Patterning and differentiation signals are often believed to drive the developmental program, including cell cycle exit of proliferating progenitors. Taking advantage of the spatial and temporal separation of proliferating and differentiated cells within the developing anterior pituitary gland, we investigated the control of cell proliferation during organogenesis. Thus, we identified a population of noncycling precursors that are uniquely marked by expression of the cell cycle inhibitor p57Kip2 and by cyclin E. In p57Kip2−/− mice, the developing pituitary is hyperplastic due to accumulation of proliferating progenitors, whereas overexpression of p57Kip2 leads to hypoplasia. p57Kip2-dependent cell cycle exit is not required for differentiation, and conversely, blockade of cell differentiation, as achieved in Tpit−/− pituitaries, does not prevent cell cycle exit but rather leads to accumulation of p57Kip2-positive precursors. Upon differentiation, p57Kip2 is replaced by p27Kip1. Accordingly, proliferating differentiated cells are readily detected in p27Kip1−/− pituitaries but not in wild-type or p57Kip2−/− pituitaries. Strikingly, all cells of p57Kip2−/−;p27Kip1−/− pituitaries are proliferative. Thus, during normal development, progenitor cell cycle exit is controlled by p57Kip2 followed by p27Kip1 in differentiated cells; these sequential actions, taken together with different pituitary outcomes of their loss of function, suggest hierarchical controls of the cell cycle that are independent of differentiation.
Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57Kip2 and p27Kip1, control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57Kip2 remain poorly defined. Using in vivo and culture approaches, we show p57Kip2 overexpression at E14.5–15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57Kip2-deficient precursors. Studies at later ages indicate p57Kip2 overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57Kip2 overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57Kip2 was twice as effective as p27Kip1 in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57Kip2 alone inhibits in vivo migration. Furthermore, p57Kip2 effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27Kip1 studies report cell cycle-independent functions. These observations suggest p57Kip2 coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27Kip1.
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27Kip1 having the broadest expression, and both p57 kip2 and p21Cip1 showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57kip2; p27kip1double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity CyclinD1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57kip2 in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.
Neurogenesis; stem cells; proliferation; neural tube; differentiation; progenitor cyclin; p57kip2; p27kip1; p21Cip1
Neuronal progenitor cells of the anterior subventricular zone (SVZa) migrate along the rostral migratory stream (RMS) to the olfactory bulb, where they exit the cell cycle and differentiate. The molecular mechanisms that regulate SVZa progenitor proliferation and cell cycle exit are largely undefined. We investigated the role of p27KIP1 in regulating cell proliferation and survival in the RMS and olfactory bulb between postnatal day 1 (P1) and P14, the peak period of olfactory bulb neuron generation. A large proportion of cells in the RMS and the olfactory bulb express cytoplasmic p27KIP1, but a small percentage display high nuclear p27KIP1 immunostaining, which exhibit a caudallow-rostralhigh gradient: lowest in the SVZa and highest in the glomerular layer of the olfactory bulb. p27KIP1 is also present in the nucleus and/or the cytoplasm of neuron-specific type III β-tubulin(+) cells. Cells with strong nuclear p27KIP1 expression are BrdU(−) and Ki67(−). The percentage of BrdU(+) cells in the SVZa, RMS and olfactory bulb is higher in p27KIP1 null than wild type (WT) mice at all ages analyzed. Consistent with these findings, p27KIP1 overexpression in cultured p27KIP1 null and WT SVZ cells reduced cell proliferation and self-renewal. Finally, in p27KIP1 null mice, the diameter of the horizontal limb of the RMS is larger than in WT mice, and development of the olfactory bulb granule cell layer is delayed, together with increased apoptotic cell density. Our results indicate that in the postnatal brain p27KIP1 regulates the proliferation and survival of neuronal cells in the RMS and olfactory bulb.
cell differentiation; cell proliferation; cell survival; cyclin dependent kinase inhibitor; neuronal progenitor cells; subventricular zone
To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27KIP1. Generation of Mad1/p27KIP1 double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27KIP1, including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27KIP1 double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27KIP1 single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27KIP1 operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.
Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.
proliferation; cell cycle; p27; Cdk inhibitor; auditory; cochlea; pituitary
The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.
Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27Kip1 is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27Kip1 transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27Kip1 transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27Kip1 transcription, G1 arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27Kip1 suppresses p27Kip1 transcription and neuronal differentiation, suggesting a causal link between p27Kip1 expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27Kip1 transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27Kip1 promoter in vivo and mediates p27Kip1-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27Kip1 transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.
The cyclin-dependent kinase inhibitor (CKI) p57Kip2 plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27Kip1 at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.
A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1.
