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CDK inhibitors, cell cycle arrest and differentiation (part 2/2) Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter
via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This
adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Stable transfection and
overexpression of the integrin-linked kinase (ILK), which interacts with the beta1 and beta3 integrin cytoplasmic domains (See Drosophila Myospheroid), induces
anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK
overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and
hyperphosphorylation of the retinoblastoma protein. In addition, ILK overexpression results in the expression of p21 and p27 Cdk
inhibitors with altered electrophoretic mobilities, with the p27 from ILK-overexpressing cells having reduced inhibitory activity. The
transfer of serum-exposed IEC18 cells from adherent cultures to suspension cultures results in a rapid down-regulation of expression
of cyclin D1 and cyclin A proteins, as well as in retinoblastoma protein dephosphorylation. In marked contrast, transfer of
ILK-overexpressing cells from adherent to suspension cultures results in continued high levels of expression of cyclin D1 and cyclin A
proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These results indicate that,
when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S cyclin-Cdk activities, which are normally
regulated by cell adhesion and integrin engagement (Radeva, 1997).
The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by
binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and
cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell
shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects
during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled
by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic
dishes coated with defined ECM molecular coating densities. Cells that are prevented from spreading in medium containing soluble growth factors
exhibit normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells fail to
progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlates with the failure of three activities: (1) an increase in cyclin D1 protein levels; (2) the
down-regulation of the cell cycle inhibitor p27(Kip1), and (3) the phosphorylation of the retinoblastoma protein in late G1. A similar block in cell cycle progression is
induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation
using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension have any effect
on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for
growth, these events alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation is controlled by tension-dependent changes in cell
shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition (Huang, 1998).
p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in
response to DNA damage. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases, but is much
less effective toward Cdc2/cyclin B and Cdk5/p35,
and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus,
p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes.
Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by
the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition.
With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has
slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active
Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of
exogenous p21. Reconstruction experiments using purified components indicate that multiple
molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more
than one molecule of p21. Together, these data suggest a model whereby p21 functions as an
inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle
progression (Harper, 1995).
During development of the central nervous system, oligodendrocyte progenitor cells (O-2A) undergo an
orderly pattern of cell proliferation and differentiation, culminating in the ability of oligodendrocytes to
myelinate axons. p27(Kip1), a cyclin-dependent kinase inhibitor, is an important
component of the decision of O-2A cells to withdraw from the cell cycle. In vitro, accumulation of p27
correlates with differentiation of oligodendrocytes. Only a fraction of O-2A cells derived
from p27-knockout mice differentiate successfully, as compared to controls. The inability to differentiate
correlates with continued proliferation, suggesting that p27 is an important component of the machinery
required for the G1/G0 transition in O-2A cells. In vivo, expansion of O-2A precursors before
withdrawal leads, in part, to a greater number of oligodendrocytes. Together, these data indicate that in the oligodendrocyte lineage, there is a role
for p27 during the decision to withdraw from the cell cycle (Casaccia-Bonnefil, 1997).
Strict control of cellular proliferation is required to shape
the complex structures of the developing embryo. The
organ of Corti, the auditory neuroepithelium of the inner
ear in mammals, consists of two types of terminally
differentiated mechanosensory hair cells and at least four
types of supporting cells arrayed precisely along the length
of the spiral cochlea. In mice, the progenitors of greater
than 80% of both hair cells and supporting cells undergo
their terminal division between embryonic day 13 (E13)
and E14. As in humans, these cells persist in a non-proliferative
state throughout the adult life of the animal. The correct timing of cell cycle
withdrawal in the developing organ of Corti requires
p27Kip1, a cyclin-dependent kinase inhibitor that functions
as an inhibitor of cell cycle progression. p27Kip1 expression
is induced in the primordial organ of Corti between E12
and E14, correlating with the cessation of cell division of
the progenitors of the hair cells and supporting cells. In
wild-type animals, p27Kip1 expression is downregulated
during subsequent hair cell differentiation, but it persists
at high levels in differentiated supporting cells of the
mature organ of Corti. In mice with a targeted deletion of
the p27Kip1 gene, proliferation of the sensory cell
progenitors continues after E14, leading to the appearance
of supernumerary hair cells and supporting cells. In the
absence of p27Kip1, mitotically active cells are still observed
in the organ of Corti of postnatal day 6 animals, suggesting
that the persistence of p27Kip1 expression in mature
supporting cells may contribute to the maintenance of
quiescence in this tissue and, possibly, to its inability to
regenerate. Homozygous mutant mice are severely hearing
impaired. Thus, p27Kip1 provides a link between
developmental control of cell proliferation and the
morphological development of the inner ear (Chen, 1999).
