Purkinje cells
and some cells of the internal granular layer. In our studies,
total cerebella p27 protein levels were induced 20-fold,
concurrently with differentiation of the external granular cells.
p27 and cyclin D1 did not co-localize in the proliferating external
granular cells, however, suggesting p27 is not involved in
proliferative arrest of these cells. Furthermore, p27 levels were
reduced in the adult cerebellum suggesting that p27 function is not
required for the maintenance of the terminally differentiated
neural phenotype. These findings contrast with cultured myocytes in
which the induction of p27 abundance was induced in a sustained
manner suggesting a role for p27 in both the induction and
maintenance of the differentiated phenotype w16,19 . We had
hypothesized that p27 level may bex increased in the postmitotic
layer of the external granular cells implicating p27 in the
proliferative arrest of these cells because p27 can inhibit cyclin
D1rcdk4 activity and cellular proliferation in vitro and in
cultured fibroblasts w54x. Recent observations, however, suggest
that p27 is present in active kinase complexes in proliferating
cells w50x and can bind cyclin D1rcdk4 without inhibiting its
kinase activity, functioning as an assembly factor 24,39 .w x
Alternatively, the accumulation of p27 may occur as part of an
intrinsic counting system that determines when a precursor cell
stops proliferating and terminally differentiates 13 . p27 levels
accumulated in proliferating oligoden-w x drocyte precursor cells
and p27 was also present in differentiated oligodendrocytes w13x,
thus the role of p27 in proliferative arrest may be cell-type
specific.The induction of p21 in the developing cerebellum peaked
at 9 days and then decreased in abundance. During myocyte
differentiation the abundance of p21 was increased in a sustained
manner, implicating this protein in maintenance of the
differentiated phenotype w19,35,49 .x Homozygous disruption of p21
did not block normal myocyte differentiation, however, and p21
expression occurred after myoblasts began differentiating 10 .
Althoughw x functional redundancy may contribute to the lack of
phenotype in these animals, these results suggest p21 may not be
important in the maintenance of the differentiated phenotype. In
the present studies, p21 was found in the external granular cells
and was also found in the molecular and internal granular cells.
p21 was therefore found in cell types known to be proliferating and
differentiating. The expression of p21 is known to occur in
proliferating cells with maximal synthesis during G -phase and the
nadir1 during S-phase 27,33 . In addition to binding and inhibit-w
x ing G cyclin1 rcdk complexes, p21 also binds the processivity
factor proliferating cell nuclear antigen PCNA. w19,35,49,57 . In
previous studies, PCNA was also localxized to the proliferating
external granular cells 41 and itw x will be of interest to
determine whether p21 is bound to PCNA in these cells. The presence
of p21 in the differentiated cerebellar cells may be the result of
its function to induce the differentiated phenotype, as suggested
in myocytes 19,35,49 . Alternatively, recent studies in the neu-w x
roblastoma cell line, SH-SY5Y, demonstrated a neuronal survival
function of p21, inhibiting programmed cell death w38 . p21x may
convey a similar function in the developing cerebellum.p18 levels
were relatively unchanged between days 4 to 24 and then decreased
50% in the adult. p19 increased between days 4 and 9 postnatally,
and then decreased to low levels in the adult. These results
contrast with differentiating cultured myocytes in which we
observed increased p18 levels and decreased p19 levels continually
upon differentiation as previously described 16 . As the magni-w x
tude of induction during differentiation is greater than 50-fold,
p18 is thought to function as the major CDK inhibitor involved in
myogenic differentiation. In contrast, the abundance of the other
CDKIs including p27 was only modestly altered w16x. Both p18 and
p19 inhibit cyclin D-dependent kinases, and overexpression of p18
or p19 induces G -phase arrest 18,21 . Because cyclin D1 levels1 w
x decreased from day 9, the relative abundance of p18 and p19
compared with cyclin D1 increased during the time of
differentiation after day 9, consistent with a model in which the
relative abundance of the CDKI compared with the CDK holoenzyme,
increase in paradigms of differentiation. The role of additional
cyclins such as p39, which plays a role in models of hippocampal
cell differentiation w62x, remains to be investigated in cerebellar
development.The molecular mechanisms by which protein calorie
malnutrition arrests cerebellar development is complex. In these
studies, starvation was associated with a 60% reduction in CD1K
activity. The reduction in CD1K activity associated with
malnutrition is consistent with several studies in which growth
factors induced CD1K activity reviewed in Ref. 45 . A reduction in
CD1K activity mayw x. be due to an increase in the CDKI abundance
or a decrease in cyclin D1rcdk abundance. In the current studies,
in the cerebellum of starved animals, p16 and p21 levels were
unchanged, p27 levels decreased, and in previous studies, cyclin D1
immunopositivity was decreased w2,41 . Tox gether these results
suggest the reduction in CD1K activity found in the starved animals
cerebella may be due to reduced cyclin D1 protein levels rather
than an induction in CDKI abundance. Insulin-like growth factor I
IGF-I. plays an important role in the normal growth of the
developing cerebellum, and malnutrition is associated with reduced
bioavailable IGF-I. Cyclin D1 is induced by IGF-I w2,41 and has a
short half life upon withdrawal of growthx factors 11 . The
reduction in cyclin D1 protein levels inw x the cerebella of
starved animals is consistent with a model in which cyclin D1
levels are induced in the developing cerebella by IGF-I.In summary,
these studies demonstrate that postnatal cerebellar development is
associated with an increase in cdk5 abundance and transiently
enhanced expression of each of the CDKI examined. The temporal
profile of expression of each of the CDKIs was distinct, consistent
with different roles for each CDKI in promoting but not in
maintaining the differentiated phenotype. These studies provide
useful information about the temporal and spatial profile of
expression of the CDKI proteins during cerebellar development and
provide supportive evidence that the CDKI may play a role in the
induction of differentiation of specific cell
types.AcknowledgementsWe thank Dr. Li-Huei Tsai, Dr. M. Nicolic,
Dr. B. Dynlacht and Dr. L. Binder for gifts of antibodies and cell
lines, and Dr. A. Koch and Dr. E. Mugnaini for their expert advice.
This work was supported in part by the NIH FIRST award 1R29CA70897
and grant R55CA75503 to R.G.P. and Merit Reviews from the
Department of Veter-. ans Affairs G.E.S. . Work at the Albert
Einstein College . of Medicine was also supported by Cancer Center
Core National Institute of Health grant 5-P30-CA13330-26.References
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