The expression levels of the p21Cip1 family CDK inhibitors (CKIs), p21Cip1, p27Kip1 and p57Kip2, play a pivotal role in the precise regulation of cyclin-dependent kinase (CDK) activity, which is instrumental to proper cell cycle progression. The stabilities of p21Cip1, p27Kip1 and p57Kip2 are all tightly and differentially regulated by ubiquitylation and proteasome-mediated degradation during various stages of the cell cycle, either in steady state or in response to extracellular stimuli, which often elicit site-specific phosphorylation of CKIs triggering their degradation.
phosphorylation; ubiquitylation; proteasome; p21Cip1; p27Kip1; p57Kip2
In developing brains, neural progenitors exhibit cell cycle-dependent nuclear movement within the ventricular zone [interkinetic nuclear migration (INM)] and actively proliferate to produce daughter progenitors and/or neurons, whereas newly generated neurons exit from the cell cycle and begin pial surface-directed migration and maturation. Dysregulation of the balance between the proliferation and the cell cycle exit in neural progenitors is one of the major causes of microcephaly (small brain). Recent studies indicate that cell cycle machinery influences not only the proliferation but also INM in neural progenitors. Furthermore, several cell cycle-related proteins, including p27kip1, p57kip2, Cdk5, and Rb, regulate the migration of neurons in the postmitotic state, suggesting that the growth arrest confers dual functions on cell cycle regulators. Consistently, several types of microcephaly occur in conjunction with neuronal migration disorders, such as periventricular heterotopia and lissencephaly. However, cell cycle re-entry by disturbance of growth arrest in mature neurons is thought to trigger neuronal cell death in Alzheimer's disease. In this review, we introduce the cell cycle protein-mediated regulation of two types of nuclear movement, INM and neuronal migration, during cerebral cortical development, and discuss the roles of growth arrest in cortical development and neurological disorders.
The cyclin/cyclin-dependent kinase (cdk) inhibitor p27kip1 is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27kip1 in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27kip1 on cell cycle traverse is determined by cell density. We found that ectopic expression of p27kip1 blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27kip1 expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27kip1 but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27kip1-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.
Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development.
The tumor suppressive activities of the Kip-family of cdk inhibitors often go beyond their role in regulating the cell cycle. Here, we demonstrate that p27 enhances Rad51 accumulation during repair of double-strand DNA breaks. Progression of PDGF-induced oligodendrogliomas was accelerated in mice lacking the cyclin-cdk binding activities of p27kip1. Cell lines were developed from RCAS-PDGF infection of nestin-tv-a brain progenitor cells in culture. p27 deficiency did not affect cell proliferation in early passage cell lines; however, the absence of p27 affected chromosomal stability. In p27 deficient cells, the activation of Atm and Chk2, and the accumulation of γH2AX was unaffected compared to wild type cells, and the number of phospho-histone H3 staining mitotic cells was decreased, consistent with a robust G2/M checkpoint activation. However, the percentage of Rad51 foci positive cells was decreased, and the kinase activity that targets the C-terminus of BRCA2, regulating BRCA2/Rad51 interactions, was increased in lysates derived from p27 deficient cells. Increased numbers of chromatid breaks in p27 deficient cells that adapted to the checkpoint were also observed. These findings suggest that Rad51-dependent repair of double stranded breaks was hindered in p27 deficient cells, leading to chromosomal instability, a hallmark of cancers with poor prognosis.
p27; tumor suppressor; DNA repair; glioma; mouse model
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.
Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.
Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.
Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
cell cycle regulator; cyclin-dependent kinase inhibitor; Kip1; compensatory growth; liver regeneration; rat hepatocytes; cell proliferation
p27Kip1 plays a major role as a negative regulator of the cell cycle. The regulation of p27Kip1 degradation is mediated by its specific ubiquitin ligase subunits S-phase kinase protein (Skp) 2 and cyclin-dependent kinase subunit (Cks) 1. However, little is known regarding the prognostic utility of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma.
Immunohistochemistry was performed for p27Kip1, Skp2 and Cks1 in tissue microarrays of 482 renal cell carcinomas with follow-up. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.
Immunoreactivity of p27Kip1, Skp2 and Cks1 was noted in 357, 71 and 82 patients, respectively. Skp2 and Cks1 expression were not noted in chromophobe cancers. A strong correlation was found between Skp2 and Cks1 expression (P < 0.001), both of which were inversely related to p27Kip1 levels (P = 0.006 and P < 0.001), especially in primary and clear-cell cancers. Low p27Kip1 expression and Skp2 expression were correlated with larger tumor size and higher stage, as well as tumor necrosis. Cks1 expression was only correlated with tumor size. In univariate analysis, low p27Kip1 expression, Skp2 and Cks1 expression were all associated with a poor prognosis, while in multivariate analysis, only low p27Kip1 expression were independent prognostic factors for both cancer specific survival and recurrence-free survival in patients with RCC.
Our results suggest that immunohistochemical expression levels of p27Kip1, Skp2 and Cks1 may serve as markers with prognostic value in renal cell carcinoma.