Different members of the Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S
phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promotes cell cycle progression, activation of
deltaRaf-1:ER and deltaB-Raf:ER elicit a G1 arrest that is not overcome by exogenously added growth factors.
Activation of all three deltaRaf:ER kinases leads to elevated expression of cyclin D1 and cyclin E and reduced expression of
p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induces the expression of p21Cip1, whereas activation of
deltaA-Raf:ER does not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induces
p21Cip1 expression and elicits cell cycle arrest similar to deltaB-Raf:ER and deltaRaf-1:ER. These data suggest that the
strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By
titration of deltaB-Raf:ER activity it was demonstrated that low levels of Raf activity lead to activation of cyclin D1-cdk4 and cyclin
E-cdk2 complexes and to cell cycle progression, whereas higher Raf activity elicits cell cycle arrest correlating with p21Cip1
induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse
embryo fibroblasts (MEFs) it was demonstrated that p21Cip1 is induced by Raf in a p53-independent manner, leading to cell
cycle arrest. By contrast, activation of Raf in p21Cip1-/- MEFs leads to a robust mitogenic response that is similar to that
observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf
can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is
strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that
bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both
proliferation and differentiation of mammalian cells (Woods, 1997).
Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and
terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the
retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by
cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a
skeletal muscle-specific transcriptional regulator, activates the expression of the Cdk inhibitor
p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated
induction of p21 does not require the tumor suppressor protein p53 and correlates with cell cycle
withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle
differentiation by increasing the expression of p21 (Halevy, 1995).
Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors, which are thought to be responsible for preventing these cells from reentering the cell cycle. The cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ (Lowenheim, 1999).
The proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system, are subject to regulation by the cyclin-dependent kinase inhibitor p27/Kip1. Two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes.The cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate
and is present at high levels in oligodendrocytes. These findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins, and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. Others have recently found that p27-deficient mice have an increased number of cells in all of the organs examined; this suggests that this
function of p27 is not restricted to the oligodendrocyte cell lineage (Durand, 1997).
gax is a relatively divergent Antp class homeobox gene with a homeodomain nearly identical to that of Mox-1, a recently described homeobox gene restricted to mesoderm and mesodermally derived tissue. Gax is less homologous to either proboscipedia (66% identity in the homeodomain) or to Deformed (61% identity in the homeodomain. gax is expressed in vascular smooth muscle cells (VSMCs) and
is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular
injury that leads to cellular proliferation. Recombinant Gax protein microinjected into
VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when
introduced either during quiescence or early stages of G1. Overexpression of gax with
a replication-defective adenovirus vector results in G0/G1 cell cycle arrest of
VSMCs and fibroblasts. The gax-induced growth inhibition correlates with a
p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax
overexpression also leads to an association of p21 with cdk2 complexes and a decrease
in cdk2 activity. Fibroblasts deficient in p21 are not susceptible to a reduction in cdk2
activity or growth inhibition by gax overexpression. Localized delivery of the virus to
denuded rat carotid arteries significantly reduces neointima formation and luminal
narrowing. These data indicate that gax overexpression can inhibit cell proliferation in
a p21-dependent manner and can modulate injury-induced changes in vessel wall
morphology that result from excessive cellular proliferation (Smith, 1997).
Much of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in the BRCA1
tumour-suppressor gene. The nuclear protein BRCA1 has the properties of a transcription factor, and can interact with the
recombination and repair protein RAD51. Young women with germline alterations in BRCA1 develop breast cancer at rates
100-fold higher than the general population, and BRCA1-null mice die before day 8 of development. However, the
mechanisms of BRCA1-mediated growth regulation and tumour suppression remain unknown. BRCA1
transactivates expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in a p53-independent manner, and
BRCA1 inhibits cell-cycle progression into the S-phase following its transfection into human cancer cells. BRCA1 does not
inhibit S-phase progression in p21-/- cells, unlike p21+/+ cells. Tumour-associated, transactivation-deficient mutants of
BRCA1 are defective in both transactivation of p21 and cell-cycle inhibition. These data suggest that one mechanism by which
BRCA1 contributes to cell-cycle arrest and growth suppression is through the induction of p21 (Somasundaram, 1997).
The cyclin-dependent kinase inhibitor p21(Cip1/WAF1) has been implicated as an inducer of differentiation. However, although expression of p21 is increased in postmitotic cells immediately adjacent to the proliferative compartment, its expression is decreased in cells further along the differentiation program. Expression of the p21 protein is decreased in terminally differentiated primary keratinocytes of mice, and this occurs by a proteasome-dependent pathway. Forced expression of p21 in these cells inhibits the expression of markers of terminal differentiation at both the protein and messenger RNA levels. These inhibitory effects on differentiation are not observed with a carboxyl-terminal truncation mutant or with the unrelated cyclin-dependent kinase inhibitor p16(INK4a), although all these proteins exert similar inhibition of cell growth. These findings reveal an inhibitory role for p21 in the late stages of differentiation that does not result from the effects of p21 on the cell cycle. An attractive model for future studies is that p21 may function as a specific bridge between signaling complexes (such as cyclin-CDK complexes and SAPKs) and other multiprotein apparatuses, such as the transcription machinery involved in differentation (Di Cunto, 1998).
Transient transfection of vectors
expressing neuroD2, MASH1, ngn1 or related neural
bHLH proteins, with their putative dimerization partner
E12, can convert mouse P19 embryonal carcinoma cells
into differentiated neurons. Transfected cells express
numerous neuron-specific proteins, adopt a neuronal
morphology and are electrically excitable. Pan-neuronal
markers such as neurofilament-M, the HuC/D RNA-binding
proteins, M6 and synapsin I are all present in most or all cells with
neuronal morphology. Moreover,
subsets of the transfected cells are immunoreactive for the
neurotransmitters GABA and glutamate, the GABA
synthetic enzyme glutamatic acid decarboxylase (GAD), the
neuropeptide substance P, NMDA receptor 1, and the
transcription factors Islet-1 or LIM1. At present,
it is not known if the subsets of cells that express these
proteins overlap. The same constellation of
neurotransmitters, receptors and other markers of mature
neuronal subtypes have been observed regardless of which neural bHLH vector is
transfected. No expression of glial fibrillary
acidic protein (GFAP) or markers of radial glia have been observed in any
transfected cells. Thus, the
expression of neural bHLH proteins is sufficient to confer
a neuronal fate on uncommitted mammalian cells.
Neuronal differentiation of transfected cells is preceded
by elevated expression of the cyclin-dependent kinase
inhibitor p27 Kip1 and cell cycle withdrawal. This
demonstrates that the bHLH proteins can link neuronal
differentiation to withdrawal from the cell cycle, possibly
by activating the expression of p27 Kip1. The ability to
generate mammalian neurons by transient expression of
neural bHLH proteins should create new opportunities
for studying neurogenesis and devising neural repair
strategies (Farah, 2000).
Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have
dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of
stem cells has been conjectured to be of critical biologic importance in protecting the stem cell
compartment; this has been directly assessed using mice engineered to be deficient in the G1 checkpoint
regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic
stem cell proliferation and absolute number are increased under normal homeostatic conditions.
Exposing the animals to cell cycle-specific myelotoxic injury results in premature death due to hematopoietic cell depletion. Further, self-renewal of
primitive cells is impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21
is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion.
Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death (Cheng, 2000).