To investigate how overexpression of p27KIP1, a downstream effector of TGF-beta and a universal cyclin-dependent kinase (CDK) inhibitor could influence the malignant phenotype of malignant human brain tumor cells, an adenovirus vector system was used to transfer the human p27KIP1 gene (Adp27KIP1) into the human astrocytoma cell line, U-373MG. Inhibition of CDK activity in Adp27KIP1-infected cells was indicated by inhibition of [3H]thymidine incorporation, an increase in cell doubling time and by cell cycle arrest in G1. Notably, ectopic overexpression of p27KIP1 was associated with a marked decrease in the accumulation of aneuploid cells. Diminished malignant potential of Adp27KIP1-infected cells was manifested by the loss of anchorage-independent growth in soft agar and by the inability to induce tumorgenesis in a xenograft model. These studies suggest that p27KIP1 is a tumor suppressor gene and supports the use of Adp27KIP1 for gene therapy of human brain tumors.
p27kip1 is a cyclin-dependent kinase inhibitor that regulates progression from G1 into S phase. Aberrations in cell cycle control are often observed in tumors and might even be necessary in tumor development. Recent reports showed that low p27kip1 expression is associated with poor prognosis in several tumors and leukemia. To investigate the expression of p27kip1 in malignant lymphomas and elucidate the role of p27kip1 as a possible prognostic indicator, the authors performed an immunohistochemical staining of p27kip1 correlated with Ki-67 labelling index and clinical parameters. p27kip1 expression was reduced variably in most malignant lymphomas and inversely correlated with Ki-67 labelling index (p=0.0151). Regarding chemotherapeutic response, p271kip1 expression in the complete remission group showed statistically significant difference in expression compared to the progressive disease group (p=0.0021). There were significant differences in survival between cases with low and high p27kip1 expression (p=0.0071). In a multivariate Cox analysis, p27kip1 expression was independent prognostic factors as well as other known prognostic factors including age, grade, stage and chemotherapeutic response. In conclusion, the study suggests that reduced expression of p27kip1 protein may play a role in the pathogenesis and biologically aggressive behavior of malignant lymphomas.
Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. The loss of Cks1 results in p27Kip1 accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27Kip1. Specifically, Cks1−/−; p27Kip1−/− mouse embryonic fibroblasts retain defects in the G1-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27Kip1 that is manifest in various organs of p27Kip1−/− mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1−/− cells are p27Kip1 dependent. Collectively, these findings establish p27Kip1-independent functions of Cks1 in regulating the G1-S transition.
The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.
p27Kip1 levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27Kip1 levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27Kip1 enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27Kip1. Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27Kip1 can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27Kip1 is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27Kip1 increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter. p27Kip1 leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27Kip1 involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.
Myocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.
Transgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 ± 15 vs 30 ± 6 mmHg, p = 0.05), rate pressure product (22.8 ± 4.86 vs 10.4 ± 2.1 × 103bpm × mmHg, p < 0.05) and coronary flow (3.5 ± 0.5 vs 2.38 ± 0.24 ml/min, p <0.05).
These data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.
A delicate balance between proliferation and differentiation must be maintained in the developing pituitary to ensure the formation of the appropriate number of hormone producing cells. In the adult, proliferation is actively restrained to prevent tumor formation. The cyclin dependent kinase inhibitors (CDKIs) of the CIP/KIP family, p21, p27 and p57, mediate cell cycle inhibition. Although p21 is induced in the pituitary upon loss of Notch signaling or initiation of tumor formation to halt cell cycle progression, its role in normal pituitary organogenesis has not been explored. In wildtype pituitaries, expression of p21 is limited to a subset of cells embryonically as well as during the postnatal proliferative phase. Mice lacking p21 do not have altered cell proliferation during early embryogenesis, but do show a slight delay in separation of proliferating progenitors from the oral ectoderm. By embryonic day 16.5, p21 mutants have an alteration in the spatial distribution of proliferating pituitary progenitors, however there is no overall change in proliferation. At postnatal day 21, there appears to be no change in proliferation, as assessed by cells expressing Ki67 protein. However, p21 mutant pituitaries have significantly less mRNA of Myc and the cyclins Ccnb1, Ccnd1, Ccnd2 and Ccne1 than wildtype pituitaries. Interestingly, unlike the redundant role in cell cycle inhibition uncovered in p27/p57 double mutants, the pituitary of p21/p27 double mutants has a similar proliferation profile to p27 single mutants at the time points examined. Taken together, these studies demonstrate that unlike p27 or p57, p21 does not play a major role in control of progenitor proliferation in the developing pituitary. However, p21 may be required to maintain normal levels of cell cycle components.
pituitary; cell cycle; p21; p27
Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27Kip1, which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27Kip1 levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27Kip1 cDNA inhibited serum-stimulated [3H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [3H]leucine/[3H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27Kip1 in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27Kip1 expression. Ang II stimulated an increase in [3H]thymidine incorporation and the percentage of S-phase cells in antisense ODN–transfected cells but not in control ODN–transfected cells. We conclude that p27Kip1 plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27Kip1 levels are high results in hypertrophy but promotes hyperplasia when levels of p27Kip1 are low, as in the presence of other growth factors.