A precise balance between proliferation and differentiation
must be maintained during retinal development to obtain
the correct proportion of each of the seven cell types found
in the adult tissue. Cyclin kinase inhibitors can regulate
cell cycle exit coincident with induction of differentiation
programs during development. The
p57 Kip2 cyclin kinase inhibitor is upregulated during G1/G0
in a subset of retinal progenitor cells exiting the cell
cycle between embryonic day 14.5 and 16.5 of mouse
development. Retroviral mediated overexpression of
p57 Kip2 in embryonic retinal progenitor cells leads to
premature cell cycle exit. Retinae from mice lacking
p57 Kip2 exhibit inappropriate S-phase entry and
apoptotic nuclei were found in the region where p57 Kip2 is
normally expressed. Apoptosis precisely compensates for
the inappropriate proliferation in the p57 Kip2-deficient
retinae to preserve the correct proportion of the major
retinal cell types. Postnatally, p57 Kip2 is
expressed in a novel subpopulation of amacrine
interneurons. At this stage, p57 Kip2 does not regulate
proliferation. However, perhaps reflecting its role during
this late stage of development, animals lacking p57 Kip2
show an alteration in amacrine subpopulations. p57 Kip2
is the first gene to be implicated as a regulator of amacrine
subtype/subpopulation development. Consequently, it is
proposed that p57 Kip2 has two roles during retinal
development, acting first as a cyclin kinase inhibitor in
mitotic progenitor cells, and then playing a distinct role in
neuronal differentiation (Dyer, 2000).
Several lines of evidence suggest that Cip/Kip family members
in general, and p57 Kip2 in particular, may be involved in
developmental processes beyond their prescribed roles as
cyclin kinase inhibitors. The Cip/Kip family members are
promiscuous kinase inhibitors and may regulate kinases
required for cell fate specification and/or differentiation during
development. In addition to their shared
cyclin kinase binding domains, the Cip/Kip proteins have
distinct, structurally complex domains that may prove to
contain novel biochemical properties.
This is especially true for p57 Kip2 , which is the most
structurally complex family member.
Several of the abnormalities observed in the p57 Kip2 knockout
mice (including defects in muscle, kidney, palate and
chondrocyte development) appear to not be linked to
proliferation defects. It has therefore been proposed that this protein
directly influences cell fate specification and/or differentiation
in these tissues. The Xenopus
cyclin kinase inhibitor p27 Xic1, which is a member of the
Cip/Kip family and is believed to be related to the mammalian
protein p27 Kip1, has been shown to induce the M¨ller glial cell fate
during retinal development. Consistent
with the idea that cyclin kinase inhibitors may be bifunctional
molecules, the Müller-inducing portion of the p27 Xic1 is
found to be separable from the portion of protein that induces
cell cycle exit. Cyclin kinase inhibitors
are not the only molecules that may regulate both cell cycle
progression and cell fate specification. A novel protein cloned
from Xenopus called geminin was found to contain a
neuralizing domain that was separable from a domain that has
been shown to be involved in the regulation of DNA replication. Taken together, these reports indicate that
cell cycle exit and cell fate specification are not only
coordinated temporally during development but that individual
molecules can play roles in both processes through distinct
protein domains (Dyer, 2000 and references therein).
The molecular basis of the antagonism between cellular proliferation and differentiation is poorly understood. The role of the cyclin-dependent kinase inhibitor p27Xic1 has been investigated in the co-ordination of cell cycle exit and differentiation during early myogenesis in vivo using Xenopus embryos. p27Xic1 is highly expressed in the developing myotome; ablation of p27Xic1 protein prevents muscle differentiation, and that p27Xic1 synergizes with the transcription factor MyoD to promote muscle differentiation. Furthermore, the ability of p27Xic1 to promote myogenesis resides in an N-terminal domain and is separable from its cell cycle regulation function. This data demonstrates that a single cyclin-dependent kinase inhibitor, p27Xic1, controls in vivo muscle differentiation in Xenopus and that regulation of this process by p27Xic1 requires activities beyond cell cycle inhibition (Vernon, 2003a).
The role of the cyclin-dependent kinase inhibitor, p27Xic1, has been investigated in the coordination of cell cycle exit and differentiation during early neurogenesis. p27Xic1 is highly expressed in cells destined to become primary neurons and is essential for an early stage of neurogenesis. Ablation of p27Xic1 protein prevents differentiation of primary neurons, while overexpressing p27Xic1 promotes their formation. p27Xic1 may enhance neurogenesis by stabilizing the bHLH protein, neurogenin, although the molecular mechanism of this stabilization is unknown. Moreover, the ability of p27Xic1 to stabilize neurogenin and enhance neurogenesis localizes to an N-terminal domain of the molecule and is separable from its ability to inhibit the cell cycle (Vernon, 2003b).
Precursors of cochlear and vestibular hair cells of the inner ear exit the
cell cycle at midgestation. Hair cells are mitotically quiescent during
late-embryonic differentiation stages and postnatally. The
retinoblastoma gene Rb and the encoded protein pRb are expressed in
differentiating and mature hair cells. In addition to Rb, the cyclin
dependent kinase inhibitor (CKI) p21 is expressed in developing hair
cells, suggesting that p21 is an upstream effector of pRb activity. p21
apparently cooperates with other CKIs, since p21-null mice exhibit an
unaltered inner ear phenotype. By contrast, Rb inactivation leads to
aberrant hair cell proliferation, as analysed at birth in a
loss-of-function/transgenic mouse model. Supernumerary hair cells express
various cell type-specific differentiation markers, including components of
stereocilia. The extent of alterations in stereociliary bundle morphology
ranges from near-normal to severe disorganization. Apoptosis contributes to
the mutant phenotype, but does not compensate for the production of
supernumerary hair cells, resulting in hyperplastic sensory epithelia. The
Rb-null-mediated proliferation leads to a distinct pathological
phenotype, including multinucleated and enlarged hair cells, and infiltration
of hair cells into the mesenchyme. These findings demonstrate that the pRb
pathway is required for hair cell quiescence and that manipulation of the cell
cycle machinery disrupts the coordinated development within the inner ear
sensory epithelia (Mantela, 2005).
These data show that the CKI p21 is expressed in the differentiating
cochlear and vestibular HCs, and that the expression is induced at the
initiation of HC differentiation. In the auditory sensory epithelium,
p21 expression is initiated at E14.5, at the stage when
Math1 expression is first detected. It is
possible that p21 induction in HCs is regulated by Math1, by
analogy to the positive role of bHLH proteins such as Myod1 and myogenin in
skeletal myogenesis. Thereafter, p21 together with other CKI(s)
might have an active role in keeping pRb in a hypophosphorylated form. Thus,
negative regulation at the level of both pRb and CKIs seems to be responsible
for the maintenance of HC quiescence. No phenotypic alterations or aberrant mitoses
were found in the inner
ears of developing or adult p21-/- mice. Interestingly, in
addition to p21, another CKI, p19, has been shown to be
expressed in the late-embryonic organ of Corti, but its inactivation does not
result in developmental abnormalities. Thus, functional redundancy may exist between
p21 and p19 in developing cochlear HCs. In addition,
developing vestibular HCs express p21, but do not show phenotypic
changes following targeted gene disruption, most probably owing to functional
compensation. The identity of the CKI that may cooperate with p21 in
vestibular HCs remains to be identified, since p19 expression and the
consequences of p19 inactivation have not been reported in vestibular
organs (Mantela, 2005).
Integrin-extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of beta1 integrin in the mammary gland, Itgbeta1flox/flox mice were crossed with WAPiCre transgenic mice, which led to specific ablation of beta1 integrin in luminal alveolar epithelial cells. In the beta1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell-basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in beta1 integrin mutant glands, which correlated with a specific increase of p21 Cip1 expression. In a p21 Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21 Cip1 to the proliferative defect observed in beta1 integrin mutant glands. A connection between p21 Cip1 and beta1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of beta1 integrin signaling in mammary epithelial cell proliferation (Li, 2005).
A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. This study addresses how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21cip1 and p57kip2), the Notch pathway and myogenic regulatory factors (MRFs) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. It was first shown that cell cycle exit and myogenic differentiation can be uncoupled. In addition, it was establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, it was demonstrated that Notch signaling is required to repress p21cip1 and p57kip2 expression in muscle progenitor cells. Finally, a muscle-specific regulatory element of p57kip2 directly activated by MRFs was identified in myoblasts but was found to be repressed by the Notch targets Hes1/Hey1 in progenitor cells. A molecular mechanism is proposed whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57kip2 enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation (Zalc, 2014).
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